Mutant PSII D1 or psbA D1 protein, nucleic acid encoding same and application thereof
阅读说明：本技术 突变型PSII D1或psbA D1蛋白、其编码核酸以及应用 (Mutant PSII D1 or psbA D1 protein, nucleic acid encoding same and application thereof ) 是由 连磊 李华荣 莫苏东 彭学岗 于 2018-07-09 设计创作，主要内容包括：本发明属于农业基因工程领域,具体涉及突变型PSII D1或psbA D1蛋白、其编码核酸以及应用。所述突变型PSII D1或psbA D1蛋白的氨基酸序列与野生型水稻PSII D1或psbA D1蛋白的氨基酸序列相比,在对应于SEQ ID.04所示的野生型水稻PSII D1或psbA D1蛋白氨基酸序列中的第219、249、251、255、256、264、266和275位中的一个或多个位置处具有选自下述中的一个或多个突变：219I、249A、251V、255Y、256A、264G、264A、264T、266T和275F。本发明通过上述突变位点来赋予水稻抗PSII抑制性除草剂的能力,以解决严重的杂草稻及其他作物受除草剂药害的问题。(The invention belongs to the field of agricultural genetic engineering, and particularly relates to a mutant PSII D1 or psbA D1 protein, and a coding nucleic acid and application thereof. The amino acid sequence of the mutant PSII D1 or psbA D1 protein has one or more mutations selected from the group consisting of at positions corresponding to one or more of positions 219, 249, 251, 255, 256, 264, 266, and 275 in the amino acid sequence of the wild-type rice PSII D1 or psbA D1 protein set forth in SEQ id No. 04, as compared to the amino acid sequence of the wild-type rice PSII D1 or psbA D1 protein: 219I, 249A, 251V, 255Y, 256A, 264G, 264A, 264T, 266T, and 275F. The invention endows the rice with the PSII inhibition herbicide resistance through the mutation sites so as to solve the problem that severe weedy rice and other crops are phytotoxicity to the herbicide.)
1. A mutant PSII D1 or psbA D1 protein, or a biologically active fragment thereof, wherein the amino acid sequence of said mutant PSII D1 or psbA D1 protein has one or more mutations selected from the group consisting of: 219I, 249A, 251V, 255Y, 256A, 264G, 264A, 264T, 266T, and 275F.
2. The mutant PSII D1 or psbA D1 protein or biologically active fragment thereof of claim 1, wherein the amino acid sequence of the mutant PSII D1 or psbA D1 protein further has at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the amino acid sequence set forth in SEQ ID.04.
3. The mutant PSII D1 or psbA D1 protein or biologically active fragment thereof of claim 1 or 2, wherein the mutant PSII D1 or psbA D1 protein has the amino acid sequence set forth in SEQ ID.04, except for having one or more amino acid mutations defined in claim 1.
4. The mutant PSII D1 or psbA D1 protein or biologically active fragment thereof according to any one of claims 1-3, wherein the amino acid sequence of said mutant PSII D1 or psbA D1 protein has one or more mutations selected from the group consisting of: V219I, V249A, a251V, F255Y, G256A, S264G, S264A, S264T, N266T and L275F.
5. The mutant PSII D1 or psbA D1 protein or biologically active fragment thereof according to claim 4, wherein the amino acid sequence of said mutant PSII D1 or psbA D1 protein has one or more mutations selected from the group consisting of: V219I, a251V and S264G, preferably the amino acid sequence of said mutant PSII D1 or psbA D1 protein has the following amino acid mutations: S264G/A251V or S264G/V219I.
6. The mutant PSII D1 or psbA D1 protein or biologically active fragment thereof of any one of claims 1-5, wherein the mutant PSII D1 or psbA D1 protein has the amino acid sequence set forth in SEQ ID.06 or SEQ ID.08 or SEQ ID.10 or SEQ ID.12 or SEQ ID.14 or SEQ ID.16 or SEQ ID.18 or SEQ ID.20 or SEQ ID.22 or SEQ ID.24 or SEQ ID.26 or SEQ ID.28.
7. A fusion protein comprising a mutant PSII D1 or psbA D1 protein or biologically active fragment thereof according to any one of claims 1-6, fused to another component, e.g. a tag peptide such as 6 xhis, or a plastid targeting peptide such as a peptide that targets chloroplasts.
8. An isolated polynucleotide comprising a nucleic acid sequence encoding the PSII D1 or psbAD1 protein of any one of claims 1-6 or a biologically active fragment thereof, or the fusion protein of claim 7, or a complement thereof, preferably wherein said polynucleotide is DNA, RNA, or a hybrid thereof; the polynucleotide is single-stranded or double-stranded.
