阅读说明：本技术 突变型对羟苯基丙酮酸双氧化酶、其编码核酸以及应用 (Mutant p-hydroxyphenylpyruvate dioxygenase, nucleic acid encoding same and use thereof ) 是由 连磊 莫苏东 李华荣 苑广迪 李振国 张俊杰 丁德辉 陈波 刘桂智 宋超 王蕾 于 2019-01-28 设计创作，主要内容包括：本发明涉及突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白、其生物活性片段以及包含编码所述蛋白或片段的核酸序列的分离的多核苷酸,其中所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段保留或加强了其催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质,且对HPPD抑制剂的敏感性明显低于野生型HPPD。本发明还涉及包含所述多核苷酸的核酸构建体、表达载体和宿主细胞,以及生产具有催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质、同时对HPPD抑制性除草剂的敏感性明显降低的植物的方法。(The present invention relates to mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) proteins, biologically active fragments thereof, and isolated polynucleotides comprising nucleic acid sequences encoding the proteins or fragments, wherein the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) proteins or biologically active fragments thereof retain or potentiate their property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate and are significantly less sensitive to HPPD inhibitors than the wild-type HPPD. The invention also relates to nucleic acid constructs, expression vectors, and host cells comprising the polynucleotides as well as methods of producing plants having the property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate with substantially reduced sensitivity to HPPD inhibiting herbicides.)

1. A mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein, or a biologically active fragment thereof, wherein the amino acid sequence of said mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein has, in comparison with the amino acid sequence of a wild-type rice hydroxyphenylpyruvate dioxygenase protein, one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L.

2. The mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or biologically active fragment thereof of claim 1, wherein the amino acid sequence of said mutant hydroxyphenylpyruvate dioxygenase protein further has an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence indicated in SEQ ID NO. 2.

3. A mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or a biologically active fragment thereof as claimed in claim 1, wherein the mutant hydroxyphenylpyruvate dioxygenase protein has the amino acid sequence shown in SEQ ID NO:2, with the difference that it has one or more amino acid mutations as defined in claim 1.

4. A mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or a biologically active fragment thereof as claimed in any one of claims 1 to 3, wherein the amino acid sequence of the mutant hydroxyphenylpyruvate dioxygenase protein has one or more mutations selected from the group consisting of: R93S, a103S, H141R, H141K, H141T, a165V, V191I, R220K, G226H, L276W, P277N, P336D, P337A, N338D, N338S, N338Y, G342D, R346C, R346D, R346H, R346S, R346Y, D370N, I377C, P386T, L390I, M392I, E36403, K410I, K418I, G419I, N420I, E36430 and Y431I.

5. The mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or biologically active fragment thereof as claimed in claim 4, wherein the mutant hydroxyphenylpyruvate dioxygenase protein has the amino acid sequence SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 48, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82 or 84.

6. The mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or biologically active fragment thereof of claim 4, wherein the amino acid sequence of said mutant hydroxyphenylpyruvate dioxygenase protein has the following amino acid mutations: H141R/G342D, H141R/D370N, G342D/D370N, H141R/N338D, H141R/G342D, N338D/G342D, K418P/G419F, G419F/N420S, G342D/R346C, G342C/R346C, H141/N420C, G338C/K418C, P277C/N338C, L276C/P277 346, H141/G C/D C, H141C/N338C/N420C, H141/N C/N338C, P336/N338/G342/G C/N338, P C/N336/C, P C/N338/N336/C, P342/N338/N342, P342/N C/N338/N342/N C, N C/N338/N342/N C, N C/N C, N C/C, N C/36342/C, N C/N338/C/36342, N338/C/, P336D/N338D/R346C, P336D/N338D/R346H, P336D/N338D/R346S, P336D/N338S/R346C, P336D/N338S/R346H, P336D/N338S/R346S, P336D/N338Y/R346C, P336D/N338D/R36346 72, P336D/N338D/R36346D/R346D/36346 72, H141/N338/G342/G D, H141/G D/K418D, H141/G342/G D/G419/G36342, H D/36342/D, K36392/G D/36342/D/36342, H D/36342/D/36141/D/36342, H D/36342/D/36342, H D/36342/D/36342, H/36141/36342/D/36342, H/D/36342/D/36342, H/36342/D/36141/36342, H/D/36342/D, H/D/36342/D, H/36141/D/36342/D, H141/N338/G342, P277/G342/R346, P277/N338/N420, N338/G342/K418, H141/N338/G342/G419, H141/N338/G342/P386, H141/N338/G342/R346, H141/G342/K418/G419, H141/G342/L276/P277, P336/N338/G/R346, P336/N338/G342/R346, P336/N338/G346, P336/N336/G342/R346, P336/N338/G342/R342, P342/N342/G/R342, and P141/G342/P342/G/P342/L346, P336D/N338Y/G342D/R346S, P277N/P336D/N338D/G342D, P277N/N338D/G342D/R346C, P277N/N338D/K418P/G419F, H141R/N338D/G342D/K418P/G419F, H141R/N338D/G342D/G419F/N420S, H141R/G336D/G342D/K418P/G419F/N420S, H141R/N338D/G342D/K418P/G419F/N420S, H141R/N338D/G342D/K418P/G419F/N420T, H141R/N338D/G342D/R346C/K418P/G419F/N420F, H141F/N338F/G342F/R346F/K418F/G419F/N420F, H141F/P277F/G F/K338F/G419F/N F/G36342/K419F/N F/G F/K418/G419F/N419F/G F/K419F/G36419F/N F/G36419/N F/G36419F/N F/G F/.

7. The mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or biologically active fragment thereof as claimed in claim 6, wherein the mutant hydroxyphenylpyruvate dioxygenase protein has SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 86, SEQ ID NO 88, SEQ ID NO 90, SEQ ID NO 92, SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 112, SEQ ID NO 114, SEQ ID NO 116, SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 122, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO 86, SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 112, SEQ ID NO 114, SEQ ID NO 116, SEQ ID, SEQ ID NO 130, SEQ ID NO 132, SEQ ID NO 134, SEQ ID NO 136, SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 142, SEQ ID NO 144, SEQ ID NO 146, SEQ ID NO 148, SEQ ID NO 150, SEQ ID NO 152, SEQ ID NO 154, SEQ ID NO 156, SEQ ID NO 158, SEQ ID NO 160, SEQ ID NO 162, SEQ ID NO 164, SEQ ID NO 166, SEQ ID NO 168, SEQ ID NO 170, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 176, SEQ ID NO 178, SEQ ID NO 180, SEQ ID NO 182, SEQ ID NO 184, SEQ ID NO 186, SEQ ID NO 188, SEQ ID NO 190, SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO, SEQ ID NO: 196. SEQ ID NO: 198. SEQ ID NO: 200. SEQ ID NO: 202. SEQ ID NO: 204. SEQ ID NO: 206. SEQ ID NO: 208. SEQ ID NO: 210. SEQ ID NO: 212. SEQ ID NO: 214. SEQ ID NO: 216. SEQ ID NO: 218. SEQ ID NO: 220. SEQ ID NO: 222. SEQ ID NO: 224. SEQ ID NO: 226. SEQ ID NO: 228. SEQ ID NO: 230. SEQ ID NO: 232. SEQ ID NO: 234. SEQ ID NO: 236. SEQ ID NO: 238. SEQ ID NO: 240. SEQ ID NO: 242. SEQ ID NO: 244. SEQ ID NO: 246. SEQ ID NO: 248. SEQ ID NO: 250. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 256. SEQ ID NO:258 or SEQ ID NO:260, or a pharmaceutically acceptable salt thereof.

8. A fusion protein comprising a mutated HPPD protein according to any one of claims 1 to 7, or a biologically active fragment thereof, and further components fused thereto, such as a tag peptide, e.g. 6 xhis, or a plastid targeting peptide, e.g. a peptide that targets chloroplasts.

9. An isolated polynucleotide comprising a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein of any one of claims 1 to 7 or a biologically active fragment thereof, or a fusion protein as set forth in claim 8, or a complement thereof; said polynucleotide is preferably DNA, RNA or a hybrid thereof; the polynucleotide is preferably single-stranded or double-stranded.

10. The polynucleotide of claim 9 having a nucleic acid sequence selected from the group consisting of:

(1) encoding SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 140, 142, 144, 132, 134, 136, 138, 140, 142, 144, SEQ ID NO 146, SEQ ID NO 148, SEQ ID NO 150, SEQ ID NO 152, SEQ ID NO 154, SEQ ID NO 156, SEQ ID NO 158, SEQ ID NO 160, SEQ ID NO 162, SEQ ID NO 164, SEQ ID NO 166, SEQ ID NO 168, SEQ ID NO 170, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 176, SEQ ID NO 178, SEQ ID NO 180, SEQ ID NO 182, SEQ ID NO 184, SEQ ID NO 186, SEQ ID NO 188, SEQ ID NO 190, SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 196, SEQ ID NO 198, SEQ ID NO 200, SEQ ID NO 202, SEQ ID NO 204, SEQ ID NO 206, SEQ ID NO 208, SEQ ID NO 210, SEQ ID NO, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258 or 260 amino acid sequences or the complementary sequences thereof;

(2) 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 61, 63, 65, 67, 53, 55, 57, 59, 61, 63, 65, 67, 17, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 111, 113, 115, 133, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 175, 177, 179, 181, 183, 181, 175, 177, 179, 181, 183, 185, 187, 193, 191, 195, 197, 199, 195, 197, 199, and, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 or 259 or the complement thereof;

(3) a nucleic acid sequence which hybridizes with the sequence shown in (1) or (2) under a strict condition; and

(4) a nucleic acid sequence which encodes the same amino acid sequence as that of the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or a complementary sequence thereof.

11. The polynucleotide of claim 10, wherein the nucleic acid sequence is optimized for expression in a plant cell.

12.A nucleic acid construct comprising the polynucleotide of any one of claims 9-11 operably linked to regulatory elements.

13. An expression vector comprising the polynucleotide of any one of claims 9-11 operably linked to an expression control element.

14. A host cell comprising the polynucleotide of any one of claims 9-11, the nucleic acid construct of claim 12 or the expression vector of claim 13, preferably said host cell is a plant cell.

15. A method of producing a plant with increased resistance or tolerance to an herbicide comprising regenerating the plant cell of claim 14 into a plant.

16. A plant produced by the method of claim 15.

17.A method of increasing HPPD-inhibiting herbicide resistance or tolerance of a plant cell, plant tissue, plant part or plant comprising expressing in the plant cell, plant tissue, plant part or plant a mutant p-hydroxyphenyl pyruvate dioxygenase (HPPD) protein or biologically active fragment thereof according to any one of claims 1 to 7 or a fusion protein according to claim 8;

or which comprises crossing a plant expressing a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein or a biologically active fragment thereof according to any of claims 1 to 7 or a fusion protein according to claim 8 with another plant and selecting plants or parts thereof which have an increased resistance or tolerance to HPPD-inhibiting herbicides;

or wherein a gene editing of an endogenous HPPD protein of said plant cell, plant tissue, plant part or plant is comprised to achieve expression therein of a mutant p-hydroxyphenylpyruvate dioxygenase protein, or a biologically active fragment thereof, according to any one of claims 1 to 7, or a fusion protein according to claim 8.

18. Use of a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein or a biologically active fragment thereof according to any one of claims 1 to 7, a fusion protein according to claim 8 or a polynucleotide according to any one of claims 9 to 11 for increasing the resistance or tolerance of a host cell or plant cell, plant tissue, plant part or plant to a HPPD-inhibiting herbicide, preferably the host cell is a bacterial cell or a fungal cell.

19. A method of controlling weeds at a plant locus, wherein the plants comprise the plant of claim 16 or a plant prepared by the method of any one of claims 15 and 17, the method comprising applying to the locus a weed controlling effective amount of one or more HPPD inhibiting herbicides; preferably, the HPPD-inhibiting herbicide comprises at least one of the following active ingredients: 1) triketones: sulcotrione, mesotrione, flurtamone, tembotrione, benzofuranone, and benzobicyclon; 2) diketonitriles: 2-cyano-3-cyclopropyl-1- (2-methylsulfonyl-4-trifluoromethylphenyl) propane-1, 3-dione, 2-cyano-3-cyclopropyl-1- (2-methylsulfonyl-3, 4-dichlorophenyl) propane-1, 3-dione, 2-cyano-1- [4- (methylsulfonyl) 2-trifluoromethylphenyl ] -3- (1-methylcyclopropyl) propane-1, 3-dione; 3) isoxazoles: isoxaflutole, isoxaclomazone, clomazone; 4) pyrazoles: topramezone, pyraclostrobin, pyraflutole, topramezone, bicyclopyrone and tembotrione; 5) benzophenones; 6) other classes: lancotrione, fenquinolone; more preferably, the HPPD-inhibiting herbicide comprises at least one of the following active ingredients: tembotrione, topramezone, bicyclopyrone, topramezone, mesotrione and topramezone.

20. The plant of claim 16, the method of any one of claims 17 and 19, or the use of claim 18, wherein the plant is a dicot or monocot, such as a food crop, a legume crop, an oil crop, a fiber crop, a fruit crop, a root crop, a vegetable crop, a flower crop, a pharmaceutical crop, a raw crop, a pasture crop, a sugar crop, a beverage crop, a lawn plant, a tree crop, a nut crop, and the like.

21. A method of making a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein which retains or potentiates the property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate and which is significantly less sensitive to an HPPD-inhibiting herbicide than the wild-type HPPD, which comprises mutating a nucleic acid encoding a wild-type HPPD, fusing and ligating the mutated nucleic acid in an expression vector in frame with a nucleic acid sequence encoding a solubility-enhancing component to form a fusion protein coding sequence, transforming the resulting recombinant expression vector into a host cell, expressing the fusion protein under suitable conditions containing the HPPD-inhibiting herbicide and an HPPD enzymatic substrate and screening for a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein which retains or potentiates the property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate and which has a significantly reduced sensitivity to an HPPD-inhibiting herbicide; the solubility enhancing component is preferably NusA, which forms a fusion protein with the mutated HPPD protein of the invention; the expression vector is preferably a pET-44a vector; the host cell is preferably a bacterial cell, a fungal cell or a plant cell.

Technical Field

The invention belongs to the field of agricultural genetic engineering, and particularly relates to a novel mutant p-hydroxyphenyl pyruvate dioxygenase (HPPD) for endowing plants with HPPD inhibitory herbicide resistance or tolerance, a nucleic acid encoding the same and application thereof.

Background

Hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme that catalyzes the reaction of Hydroxyphenylpyruvate (HPP) to convert it into a homogentisate. This reaction occurs in the presence of enzyme-bound iron and oxygen. Herbicides that act by inhibiting HPPD are well known and include various types such as isoxazoles, diketonitriles, triketones, and pyrazoline salts, among others. Inhibition of HPPD blocks the biosynthesis of Plastoquinone (PQ) from tyrosine. PQ is an essential cofactor in the biosynthesis of carotenoid pigments, which are essential for photoprotection in photosynthetic centers. HPPD-inhibiting herbicides are bleaches that are mobilized through the phloem, causing new meristems and leaves exposed to light to appear white. In the absence of carotenoids, chlorophyll is photo-destructive and itself becomes a photo-lytic agent by the photosensitization of singlet oxygen.

Technical routes and methods for providing plants tolerant to HPPD inhibiting herbicides are also known, including over-expressing HPPD enzymes to produce large quantities of HPPD enzymes in plants that are sufficiently related to a given herbicide to have enough available functional enzyme (despite its inhibitor present), or mutating the target HPPD enzyme to a functional HPPD that is less sensitive to the herbicide. HPPD inhibiting herbicides are a broad class which covers many different types. While a given mutant HPPD enzyme may provide a useful level of tolerance to one or some HPPD-inhibiting herbicides, the same or a single mutant HPPD may not be sufficient to provide a commercial level of tolerance to another or another different, more desirable HPPD-inhibiting herbicide (see, e.g., U.S. application publication No. 2004/0058427; and PCT application publications nos. WO98/20144 and WO 02/46387; also see U.S. application publication No. 2005/0246800, which relates to the identification and labeling of soybean varieties that are relatively tolerant to HPPDs). Moreover, different HPPD-inhibiting herbicides can vary in the range of weeds they control, the crop objects used, the cost of manufacture, and the environmental benefits. Thus, there remains a need in the art for novel mutant HPPDs for conferring resistance/tolerance to HPPD-inhibiting herbicides to different crops and crop varieties.

In the aspect of creating herbicide tolerant crops and crop varieties, transgenic technology is widely applied. However, the use of transgenic crops has been limited by the high cost of registration. This current situation can be changed by the advancement of gene editing technology represented by CRISPR/Cas 9. CRISPR/Cas9 is a new gene site-directed editing technique that has emerged since 2012 (Jinek, m., chlylinski, k., Fonfara, i., Hauer, m., Doudna, j.a., and charpienter, e.2012.aprogrammable dual-RNA-guided DNA endlinking, i.e. science.337: 816. 821), cog, l.a., Ran, f.a., Cox, d.line, s.barretto, r, libob, n.n., p.d., wu.wu, wu.x., wiring, w.w., raffini, l.a, and Zhang, f.2013.multigene engineering, g.g., gene, r.g.m. wo., coding, r.g., gene, n.12, r, n.g., gene, g.12, and g., gene, g.2013. multigene engineering, g.g., gene, g.12, gene, g.g.12, gene. The recognition of the editing target by the CRISPR/Cas9 system depends on the complementary base pairing between nucleic acid molecules, and any target sequence of 20bp which is followed by PAM (NGG) can be edited. In addition, the CRISPR/Cas9 system is simple to operate, only 20-30bp of target nucleotide sequence on the original vector needs to be replaced for each targeting, and the method is suitable for high-throughput operation. Multiple sites of the same gene can be edited simultaneously as well as multiple different genes. At present, the technology has a great application prospect in the aspects of biomedicine, Functional Genomics, animal and Plant trait Improvement, new trait creation and the like, and is generating revolutionary promotion effect on animal and Plant breeding (Hui Zhang, jin shan Zhang, Zhaobolang, Jose Ramon Botelled, and Jian-Kang Zhu.2017 genome edition-principles and Applications for Functional Genomics Research and Crop Improvement, clinical Reviews in Plant Sciences 36:4,291 309 and DOI 10.1080/07352689.2017.1402989).

The CRISPR/Cas9 is used as a third-generation gene editing tool, and the site-specific editing is realized mainly through three modes. The first is the site-directed knockout of the gene to obtain mutants. Specifically, Cas9 recognizes and cleaves a target under the guidance of a targeting rna (grna), generating a double-stranded DNA break; fragmented DNA is usually repaired by non-homologous end joining (NHEJ); it is easy to generate frame shift mutation to destroy the gene during repair. The efficiency of fixed point knockout is high. The second is homologous substitution of the target to replace the target sequence or site-directed insertion. When a double-stranded DNA break is created, homologous substitution or site-directed insertion may occur if a homologous repair template is present nearby. Homologous substitution is less efficient and becomes even less as the length of the sequence to be substituted increases. The third is single base editing (Komor AC, Kim YB, Packer MS, Zuris JA, LiuDR. Programming editing of a target base in genomic DNA without double-stranded DNA clean. Nature.2016May 19; 533(7603):420-4.doi:10.1038/nature 17946; Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, LiuDR. Programming base editing of A.T.G.C in genomic DNA without DNAxis. Nature.2017Nov 23; 551 7681: 464-471.doi:10.1038/nat 644. Epubu 2017 ct 25. Er8um. Nature. 2). Single base editing is a gene editing method that uses the CRISPR/Cas9 system to target deaminase to a specific site in the genome, thereby modifying a specific base. This method has been successfully practiced in rice. Such as: yan f., Kuang y., Ren b., Wang j., Zhang d., Lin h., Yang b., zhoux, and Zhou h. (2018). High-efficient a.t to g.c base injection by Cas9n-guided trna adenine deaminase in rice.mol.plant.doi:10.1016/j.mol p.2018.02.008.

In addition, CRISPR/Cpf1 may also be used for gene editing (Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., Joung, J.Oost, J.Gev., A.Koonin, E.V., and Zhang, F.2015.Cpf1a single RNA-bound end effector of a Class 2CRISPR Cas. cell.163: 759. gene 771; Endo, A.A., Masafumi, M.Kaya, H.and Toki, S.2016a.Efficiend target bacterium of Francinge.169). CRISPR/Cpf1 has two main components: cpf1 enzyme and crRNA that determines system specificity. Although the CRISPR/Cpf1 and CRISPR/Cas9 systems are similar, there are some important differences (Hui Zhang, Jinshan Zhang, ZhaoboLang, Jos re Ram Lou n Botella & Jian-Kang Zhu (2017) Genome Editing-Principles and Applications for Functional Genomics Research and Crop Improvement, Critical reviews in Plant Sciences,36:4, 291-. First, the CRISPR/Cpf 1system does not require transactivation of crrna (tracrrna), but instead CRISP/Cas9 is necessary. Therefore, it is relatively short, having only 42-44 nucleotides, including a 19 nucleotide repeat and a 1-23-25 nucleotide long spacer. Third, unlike Cas9 which cleaves DNA double strands at the same position (3-4 bp upstream of PAM) to generate blunt ends, the target sequence cleavage position for Cpf1is 23bp downstream of PAM sequence and the non-target single strand is 18bp downstream of PAM sequence to generate a 5bp overhanging sticky end. The resulting sticky ends may increase the efficiency of HDR-mediated insertion of donor DNA into the Cpf1 cleavage site. Fourth, the CRISPR/Cpf 1system only needs one promoter to drive multiple small crRNAs arrays when editing multiple targets or genes, and is very suitable for multi-target editing. Fifth, the CRISPR/Cas9 system requires a G-rich (5 '-NGG-3') PAM sequence at the 3 'end of the target sequence, and the CRISPR/Cpf 1system requires a T-rich (5' -TTTN-3 'or 5' -TTN-3 ') PAM sequence at the 5' end of the target sequence, suitable for editing multiple A/T DNA or genes. Three engineered CRISPR/Cpf1 systems have been developed, including FnCpf 1from Francisella novicida (Francisella), ascif 1from Acidaminococcus sp., and LbCpf 1from Lachnospiraceae bacteria (lachnospirillum). Three Cpf1 systems have been used as plant genome editing on the following species: rice, arabidopsis, tobacco and soybean (Endo, a., Masafumi, m., Kaya, h., and Toki, s.2016a. efficient targeted mutagenesis of rice and tobaca genes using Cpf1from Francisella novicida.sci.rep.6: 38169; Kim, h., Kim, s.t., Ryu, j., Kang, b.c., Kim, j.s., and Kim, s.g.2017.CRISPR/Cpf1-media dna-free plant injection. nat.8: 14406.; Tang, x., loder, l.g., Zhang, t.t., major, a.a., Zheng, Zhang, c., t.s.1. bulking, r.1, r.t., cement, r.d., r.1, m.r.t., r.1. m., r.m., m.m., r.d., r.d., m, m.r.d. 1, m, r.d.d. 1. r.g., r.r.g., Zhang, c., Zhang, r.g., Zhang, g., Zhang, r.t., t., ma.

The improvement of herbicide tolerance of important crops through gene editing-mediated homologous replacement, site-directed modification or single base editing is one of the hotspots in the current gene editing research field, and several successful examples have been reported, but all focused on the anti-Acetolactate synthase (ALS) inhibiting herbicides (Yongwei Sun, Xin Zhang, Chuanyin Wu, ubing He, Youzhi Ma, Han Hou, Xiuping Guo, Wenming Du, Yunde Zhao and Lanqin Xia.2016.engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous combination of acetic synthase Synthesis Plant 9, 628. quadrature. org/10.1016/j. molp.2016.01.001; Yiyu Cheng Wang, Hanwen Ni, Yong Xu, Qianjun Cheng, Linjian J.7.017/20126-76. Jiyu Cheng Wang, Hanwen Ni, Yungsu, Qianjun Cheng Huan, WO 27-19, WO 27-found in Yingqing Hua Ka-19. Yingsu Shu Hua Ka-19. Yingsu Shu Kangsu 19. Sandi-7. K-Sandi-3. K-3. Tokyo Ka-3. K-3. Yingsu Hua Kangsu Hua No. Yingsu Hua No. 27. Yingsu Hua Kangsu 19. Yingsu Hua No. 3-3. Yingsu. This requires the continued research and development of new methods for increasing the tolerance of crops to different types of herbicides.

Disclosure of Invention

Based on this, the present application provides a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) conferring HPPD-inhibiting herbicide resistance or tolerance to plants, which mutant HPPD retains or potentiates its property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate, while being significantly less sensitive to HPPD-inhibiting herbicides than the wild-type HPPD. The invention also relates to a biological active fragment of the mutant p-hydroxyphenylpyruvate dioxygenase, a polynucleotide for coding the protein or the fragment and application thereof.

Accordingly, in one aspect, the present invention provides a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein having one or more mutations selected from the group consisting of at positions 93, 103, 141, 165, 191, 220, 226, 276, 277, 336, 337, 338, 342, 346, 370, 377, 386, 390, 392, 403, 410, 418, 419, 420, 430 and 431 in the amino acid sequence of the wild-type rice p-hydroxyphenylpyruvate dioxygenase protein shown in SEQ ID NO: 2: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L. Preferably, the amino acid sequence of said mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence depicted in SEQ ID NO. 2. More preferably, the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein has the amino acid sequence shown in SEQ ID NO:2, differing only in having one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L.

In another aspect, the invention provides a biologically active fragment of a mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein, which has a deletion of a portion of one or more (e.g., 1-50, 1-25, 1-10 or 1-5, e.g., 1,2, 3,4 or 5) amino acid residues from the N-and/or C-terminus of the protein, but which still retains the desired biological activity of the full-length protein, i.e., retains or potentiates its property of catalyzing the conversion of Hydroxyphenylpyruvate (HPP) to homogentisate, while being significantly less sensitive to HPPD-inhibiting herbicides than the wild-type HPPD or its corresponding biologically active fragment.

The invention furthermore relates to a fusion protein comprising a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein according to the invention or a biologically active fragment thereof and further components, such as peptide or polypeptide components, fused thereto. Preferably, the components confer desirable properties to the fusion protein, such as facilitating its isolation, purification, improving its stability, prolonging its half-life, providing additional biological activity, directing the fused HPPD protein into a target region, e.g. a plastid such as a chloroplast, etc. The choice of the corresponding components is well known to the person skilled in the art.

In another aspect, the invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding said mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein or a biologically active fragment or fusion protein thereof.

The invention also provides nucleic acid constructs comprising the polynucleotides and regulatory elements operably linked thereto.

In a further aspect, the present invention provides an expression vector comprising the polynucleotide and an expression control element operably linked thereto.

In yet another aspect, the invention provides a host cell comprising the polynucleotide, nucleic acid construct or expression vector.

The invention also provides a method of producing a plant with increased resistance or tolerance to an HPPD-inhibiting herbicide.

The invention further relates to plants produced by the above method.

The present invention also provides a method of increasing the resistance or tolerance of a plant to a HPPD inhibiting herbicide, comprising expressing in said plant a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein, or a biologically active fragment or fusion protein thereof, of the present invention.

The present invention further provides a method of increasing the resistance or tolerance of a plant to a HPPD inhibiting herbicide, comprising crossing a plant expressing a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein, or a biologically active fragment or fusion protein thereof, of the present invention with another plant.

The invention further provides a method for increasing the resistance or tolerance of a plant to a HPPD-inhibiting herbicide, comprising genetically editing an HPPD protein endogenous to said plant cell, plant tissue, plant part or plant.

The present invention further relates to the use of a mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein, or a biologically active fragment or fusion protein thereof, of the present invention for increasing HPPD-inhibiting herbicide resistance or tolerance in plants.

The present invention further relates to a method of controlling weeds at a locus of plants, comprising applying to the locus comprising plants or seeds of the invention a weed controlling effective amount of one or more HPPD inhibiting herbicides and not significantly affecting said plants.

Drawings

FIG. 1 shows the color reaction of the culture broth of recombinant E.coli transformed with wild-type or mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli expresses one of wild type rice HPPD (WT) or single-site mutant rice HPPD, and the wild type rice HPPD or the single-site mutant rice HPPD contains herbicides of tembotrione (left) or topramezone metabolized products (right, the structural formula is as follows:) The culture under the conditions of (1) shows color change to various degrees. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 2 shows the color reaction of the culture broth of recombinant E.coli transformed with wild-type or mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli expresses wild-type rice HPPD (WT) and each of the single-site mutant rice HPPDs, and they show color changes of different degrees when cultured in a culture solution containing different concentrations of the herbicide ciclopirox or topramezone. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 3 shows the color reaction of the culture broth of recombinant E.coli transformed with wild-type or mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli expresses wild-type rice HPPD (WT) and each of the single-site mutant rice HPPDs, and they show color reactions of different degrees when cultured in a culture solution containing the herbicide mesotrione at different concentrations. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 4 shows color reaction of recombinant E.coli culture broth transformed with wild-type or mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli expresses wild rice HPPD (WT) or one of single-site mutant H141R, G342D and D370N or a combination thereof, and the wild rice HPPD (WT) or the single-site mutant H141R, G342D and D370N show color change to different degrees when cultured under the condition of containing different concentrations of herbicide tembotrione (upper) or after-metabolism products of topramezone. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 5 shows color reaction of recombinant E.coli culture broth transformed with wild-type or mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli expresses wild-type rice HPPD (WT) or single-site mutant H141R, G342D, D370N or a combination thereof (141+342 represents H141R/G342D; 141+370 represents H141R/D370N; 342+370 represents G342D/D370N; 141+342+370 represents H141R/G342D/D370N), and the recombinant Escherichia coli can show color change with different degrees when cultured in the presence of different concentrations of herbicide cyflufenac (upper) or topramezone (lower). In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 6 shows color reaction of recombinant E.coli culture broth transformed with wild-type or mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli culture fluid expresses wild-type rice HPPD (WT) or single-site mutant H141R, G342D, D370N or a combination thereof, and the wild-type rice HPPD, the single-site mutant H141R, the G342D, the D370N or the combination thereof shows color changes to different degrees when cultured under the conditions containing different concentrations of herbicide mesotrione. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 7 shows all amino acid mutations noted on the rice HPPD wild-type enzyme protein.

FIG. 8 shows color reaction of recombinant E.coli culture broth transformed with mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli culture fluid expresses various combinations of the near mutation points 336-338-342-346 and 141R +342D +370N (336D, 338S, 338Y, 342D, 346C, 346H, 346S respectively represent P336D, N338D, N338S, N338Y, G342D, R346C, R346H, R346S;141R +342D +370N for H141R/G342D/D370N) were added to a mixture containing varying concentrations of the herbicide diafentrazone metabolite (code 101, formula:) The culture under the conditions of (1) shows color changes of different degrees. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 9 shows color reaction of recombinant E.coli culture broth transformed with mutant rice HPPD gene cultured in 96-well plate. Wherein the recombinant Escherichia coli culture solution expresses three and four mutation point combinations (141R, 336D, 338S, 338Y, 342D, 346C, 346S, 346H, 370N, 418P and 419F respectively represent H141R, P336D, N338D, N338S, N338Y, G342D, R346C, R346S, R346H, D370N, K418P and G419F), and the three and four mutation point combinations are cultured under the condition containing different concentrations of herbicide diaxazone metabolites to show different degrees of color change. In a well with the same concentration of herbicide, darker color represents higher resistance/tolerance to this herbicide.

FIG. 10 shows the inhibition curves of the metabolites of topramezone against OsHPPD WT and the respective mutants, with the abscissa representing the concentration of compound 101 and the ordinate representing the reaction rate at 0 concentration of inhibitor as 100%, representing the residual activity of the enzyme at different concentrations of 101, and the numbers in the graphs represent the respective mutation sites. As can be seen from the figure, wild-type WT was very sensitive to 101, with complete inhibition of activity at a 101 concentration of about 60uM, whereas each mutant showed a strong increase in resistance. From this result, it was possible to calculate the IC50 value for 101 inhibition of activity of each mutant, which also demonstrated that each mutant exhibited significantly improved resistance to wild-type OsHPPD (where 141R, 338D, 342D, 346C, 346H, 370N, 386T, 418P, 419F, 420S represent H141R, N338D, G342D, R346C, R346H, D370N, P386T, K418P, G419F, N420S, respectively).

FIG. 11 shows sensitivity of transgenic rice (Zhonghua 11) to the HPPD inhibitor herbicide tembotrione. Expression mutant Rice O_sHPPD3M (H141R/G342D/D370N) can be contained in 3_uGreen color in M-tembotrione medium, but negative Control (CK) expressed mCherr_yThe seedlings were also severely albino (phytotoxicity) in 1.0uM tembotrione medium.

FIG. 12 shows tolerance of transgenic rice (middle flower 11) to the HPPD inhibitor herbicide, topramezone. T0 generation plant expression rice O_sPlants of HPPD3M were able to tolerate 8-16 grams of the active ingredient topramezone per acre, but died soon after severe albinism in the non-transgenic Control (CK) (a, B); plants expressing OsHPPD3M from T1 generation plants tolerated 32-64 grams of the active ingredient topramezone per acre, but died soon after severe albinism in the non-transgenic controls (C, D).

FIG. 13 shows rice HPPD single base editing vectors.

FIG. 14 shows sequence analysis of single base edited rice seedlings and their target H141R (CAC > CGC).

A: single base editing seedlings: in a culture medium with 0.4uM cyclosulfoketone, the person which is not successfully edited whitens (phytotoxicity), and the person which is successfully edited keeps green;

b: sequence of single base editing target: the wild type of the 141 th amino acid of the rice HPPD is histidine His, the codon is CAC (upper graph), the codon is arginine Arg after editing, and the codon is CGC (in the example, heterozygote, double peak appears).

FIG. 15 shows the structure of the rice hppd gene (Oshppd > NC029257.1), displaying two exons (exon), one intron (intron), three mutation sites (141, 342, 370) and designed targeted cleavage sites (gRNA1-2, gRNA 2-1).

FIG. 16 shows the structure of a template DNA. The length of the core replacement region of the three mutant amino acids 141-342-370-1056 bp, the left and right homologous arms are 350bp respectively, and after cutting from the vector, the left and right ends are respectively left with 6bp, and the total length of the template is 1768 bp; in order to facilitate rapid genotype identification of a PCR product after PCR amplification, the NcoI enzyme cutting site in the PCR product is removed; and PAM (NGG) at the original cutting site on the template is also removed to avoid re-cutting after replacement.

FIG. 17 shows homologous substitution triple mutation points of rice HPPD gene (H141R-G342D-D370N).

A: rice HPPD gene editing seedling: in the presence of 0.4uM tembotrione, unsuccessfully edited (wild type WT) whitened (phytotoxicity), and successfully edited 2 seedlings (AW2, AW3) remained green;

b: after homologous substitution, the codons at amino acids 342 and 370 were changed, GGC to GAC and GAC to AAC (heterozygote; resulting in portions G342D and D370N); H141R (CAC > CGC) was also successfully edited (sequence not shown).

Detailed Description

Some terms used in the present specification are defined as follows.

In the present invention, an "HPPD-inhibiting herbicide" is a substance which is itself herbicidally active or which is combined with other herbicides and/or additives capable of modifying its effect and which is capable of acting by inhibiting HPPD. Substances which are themselves capable of acting herbicidally by inhibiting HPPD are well known in the art and include many types, 1) triketones, for example, Sulcotrione (CAS number: 99105-77-8); mesotrione (Mesotrione, CAS number 104206-82-8); fluroxyprione (bicyclopyrone, CAS number: 352010-68-5); tembotrione (CAS number: 335104-84-2); mesotrione (tefuryltrione, CAS number 473278-76-1); benzobicylon (Benzobicyclon, CAS number: 156963-66-5); 2) diketonitriles, for example, 2-cyano-3-cyclopropyl-1- (2-methylsulfonyl-4-trifluoromethylphenyl) propane-1, 3-dione (CAS number: 143701-75-1); 2-cyano-3-cyclopropyl-1- (2-methylsulfonyl-3, 4-dichlorophenyl) propane-1, 3-dione (CAS number: 212829-55-5); 2-cyano-1- [4- (methylsulfonyl) -2-trifluoromethylphenyl ] -3- (1-methylcyclopropyl) propane-1, 3-dione (CAS number: 143659-52-3); 3) isoxazoles, for example, isoxaflutole (isoxaflutole, CAS number: 141112-29-0); isoxachlorotole (isoxachlorotolole, CAS number 141112-06-3) clomazone (CAS number 81777-89-1); 4) pyrazoles, for example, topramezone (CAS number: 210631-68-8); sulfonylopyrazole (pyrasulfotole, CAS number: 365400-11-9); benzoxazole (pyrazoxyfen, CAS number: 71561-11-0); pyrazolate (pyrazolite, CAS number: 58011-68-0); bifenac (benzofenap, CAS number: 82692-44-2); topramezone (CAS number: 1622908-18-2); tolpyralate (CAS number: 1101132-67-5); benzoxaflutole (CAS number: 1992017-55-6); bicyclopyrone (CAS number: 1855929-45-1); mesotrione triazolate (CAS number: 1911613-97-2); 5) benzophenones; 6) other classes: lancotrione (CAS number: 1486617-21-3); fenquinolones (CAS number: 1342891-70-6). Preferably, the herbicide is tembotrione, topramezone, mesotrione, topramezone, or any combination thereof, and the like.

A plant that "has increased tolerance to an HPPD-inhibiting herbicide" or "has increased resistance to an HPPD-inhibiting herbicide" refers to a plant that has increased tolerance or resistance to the HPPD-inhibiting herbicide as compared to a plant containing a wild-type HPPD gene. An HPPD enzyme that "has increased tolerance to an HPPD-inhibiting herbicide" or "has increased resistance to an HPPD-inhibiting herbicide" refers to an HPPD enzyme that exhibits at least 10%, preferably at least 15%, more preferably at least 20% greater enzyme activity than the wild-type HPPD enzyme at a concentration of herbicide known to inhibit the activity of the corresponding wild-type HPPD enzyme protein. In the present invention, the terms "HPPD-inhibiting herbicide tolerance" and "HPPD-inhibiting herbicide resistance" are used interchangeably and refer to both tolerance to HPPD-inhibiting herbicides and resistance to HPPD-inhibiting herbicides.

The term "wild-type" refers to a nucleic acid molecule or protein that can be found in nature.

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more amino acid residues are chemical analogues of a natural amino acid residue. The proteins and polypeptides of the invention may be produced recombinantly or may be chemically synthesized. The term "mutein" or "mutant protein" refers to a protein having substitutions, insertions, deletions and/or additions of one or more amino acid residues compared to the amino acid sequence of the wild-type protein.

The terms "polynucleotide" and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, whether double-stranded or single-stranded.

In the present invention, a "host organism" is understood to be any unicellular or multicellular organism into which a nucleic acid encoding a mutated HPPD protein can be introduced, including, for example, bacteria such as e.coli, fungi such as yeasts (e.g. saccharomyces cerevisiae), molds (e.g. aspergillus), plant cells and plants, and the like.

In the context of the present invention, "plant" is understood to be any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants, such as: (1) grain crops: oryza (Oryza spp.), such as rice (Oryza sativa), broadleaf rice (Oryza latifolia), rice (Oryza sativa), and palea (Oryza glaberrima); triticum spp, such as Triticum aestivum, durum wheat (t.turgidumssp. durum); hordeum spp, such as barley (Hordeum vulgare), arizona barley (Hordeum arizonicum); rye (Secale cereale); avena species (Avena spp.) such as oats (Avena sativa), wild oats (Avena fatua), barnacle oats (Avena byzantina), Avena fatuavar, sativa, hybrid oats (Avena hybrida); echinochloa spp, for example, pearl millet (Pennisetum glaucum), Sorghum (Sorghum bicolor), triticale, maize or corn, millet, rice (rice), millet, broom millet, Sorghum bicolor, millet, Fagopyrum (Fagopyrum spp.), millet (Panicum paniculatum), millet (Setaria italica), zizaniazus aquatica (Zizaniaustifolia), Icelia carinata (Eragrostis tef), millet (Panicum paniculatum), and Uncaria paniculata (Eleusco tara); (2) bean crops: glycine (Glycine spp.), for example, Glycine (Glycine max), Glycine (Soja hispida), Glycine max, Vicia (Vicia spp.), Vigna (Vigna spp.), Pisum (Pisum spp.), Pisum (field bean), Lupinus (Lupinus spp.), Vicia (Vicia), tamarind (tamarind indica), lentil (Lens culinaris), lathyrium (lathyrius spp.), physalis (Lablab lab), fava bean, mung bean, red bean, and tonka bean; (3) oil crops: peanuts (Arachis hypogaea), groundnut (Arachis spp), flax (Sesamum spp.), sunflower (Helianthus spp.), soybean (soybean annuus), oil palm (Elaeis) such as oil palm (Eiaeis guineensis, Elaeis americana (Elaeis oleifera), soybean (soybean), oilseed rape (Brassicananus), canola, sesame, mustard (Brassicajuncea), rapeseed oilseed rape (oilsedandrae), camellia oleifera, oil palm, olive, castor oil, European rape (Brassica napus L.), canola (canola); (4) fiber crops: sisal (Agave sisalana), cotton (cotton, Gossypium barbadense, Gossypium hirsutum), kenaf, sisal, abaca, flax (Linum usittissimum), jute, ramie, hemp (Cannabis sativa), or hemp; (5) fruit crops: ziziphus (Ziziphus spp.), cucurbita (Cucumis spp.), passion fruit (Passiflora edulis), Vitis (Vitis spp.), Vaccinium (Vaccinium spp.), Pyrus (Pyrus communis), Prunus (Prunus spp.), Psidium guava (Psidium spp.), Punica (Punica grantum), Malus (Malus spp.), watermelon (Citrus latus, Citrus (Citrus spp.), fig (Ficus Carica), aurantium (Fortunella spp.), strawberry (Fragaria spp.), Crataegus (Crataegus spp.), persimmon (Diospyros persica, rhododendron (mangium spp.), rhodomyrtus (mangium spp.), Prunus spp.), Carica (mangium spp.), Prunus spp., mangus (Prunus spp.), Prunus spp., mangus spp., mangium (Prunus spp.), Prunus spp. (cornus spp.), Prunus spp.) (cornus spp.) (guava (mangus spp.), Prunus spp.) (mangus spp.) (cornus spp.), Guava (Psidium guajava), apple peel (Mammea americana), mango (Mangifera indica), olive (oleaeuropaa), papaya (cariapaya), coconut (Cocos nucifera), acerola (Malpighia emarginata), naseberry (manikala zapota), pineapple (anaas comosus), Annona (Annona spp.), Citrus tree (Citrus spp.), aronia (Artocarpus spp.), Litchi (lichenis spp.), scirpus (triquetrum), scirpus (Ribes spp.), Rubus (russpp), pear, apricot, plum, bayberry, lemon, orange, rose, strawberry (strawberries, melon, coconut, blueberry, peach, walnut; (6) root crops: cassava (Manihot spp.), sweet potato (Ipomoea batatas), taro (Colocasia esculenta), tuber mustard, onion, water chestnut, nutgrass flatsedge, yam; (7) vegetable crops: spinach (Spinacia spp.), Phaseolus (Phaseolus spp.), lettuce (Lactuca sativa), Momordica charantia (Momoracia spp.), parsley (Petroselinum crispum), Capsicum (Capsicum spp.), Solanum (Solanum spp.) (such as potato (Solanum tuberosum), red tomato (Solanum integrifolium) or tomato (Solanum lycopersicum)), Lycopersicon (Lycopersicon spp.) (such as tomato (Lycopersicon esculentum), tomato (Lycopersicon esculentum), tomato shaped tomato (Lycopersicon esculentum), potato shaped (Lycopersicon esculentum), potato (Brassica oleracea), potato (Brassica oleracea), potato (Brassica ole, Asparagus (Asparagus officinalis), celery (Apium graveolens), amaranth (Amaranthus spp.), Allium (Allium spp.), okra (abelmoshus spp.), endive (Cichorium endivia), Cucurbita (Cucurbita spp.), coriander (coriander sativum), eruca sativa (b. carinata), radish (Rapbanus sativus), Brassica species (Brassica) such as Brassica napus (Brassica napus), Brassica subspecies (Brassica rapa ssp), canola (canola), Brassica napus), Brassica campestris (Brassica rapa sativa), Brassica oleracea, Brassica campestris, Brassica juncea, Brassica oleracea, Brassica juncea, Brassica oleracea, Brassica campestris, Brassica oleracea, Brassica napus, Brassica juncea, Brassica oleracea, Brassica napus, Brassica; (8) flower crop: tropaeolum (Tropaeolum minus), trollflower (Tropaeolum maju), Canna indica (Canna indica), Opuntia (Opuntia spp.), Tagetes (Tagetes spp.), orchid, Crinum asiaticum, kaffir lily, hippeastrum roseum, rose, China rose, jasmine, tulip, cherry blossom, morning glory, calendula, lotus, daisy, carnation, petunia, tulip, lily, plum blossom, narcissus, winter jasmine, primrose, daphne, magnolia liliifolia, jojoba, kaffir, peony, clove, rhododendron, michelia figo, malus chinensis, juniper berry, chinaberry, calanthe, iris, yunnan jasmine, azalea, gordonia, azalea, michelia flabellina, michelia, begonia fortuneana, dendrobium, calanthera, malus, calyx seu fructus forsythiae, calyx cantoniae, calyx seu fructus calophyllanthi, calyx seu fructus physalis, calyx seu fructus; (9) medicinal crops: safflower (Carthamus tinctorius), Mentha (Mentha spp.), Rheum undulatum (Rheum rhabararum), Crocus sativus (Crocus sativus), medlar, polygonatum odoratum, rhizoma polygonati, rhizoma anemarrhenae, radix ophiopogonis, bulbus fritillariae cirrhosae, radix curcumae, fructus amomi, polygonum multiflorum, Rheum officinale, liquorice, radix astragali, ginseng, pseudo-ginseng, acanthopanax, angelica sinensis, ligusticum wallichii, radix bupleuri, stramonium, flos daturae, mint, leonurus, wrinkled gianthyssop, scutellaria baicalensis, selfheal, pyrethrum, ginkgo biloba, cinchona japonica, natural rubber trees, alfalfa and pepper; (10) raw material crops: rubber, castor (Ricinus communis), tung tree, mulberry, rose, birch, alder, sumac; (11) pasture crops: agropyron spp, axyrium spp, Miscanthus (Miscanthus sinensis), Pennisetum (Pennisetum sp.), Phalaris (Phalaris arundinacea), switchgrass (Panicum virgatum), grassland (prairie grass), Indian grass (Indian grass), Big-bristlegrass (Big bluestem grass), Phleum pratense, turfgrasses (turf), Cyperaceae (tall-fleabane, sedge (Carex pedioformis), low-bristlegrass, alfalfa, ladder grass, lucerne, tamarisk, field-grass, red duckweed, water tassel, lupine, trefoil, sargentgloryvine, water lettuce, peanut, black grass; (12) sugar crops: sugar cane (Saccharumspp.), sugar beet (Beta vulgaris); (13) beverage crops: big leaf tea (Camellia sinensis), tea tree (tea), coffee (Coffea spp.), cocoa (Theobroma cacao), hops (hops); (14) lawn plants: grass of the genus Poa (Poa spp.) (Poa pratensis (blue grass)), Agrostis species (Agrostis spp.) (grass of the species Agrostis cut (Agrostis pulustris), Agrostis stolonifera (Agrostis pulustris), Hami grass species (Lolium spp.), Imperata species (Festusque pp.) (Lesper. fescue), Hamamelis species (Zoyssia spp.) (Hamames grass (Zoysiajaponicum)), Cynodon species (Cynodon spp.) (Bermuda grass ), Bluey grass (Stenoperum seguinum) (Octagia stolonifera), Pasteur grass species (Paulo spp.) (Dinopsis sp.), Erythropia grass (Erythropia grass), Selaginella (Eupatorium), Selaginella sp. (grass of the species (Octagia), Selaginella (Octagia sp.), Nepalustris (Populus sp.), Nepalusta) Shortleaf kyllinga (Kylingbraflifolia), Amur sedge (Cyperusaamuricius), erigeron canadensis (Erigerontacanderensis), Hydrocotyle sativa (Hydrocotyle polytrichoides), Orthosiphon aristatus (Kummerowiata), Euphorbia humifusa (Euphorbia humifusa), Viola odorata (Violaarvensis), Carex alba (Carex alba), Carex isoprocarpus, and Triperus viridis (turf); (15) and (3) tree crops: pinus (Pinus spp.), Salix sp., Acer spp., Hibiscus spp., Eucalyptus sp., Ginkgo biloba (Ginko biloba), Bambusa (Bambusa sp.), Populus spp., Mucuna (Populus spp.), Psophora (Prosopis spp.), Quercus spp., Davidia (Quercussp.), Abelmoschus spp., Phoenix (Phoenix spp.), Fagus spp, Melaleuca spp., Pinus (Fabryanus spp.), Pinus tanpendra, Cinnamomum camphora (Cinnamomum spp.), Potentilla (Corchorus sp.), Melongrass Reevesii (Phragus spp.), Aconitus spp., Physalsa (Phyllanthus spp.), Populus spp., Populus nigra (Cinnamomum spp.), Populus spp., Populus nigra, Populus spp., Populus nigra Sprensis, Populus spp., Eupatula spp., Populus nigra Sprensis, Populus spp., Populus chinensis, kapok, kapok java, cercis negundo, bauhinia variegata, rain tree, albizia julibrissin, densefruit pittosporum, erythrina indica, southern magnolia, cycas revolute, crape myrtle, conifer, arbor and shrub; (16) nut crop: brazil chestnut (bertholetia excelsea), Castanea (Castanea spp.), Corylus (Corylus spp.), pecan (Caryaspp.), Juglans (Juglans spp.), pistachio (Pistacia vera), cashew (Anacardium occidentale), Macadamia nut (Macadamia integrifolia), pecan nut, Macadamia nut, pistachio nut, badam and nut-producing plants; (17) and others: arabidopsis thaliana, brachiaria, tribulus terrestris, setaria viridis, goosegrass, Cadaba farinosa, algae (algae), Carex elata, ornamental plants, pseudodamnacanthus macrophyllus (carissaceae), Cynara (Cynara spp.), wild carrot (Daucus carota), dioscorea (Dioscoreaspp.), saccharum sp.), Festuca (Erianthus sp.), Festuca (Festuca arundinacea), daylily (hemerallis fulva), Lotus spp (Lotus spp.), Luzula sylvatica, alfalfa (Medicago sativa), sweet clover (Melilotus spp.), black mulberry (Morus nigra), tobacco (Nicotiana spp.), olea spp., olea (olea spp.), yellow clover (olea spp.), yellow pepper (olea spp.), garden balsam), yellow pepper (olea spp.), garden balsam (olea spp.), eupatula (silla, grasses (olea spp.), eupatula (silla, etc.).

In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant calli, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers and the like.

In the present invention, "plant cell" is understood to be any cell derived from or found in a plant, which is capable of forming, for example: undifferentiated tissue such as callus, differentiated tissue such as embryos, plant parts, plants or seeds.

For the terms used in the specification with respect to amino acid substitutions, the first letter represents the naturally occurring amino acid at a position in the specified sequence, the following numbers represent the position relative to SEQ ID NO. 2, and the second letter represents the different amino acid that substituted the natural amino acid. For example, A103S shows that the alanine in position 103 is replaced by a serine in relation to the amino acid sequence of SEQ ID NO. 2. By amino acid substitution where the first letter is absent, it is meant that the natural amino acid is substituted by the amino acid represented by the letter following the number at the position corresponding to SEQ ID NO:2, relative to the amino acid sequence of its wild-type protein. For double or multiple mutations, each mutation is separated by a "/". For example, H141R/G342D/D370N indicates that, relative to the amino acid sequence of SEQ ID NO:2, histidine at position 141 is substituted with arginine, glycine at position 342 is substituted with aspartic acid, and aspartic acid at position 370 is substituted with asparagine, all three mutations being present within the particular mutant HPPD protein.

In one aspect, the present invention discloses a mutant HPPD protein or biologically active fragment thereof that retains the activity of catalyzing the conversion of Hydroxyphenylpyruvate (HPP) to homogentisate while having improved resistance or tolerance to HPPD-inhibiting herbicides as compared to the wild-type p-hydroxyphenylpyruvate dioxygenase protein. Specifically, the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein of the invention has one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L. Preferably, the amino acid sequence of said mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence depicted in SEQ ID NO. 2. More preferably, the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein has the amino acid sequence shown in SEQ ID No. 2, differing only in that it has one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L.

In one embodiment, the amino acid sequence of a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein of the invention has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the wild-type rice p-hydroxyphenylpyruvate dioxygenase protein amino acid sequence shown in SEQ ID No. 2 and has one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L. Preferably, the mutant P-hydroxyphenylpyruvate dioxygenase (HPPD) protein of the invention has the amino acid sequence shown in SEQ ID NO:2, differing only in that at one or more of the positions 93, 103, 141, 165, 191, 220, 226, 276, 277, 336, 337, 338, 342, 346, 370, 377, 386, 390, 392, 403, 410, 418, 419, 420, 430 and 431 in the amino acid sequence of the wild-type rice P-hydroxyphenylpyruvate dioxygenase protein shown in SEQ ID NO:2 there are one or more mutations selected from the group consisting of R93, A103, H141, A165, V191, R220, G226, L276, P277, P336, P337, N338, G342, R346, D370, I377, P390, L403, M392, E403, K410, K420, K419, G419, Y and Y386.

The specific amino acid positions (numbering) within the proteins of the invention are determined by aligning the amino acid sequence of the protein of interest with SEQ ID NO. 2 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is highest. Alignment scores can be calculated according to the method described in Wilbur, W.J. and Lipman, D.J, (1983) Rapid similarity searches of nucleic acid and protein data bases, Proc.Natl.Acad.Sci.USA,80: 726-730. Default parameters are preferably used in the ClustalW2(1.82) algorithm: protein gap opening penalty of 10.0; protein gap extension penalty of 0.2; protein matrix Gonnet; protein/DNA end gap-1; protein/DNAGAPDIST ═ 4.

The position of a particular amino acid within a protein according to the invention is preferably determined by aligning the amino acid sequence of the protein with SEQ ID NO:2 using the AlignX program (part of the vectorNTI set) with default parameters for multiple alignments (gap open penalty: 10og gap extension penalty 0.05).

The identity of amino acid sequences can be determined by conventional methods, see, e.g., Smith and Waterman,1981, adv.Appl.Math.2:482, Pearson&Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, Thompson et al, 1994, Nucleic Acids Res 22:467380, et al, determined by computerized operating algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics computer group). The National Center for Biotechnology Information available from the United states of America may also be usedwww.ncbi.nlm.nih.gov/) The BLAST algorithm obtained (Altschul et al, 1990, mol. biol.215:403-10), was determined using default parameters.

In a further embodiment, the mutant p-hydroxyphenyl pyruvate dioxygenase protein of the invention has SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID, 70, 72, 74, 76, 78, 80, 82 or 84.

In a further embodiment, the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein of the invention has the following amino acid mutations in its amino acid sequence: H141R/G342D, H141R/D370N, G342D/D370N, H141R/N338D, H141R/G342D, N338D/G342D, K418P/G419F, G419F/N420S, G342D/R346C, G342C/R346C, H141/N420C, G338C/K418C, P277C/N338C, L276C/P277 346, H141/G C/D C, H141C/N338C/N420C, H141/N C/N338C, P336/N338/G342/G C/N338, P C/N336/C, P C/N338/N336/C, P342/N338/N342, P342/N C/N338/N342/N C, N C/N338/N342/N C, N C/N C, N C/C, N C/36342/C, N C/N338/C/36342, N338/C/, P336D/N338D/R346C, P336D/N338D/R346H, P336D/N338D/R346S, P336D/N338S/R346C, P336D/N338S/R346H, P336D/N338S/R346S, P336D/N338Y/R346C, P336D/N338D/R36346 72, P336D/N338D/R36346D/R346D/36346 72, H141/N338/G342/G D, H141/G D/K418D, H141/G342/G D/G419/G36342, H D/36342/D, K36392/G D/36342/D/36342, H D/36342/D/36141/D/36342, H D/36342/D/36342, H D/36342/D/36342, H/36141/36342/D/36342, H/D/36342/D/36342, H/36342/D/36141/36342, H/D/36342/D, H/D/36342/D, H/36141/D/36342/D, H141/N338/G342, P277/G342/R346, P277/N338/N420, N338/G342/K418, H141/N338/G342/G419, H141/N338/G342/P386, H141/N338/G342/R346, H141/G342/K418/G419, H141/G342/L276/P277, P336/N338/G/R346, P336/N338/G342/R346, P336/N338/G346, P336/N336/G342/R346, P336/N338/G342/R342, P342/N342/G/R342, and P141/G342/P342/G/P342/L346, P336D/N338Y/G342D/R346S, P277N/P336D/N338D/G342D, P277N/N338D/G342D/R346C, P277N/N338D/K418P/G419F, H141R/N338D/G342D/K418P/G419F, H141R/N338D/G342D/G419F/N420S, H141R/G336D/G342D/K418P/G419F/N420S, H141R/N338D/G342D/K418P/G419F/N420S, H141R/N338D/G342D/K418P/G419F/N420T, H141R/N338D/G342D/R346C/K418P/G419F/N420F, H141F/N338F/G342F/R346F/K418F/G419F/N420F, H141F/P277F/G F/K338F/G419F/N F/G36342/K419F/N F/G F/K418/G419F/N419F/G F/K419F/G36419F/N F/G36419/N F/G36419F/N F/G F/.

In still further embodiments, the mutant p-hydroxyphenyl pyruvate dioxygenase (HPPD) protein of the invention has SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 86, SEQ ID NO 88, SEQ ID NO 90, SEQ ID NO 92, SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 112, SEQ ID NO 114, SEQ ID NO 116, SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 122, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO 130, SEQ ID NO 132, SEQ ID NO 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258 or 260 amino acid sequences.

In the present invention, the wild-type p-hydroxyphenylpyruvate dioxygenase protein may be derived from any plant, in particular from the aforementioned monocotyledonous or dicotyledonous plants. Several sources of wild-type p-hydroxyphenylpyruvate dioxygenase protein sequences, as well as coding sequences, have been disclosed in the prior art documents, which are incorporated herein by reference.

Preferably, the wild-type p-hydroxyphenylpyruvate dioxygenase protein of the invention is derived from rice, in particular rice. More preferably, said wild-type p-hydroxyphenylpyruvate dioxygenase protein has the amino acid sequence shown in SEQ ID No. 2 or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID No. 2.

It will also be clear to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example one or more conservative amino acid substitutions may be introduced in the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional configuration of the protein molecule. Examples and embodiments of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, the amino acid residue may be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue is substituted for another nonpolar amino acid residue, a polar uncharged amino acid residue is substituted for another polar uncharged amino acid residue, a basic amino acid residue is substituted for another basic amino acid residue, and an acidic amino acid residue is substituted for another acidic amino acid residue. Conservative substitutions where one amino acid is replaced with another amino acid belonging to the same group are within the scope of the present invention, as long as the substitution does not impair the biological activity of the protein.

Thus, the mutated HPPD proteins of the invention may comprise, in addition to the above mentioned mutations, one or more further mutations, such as conservative substitutions, in the amino acid sequence. In addition, mutant HPPD proteins that also comprise one or more other non-conservative substitutions are also encompassed by the present invention, provided that the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the invention.

As is well known in the art, one or more amino acid residues may be deleted from the N-and/or C-terminus of a protein while still retaining its functional activity. Thus, in a further aspect, the present invention also relates to fragments lacking one or more amino acid residues from the N-and/or C-terminus of a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein, while retaining its desired functional activity, which are also within the scope of the present invention, referred to as biologically active fragments. In the present invention, a "biologically active fragment" refers to a part of a mutated HPPD protein of the invention which retains the biological activity of the mutated HPPD protein of the invention, while having an increased tolerance or resistance to HPPD inhibitors compared to a HPPD fragment not having said mutation. For example, a biologically active fragment of a mutant HPPD protein may be a portion of the protein lacking one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1,2, 3,4, or 5) amino acid residues at the N-and/or C-terminus, but which still retains the biological activity of the full-length protein.

The invention also provides a fusion protein comprising the mutant HPPD protein of the invention, or a biologically active fragment thereof, and further components fused thereto. In a preferred embodiment, the further component is a plastid targeting peptide, e.g. a peptide that targets the mutated HPPD protein to the chloroplast. In another embodiment, the additional component is a tag peptide, such as 6 × His. In yet another embodiment, the further component is a peptide, e.g. a NusA peptide, that contributes to increasing the solubility of the mutant HPPD protein.

In yet another aspect, the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein as described above, or a biologically active fragment thereof, or a complement thereof. The term "isolated" polynucleotide means that the polynucleotide contains substantially no components that normally accompany it in a naturally occurring environment. In one embodiment, the amino acid sequence of the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein has an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID No. 2, and further has one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L. Preferably, the mutation is one or more mutations selected from the group consisting of: R93S, a103S, H141R, H141K, H141T, a165V, V191I, R220K, G226H, L276W, P277N, P336D, P337A, N338D, N338S, N338Y, G342D, R346C, R346D, R346H, R346S, R346Y, D370N, I377C, P386T, L390I, M392I, E36403, K410I, K418I, G419I, N420I, E36430 and Y431I. More preferably, the mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein or biologically active fragment thereof is derived from a rice HPPD protein and has one or more amino acid substitutions selected from the group consisting of the above.

It will be apparent to those skilled in the art that due to the degeneracy of the genetic code, there are a variety of different nucleic acid sequences which can encode the amino acid sequences disclosed herein. It is within the ability of one of ordinary skill in the art to generate other nucleic acid sequences encoding the same protein, and thus the present invention encompasses nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism, such as a plant, the gene may be optimized for better expression using codons preferred by the host organism.

Thus, in some embodiments, the polynucleotide of the invention has a nucleic acid sequence selected from the group consisting of:

(1) encoding SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, SEQ ID NO 146, SEQ ID NO 148, SEQ ID NO 150, SEQ ID NO 152, SEQ ID NO 154, SEQ ID NO 156, SEQ ID NO 158, SEQ ID NO 160, SEQ ID NO 162, SEQ ID NO 164, SEQ ID NO 166, SEQ ID NO 168, SEQ ID NO 170, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 176, SEQ ID NO 178, SEQ ID NO 180, SEQ ID NO 182, SEQ ID NO 184, SEQ ID NO 186, SEQ ID NO 188, SEQ ID NO 190, SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 196, SEQ ID NO 198, SEQ ID NO 200, SEQ ID NO 202, SEQ ID NO 204, SEQ ID NO 206, SEQ ID NO 208, SEQ ID NO 210, SEQ ID NO 188, SEQ ID NO 190, SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 198, SEQ ID NO 200, SEQ ID NO 202, SEQ ID NO 204, SEQ ID NO 206, SEQ ID NO 208, SEQ ID NO, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258 or 260 amino acid sequences or the complementary sequences thereof;

(2) 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 17, 55, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 175, 177, 179, 181, 183, 185, 187, 189, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 257 or 259 respectively or their complements;

(3) a nucleic acid sequence which hybridizes with the sequence shown in (1) or (2) under a strict condition; and

(4) a nucleic acid sequence which encodes the same amino acid sequence as that of the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or a complementary sequence thereof.

Further preferably, the polynucleotide has a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 85, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 103, SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO 111, SEQ ID NO 113, SEQ ID NO 115, SEQ ID NO 117, SEQ ID NO 119, SEQ ID NO 121, SEQ ID NO 123, SEQ ID NO 127, SEQ ID NO 129, SEQ ID NO 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 195, 197, 193, 195, 5, 9, 24, 9, 4, 1, 4,2, SEQ ID NO: 199. SEQ ID NO: 201. SEQ ID NO: 203. SEQ ID NO: 205. SEQ ID NO: 207. SEQ ID NO: 209. SEQ ID NO: 211. SEQ ID NO: 213. SEQ ID NO: 215. SEQ ID NO: 217. SEQ ID NO: 219. SEQ ID NO: 221. SEQ ID NO: 223. SEQ ID NO: 225. SEQ ID NO: 227. SEQ ID NO: 229. SEQ ID NO: 231. SEQ ID NO: 233. SEQ ID NO: 235. SEQ ID NO: 237. SEQ ID NO: 239. SEQ ID NO: 241. SEQ ID NO: 243. SEQ ID NO: 245. SEQ ID NO: 247. SEQ ID NO: 249. SEQ ID NO: 251. SEQ ID NO: 253. SEQ ID NO: 255. SEQ ID NO:257 or SEQ ID NO:259, a degenerate sequence thereof, or a nucleic acid sequence of its complement.

Preferably, the stringent conditions may refer to 6M urea, 0.4% SDS, 0.5 XSSC or their equivalent hybridization conditions, or may refer to more stringent conditions, such as 6M urea, 0.4% SDS, 0.1 XSSC or their equivalent hybridization conditions. In various conditions, the temperature may be above about 40℃, for example, where higher stringency conditions are desired, the temperature may be about 50℃, and further about 65℃.

Still more preferably, the amino acid mutation sites correspond to the following wild-type and mutant codons:

the present invention also provides a nucleic acid construct comprising a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxygenase protein of the invention, or a biologically active fragment or fusion protein thereof, operably linked to one or more regulatory elements. The term "regulatory element" as used herein refers to a nucleic acid sequence capable of regulating the transcription and/or translation of a nucleic acid to which it is operably linked.

The regulatory element may be an appropriate promoter sequence recognized by a host cell for expression of a nucleic acid sequence encoding a protein of the invention. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. As the promoter to be expressed in plant cells or plants, a promoter native to p-hydroxyphenylpyruvate dioxygenase or a heterologous promoter active in plants may be used. The promoter may be constitutively expressed or may be inducible. Examples of the promoter include, for example, a histone promoter, a rice actin promoter, a plant virus promoter such as a cauliflower mosaic virus promoter, and the like.

The regulatory element may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein of the present invention. Any terminator which is functional in the host cell of choice may be used in the present invention.

The regulatory element may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein of the invention. Any leader sequence which is functional in the host cell of choice may be used in the present invention.

The regulatory element may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.

The regulatory element may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of the protein and directs the encoded protein into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the portion of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. A foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to facilitate secretion of the polypeptide. In any event, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., into the culture medium, may be used in the present invention.

Regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell may also be added as appropriate. Regulatory systems are, for example, those which allow gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound, such as the lac, tec, and tip operator systems, the ADH2 system or the GAL 1system, among others. Other examples of regulatory sequences are those which allow gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide will be operably linked to the control sequences.

In the present invention, the regulatory element may also be a transcriptional activator or enhancer, such as a tobacco mosaic virus translational activator as described in WO87/07644, or an intron or the like, such as the maize adh1 intron, the maize bronze 1gene (maizebronze 1gene) intron, or the rice actin intron 1. They may enhance the expression of the mutant HPPD proteins, biologically active fragments thereof or fusion proteins of the invention in transgenic plants.

The invention also provides an expression vector, which comprises a nucleic acid sequence for encoding the mutant p-hydroxyphenyl pyruvate dioxygenase protein or a biologically active fragment or fusion protein thereof and an expression control element operably connected with the nucleic acid sequence. The expression vector also contains at least one origin of replication for self-replication. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any element which ensures self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. Alternatively, the vector may be a vector for gene editing of an HPPD gene endogenous to the host cell.

Vectors may be of the type, for example, plasmids, viruses, cosmids, phages and the like, which are well known to those skilled in the art and are described extensively in the art. Preferably, the expression vector in the present invention is a plasmid. Expression vectors can include promoters, ribosome binding sites for translation initiation, polyadenylation sites, transcription terminators, enhancers, and the like. The expression vector may also contain one or more selectable marker genes for use in selecting host cells containing the vector. Such selectable markers include the gene encoding dihydrofolate reductase, or the gene conferring neomycin tolerance, the gene conferring resistance to tetracycline or ampicillin, and the like.

The vectors of the present invention may contain elements that allow the vector to integrate into the host cell genome or to replicate autonomously within the cell independent of the genome. For integration into the genome of a host cell, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector suitable for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may comprise additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at the exact location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should preferably include a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, more preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to increase the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may integrate into the genome of the host cell by non-homologous recombination. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions within the cell. The term "origin of replication" or "plasmid replicon" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase the yield of the gene product. An increase in the number of copies of a polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, in which case cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected for by artificially culturing the cells in the presence of the appropriate selectable agent.

The nucleic acid sequences of the invention may be inserted into the vector by a variety of methods, for example by ligation following digestion of the insert and vector with appropriate restriction endonucleases. A variety of cloning techniques are known in the art and are within the knowledge of those skilled in the art.

Vectors suitable for use in the present invention include commercially available plasmids such as, but not limited to: pBR322(ATCC 37017), pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1(Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pOG 1, pSG (VK 3), (pBPV, pMSG, and Strvl Pharmacia) and the like.

The invention also provides host cells comprising a nucleic acid sequence, nucleic acid construct or expression vector of the invention. The vector comprising the vector encoding the present invention is introduced into a host cell such that the vector is present as part of a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier, or the vector may be capable of gene editing an HPPD gene endogenous to the host cell. The host cell may be any host cell familiar to the person skilled in the art, including prokaryotic cells and eukaryotic food cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells or plant cells, examples of which are Escherichia coli (E.coli), Streptomyces (Streptomyces), Bacillus subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonella typhimurium), Pseudomonas (Pseudomonas), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus), Spodoptera Sf9, CHO, COS, etc. The choice of an appropriate host cell is within the ability of those skilled in the art.

In the present invention, the term "host cell" also encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

The nucleic acid sequences, nucleic acid constructs or expression vectors of the invention can be introduced into a host cell by a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid mediated gene delivery, as well as calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection or electroporation, and the like (see Davis, L., Dibner, M., Battiey, I., Basicmethods in Molecular Biology, 1986).

In a particular embodiment, the mutated HPPD proteins of the invention may be targeted to plastids, e.g. chloroplasts, within plants. This can be achieved by linking in frame a nucleic acid sequence encoding a mutant HPPD protein of the invention to a nucleic acid sequence encoding a plastid leader peptide, e.g., a chloroplast transit peptide. Alternatively, the polynucleotide, nucleic acid construct or expression vector of the invention can be directly transformed into the chloroplast genome of a plant cell. Vectors and methods useful for transforming the chloroplast genome of a plant cell will be apparent to those skilled in the art. For example, the nucleic acid sequence encoding the mutant HPPD protein of the invention may be integrated by bombarding leaves of the target plant with DNA-coated ions and by homologous or non-homologous recombination.

The transformed host cells may be cultured in conventional nutrient media, where appropriate. After transformation of a suitable host cell and cultivation of the host cell to a suitable cell density, the selected promoter may be induced by suitable means, such as temperature change or chemical induction, and the cell may be further cultivated for a period of time such that it produces the mutated HPPD protein of the invention or a biologically active fragment or fusion protein thereof.

Accordingly, the present invention also relates to a method for producing a mutated HPPD protein of the invention, or a biologically active fragment or fusion protein thereof, comprising: (a) culturing the above host cell under conditions conducive to the production of the mutated HPPD protein or biologically active fragment or fusion protein thereof; and (b) recovering the mutated HPPD protein or biologically active fragment or fusion protein thereof.

In the production methods of the invention, the cells are cultured on a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cells are cultured in laboratory or industrial fermentors by shake flask culture and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation is carried out on a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be formulated according to published compositions (e.g., on catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the culture medium, it can be recovered from the cell lysate.

The polypeptide may be detected by methods known in the art that are specific for the polypeptide. These detection methods may include the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate.

The resulting polypeptide can be recovered by methods known in the art. For example, cells can be harvested by centrifugation, physically or chemically disrupted, and the resulting crude extract retained for further purification. Transformed host cells expressing a mutated HPPD protein or biologically active fragment or fusion protein thereof of the invention may be lysed by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of a lytic agent. These methods are well known to those skilled in the art. The mutant HPPD proteins or biologically active fragments thereof of the present invention can be recovered and purified from cultures of transformed host cells by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and phytohemagglutinin chromatography, among others.

The present invention also relates to a method for producing a host organism, in particular a plant cell, plant tissue, plant part or plant, which is tolerant or resistant to HPPD-inhibiting herbicides, comprising transforming said host organism with a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxygenase protein of the invention or a biologically active fragment thereof, a nucleic acid construct or an expression vector comprising said nucleic acid sequence. Methods for transformation of host cells, such as plant cells, are known in the art and include, for example, protoplast transformation, fusion, injection, electroporation, PEG-mediated transformation, ion bombardment, viral transformation, Agrobacterium-mediated transformation, electroporation or bombardment, and the like. A series of such transformation methods are described in the prior art, for example in EP1186666 for soybean transformation, in WO 92/09696 for monocotyledonous plants, in particular rice transformation, and the like. It may also be advantageous to culture plant explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes to transfer DNA into plant cells. Whole plants can then be regenerated from infected plant material parts (such as leaf fragments, stem segments, roots and protoplasts or suspension-cultured cells) in a suitable medium, which may contain antibiotics or pesticides for selection. Transformed cells grow in the usual way in plants, they can form germ cells and transmit the transformed trait to progeny plants. Such plants can be grown in the normal manner and crossed with plants having the same transforming genetic element or other genetic elements. The resulting heterozygous individuals have the corresponding phenotypic characteristics.

The present invention also provides a method for producing a host organism, in particular a plant cell, plant tissue, plant part or plant, which is tolerant or resistant to an HPPD-inhibiting herbicide, comprising integrating into the genome of the host organism and expressing a nucleic acid encoding a mutant p-hydroxyphenylpyruvate dioxygenase protein, or a biologically active fragment thereof, according to the invention. Suitable vectors and selectable markers are well known to those skilled in the art, and one method of integration into the tobacco genome is described, for example, in WO06/108830, the disclosure of which is incorporated herein by reference. The gene of interest is preferably expressed in the plant cell by a constitutive or inducible promoter. Once expressed, mRNA is translated into protein, thereby incorporating the amino acid of interest into the protein. The gene encoding the protein expressed in the plant cell may be under the control of a constitutive promoter, a tissue specific promoter or an inducible promoter. For example, promoters of bacterial origin such as octopine synthase promoter, nopaline synthase promoter, mannopine synthase promoter; promoters derived from viruses such as cauliflower mosaic virus (35S and 19S), 35T (re-engineered 35S promoter, see U.S. Pat. No.6,166,302, especially example 7E), and the like. Plant promoter regulatory elements may also be used, including but not limited to the small subunit of the ribose-1, 6-diphosphate (RUBP) carboxylase (ssu), the β -conglycinin promoter, the β -phaseolin promoter, the ADH promoter, the heat shock promoter, and tissue-specific promoters. Constitutive promoter regulatory elements can also be used to direct sustained gene expression in all cell types and at all times (e.g., actin, ubiquitin, CaMV35S, etc.). Tissue-specific promoter regulatory elements are also useful in the present invention, which are responsible for gene expression (e.g., zein, oleosin, napin, ACP, globulin, etc.) in a particular cell or tissue type (e.g., leaf or seed). Similarly, promoter regulatory elements that are active (or inactive) at some stage of plant development may also be used. Examples of such promoter regulatory elements include, but are not limited to, pollen-specific, embryo-specific, corn ear silk-specific, cotton fiber-specific, root-specific, seed endosperm-specific, or asexual phase-specific promoter regulatory elements, and the like. In some cases it may be desirable to use inducible promoter regulatory elements which are responsible for gene expression in response to specific signals such as physical stimuli (heat shock genes), light (RUBP carboxylase), hormones (Em), metabolites, chemicals (tetracycline response) and stress. Other desirable transcription and translation elements that function in plants may be used.

The present invention also provides a method for increasing the tolerance or resistance of a plant cell, plant tissue, plant part or plant to an HPPD-inhibiting herbicide, which comprises transforming said plant or part thereof with a nucleic acid molecule comprising a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxygenase protein, or a biologically active fragment or fusion protein thereof, of the invention and allowing expression thereof. The nucleic acid molecule may be expressed as an extrachromosomal entity or may be integrated into the genome of the plant cell for expression, in particular by homologous recombination at the location of an endogenous gene in the plant cell. These embodiments are within the scope of the present invention.

The present invention also provides a method of increasing HPPD-inhibiting herbicide tolerance or resistance in a plant or part thereof, comprising crossing a plant expressing a mutant hydroxyphenylpyruvate dioxygenase (HPPD) protein, or a biologically active fragment or fusion protein thereof, according to the invention, with another plant, and screening for plants or parts thereof having increased HPPD-inhibiting herbicide tolerance or resistance.

The present invention also provides a method of increasing HPPD-inhibiting herbicide tolerance or resistance in a plant cell, plant tissue, plant part or plant comprising genetically editing an endogenous HPPD protein of said plant cell, plant tissue, plant part or plant to effect expression therein of a mutant p-hydroxyphenylpyruvate dioxygenase protein, or a biologically active fragment or fusion protein thereof, of the present invention.

The present invention also relates to a method for producing a plant with increased herbicide tolerance or resistance by conventional breeding techniques, which comprises selfing or crossing a plant with a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxygenase protein according to the invention or a biologically active fragment thereof integrated in its genome and selecting progeny which comprise said encoding nucleic acid sequence heterozygously or homozygously.

The invention further relates to plant cells, plant tissues, plant parts and plants, and progeny thereof, obtained by the above method.

Preferably, plant cells, plant tissues or plant parts transformed with a polynucleotide of the present invention can be regenerated into whole plants. The invention includes cell cultures, including tissue cell cultures, liquid cultures, and solid plate cultures. Seeds produced by and/or used to regenerate the plants of the invention are also included within the scope of the invention. Other plant tissues and parts are also encompassed by the present invention. The invention likewise includes methods for producing plants or cells which contain the nucleic acid molecules according to the invention. One preferred method of producing such plants is by planting the seeds of the invention. Plants transformed in this way can acquire resistance to a variety of herbicides with different modes of action.

For example, for transforming a plant cell with Agrobacterium, the explant can be mixed with the transformed Agrobacterium and incubated for a sufficient time to allow transformation thereof. After transformation, the Agrobacterium is killed by selection with the appropriate antibiotic and the plant cells are cultured in the appropriate selection medium. Once callus is formed, shoot formation can be promoted by the use of appropriate plant hormones according to methods well known in the art of plant tissue culture and plant regeneration. However, the callus intermediate stage is not always necessary. After bud formation, the plant cells may be transferred to a medium that promotes root formation, thereby completing plant regeneration. The plant may then be grown to produce seeds that may be used to establish future generations. Regardless of the transformation technique, it is preferred that the gene encoding the bacterial protein be incorporated into a gene transfer vector adapted for expression of the gene in a plant cell by incorporating into the vector plant promoter regulatory elements and a 3' untranslated transcription termination region (e.g., Nos, etc.).

The present invention also provides a method of controlling weeds at a plant locus comprising applying to the locus containing plants or seeds of the invention a weed controlling effective amount of one or more HPPD inhibiting herbicides.

In the present invention, the term "locus" includes a field for cultivating the plant of the present invention such as soil, and also includes, for example, plant seeds, plant seedlings and grown plants. The term "weed-controlling effective amount" refers to an amount of herbicide sufficient to affect the growth or development of a target weed, e.g., to arrest or inhibit the growth or development of a target weed, or to kill the weed. Advantageously, the weed controlling effective amount does not significantly affect the growth and/or development of the plant seeds, plant seedlings or plants of the present invention. Such an effective weed-controlling amount can be determined by one skilled in the art by routine experimentation.

The present invention also provides a method for producing a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein which retains or potentiates the property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate and which is significantly less sensitive to HPPD-inhibiting herbicides than the wild-type HPPD, which comprises mutating a nucleic acid encoding a wild-type HPPD, fusing and ligating the mutated nucleic acid in an expression vector in frame with a nucleic acid sequence encoding a solubility-enhancing component to form a fusion protein coding sequence, transforming the resulting recombinant expression vector into a host cell, expressing the fusion protein under suitable conditions comprising the HPPD-inhibiting herbicide and an HPPD enzymatic substrate and screening for a mutant p-hydroxyphenylpyruvate dioxygenase (HPPD) protein which retains or potentiates the property of catalyzing the conversion of p-Hydroxyphenylpyruvate (HPP) to homogentisate and which has a significantly reduced sensitivity to HPPD-inhibiting herbicides. Preferably, the solubility-enhancing component is NusA, which constitutes a fusion protein with the mutated HPPD protein of the invention. More preferably, the expression vector is a pET-44a vector. The host cell may be a bacterial cell, a fungal cell or a plant cell.

As used herein, the terms "a", "an" and "the" mean "at least one" unless specifically stated or implied. All patents, patent applications, and publications mentioned or cited herein are incorporated by reference in their entirety to the same extent as if individually incorporated.

Detailed Description

The present invention is further illustrated by the following examples. All methods and operations described in these embodiments are provided by way of example and should not be construed as limiting. Reference is made to Current protocols in Molecular Biology, volumes 1 and 2, Ausubel F.M., Greene publishing associates and Wiley Interscience,1989, Molecular Cloning, T.Maniatis et al, 1982, or Sambrook J.and Russell D.2001, Molecular Cloning: a laboratory, version 3.