9. The polynucleotide of claim 8 having a nucleic acid sequence selected from the group consisting of:
(1) a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID.06 or SEQ ID.08 or SEQ ID.10 or SEQ ID.12 or SEQ ID.14 or SEQ ID.16 or SEQ ID.18 or SEQ ID.20 or SEQ ID.22 or SEQ ID.24 or SEQ ID.26 or SEQ ID.28 or a complementary sequence thereof;
(2) any one of the nucleic acid sequences shown in SEQ ID.05 or SEQ ID.07 or SEQ ID.09 or SEQ ID.11 or SEQ ID.13 or SEQ ID.15 or SEQ ID.17 or SEQ ID.19 or SEQ ID.21 or SEQ ID.23 or SEQ ID.25 or SEQ ID.27 or the complementary sequence 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.
10. The polynucleotide of claim 8 or 9, said nucleic acid sequence being optimized for expression in a plant cell.
11. An expression cassette comprising the polynucleotide of any one of claims 8-10.
12. A vector comprising the polynucleotide of any one of claims 8-10.
13. A host cell comprising the PSII D1 or psbA D1 protein of any one of claims 1-6 or a biologically active fragment thereof, the fusion protein of claim 7, the polynucleotide of any one of claims 8-10, the expression cassette of claim 11, or the vector of claim 12, preferably wherein said host cell is a plant cell.
14. A method of obtaining a plant resistant or tolerant to any one of the PSII herbicides comprising regenerating the plant cell of claim 13 into a plant, preferably wherein the method is performed by transgenic, gene editing, crossing, backcrossing, selfing or asexual propagation.
15. A plant produced by the method of claim 14.
16. A method of conferring resistance or tolerance to a PSII-type herbicide to a plant cell, plant tissue, plant part, or plant, wherein (1) comprises expressing in the plant cell, plant tissue, plant part, or plant the PSII D1 or psbA D1 protein or biologically active fragment thereof of any one of claims 1-6 or the fusion protein of claim 7; or (2) comprising crossing a plant expressing the PSII D1 or psbA D1 protein or biologically active fragment thereof of any one of claims 1-6 or the fusion protein of claim 7 with another plant and selecting plants or parts thereof with increased resistance or tolerance to PSII class herbicides; or (3) comprising gene editing of a PSII D1 or psbA D1 protein of said plant cell, plant tissue, plant part or plant to effect expression therein of a PSII D1 or psbA D1 protein of any one of claims 1-6 or a biologically active fragment thereof, or a fusion protein of claim 7.
17. Use of the PSII D1 or psbA D1 protein of any one of claims 1-6, or a biologically active fragment thereof, the fusion protein of claim 7, the polynucleotide of any one of claims 8-10, the expression cassette of claim 11, or the vector of claim 12 for conferring resistance or tolerance to any one PSII class herbicide on a plant cell, plant tissue, whole plant.
18. A method of controlling weeds at a plant locus, wherein the plants comprise the plant of claim 15 or the plant prepared by the method of claim 14 or 16, the method comprising applying to the locus a weed controlling effective amount of one or more PSII-type herbicides.
19. A method for identifying a plant comprising the steps of:
a) determining whether the plant comprises a polynucleotide of any one of claims 8-10; or the like, or, alternatively,
b) determining whether said plant expresses the PSII D1 or psbA D1 protein or biologically active fragment thereof according to any one of claims 1-6 or the fusion protein according to claim 7.
20. The method of any one of claims 14, 16 and 18 or the use of claim 17, wherein the PSII-class herbicide comprises at least one of the following active ingredients:
(1) amides: mechloranilide, propanil;
(2) benzopyrazoles: bentazone;
(3) oximes/hydroxybenzonitriles: phytocide, bromoxynil and ioxynil;
(4) carbamates: desmedipham, diphenoxylate;
(5) pyridazinones: killing phytocide;
(6) triazolinones: amicarbazone;
(7) uracils: weeding, weeding and weeding;
(8) phenylpyridazines: pyridazinol, pyridate, QYP 02410;
(9) triazinones: hexazinone, metamitron, metribuzin;
(10) triazines: atrazine, ametryn, cyanazine, dimethomon, pentrazine, prometon, prometryn, simazine, simetryn, terbuthylazine, terbutryn, and prodazine;
(11) ureas: chlorsulfuron, chlortoluron, chlorsulfuron, oxazolone, diuron, sulfosulfuron, fensulfuron, fluometuron, isoproturon, clomazone, linuron, bromuron, thiazoluron, metoxuron, chlorsulfuron, glusulfuron, cycuron, tebuconazole, thidiazuron;
(12) other classes: kacaoling.
21. The method of any one of claims 14, 16 and 18 to 19, the plant of claim 15 or the use of claim 17, wherein the plant is a graminaceous plant such as rice, wheat, maize, millet or barley.
Technical Field
The invention belongs to the field of agricultural genetic engineering, and particularly relates to a mutant PSII D1 or psbA D1 protein, and a coding nucleic acid and application thereof.
Background
Rice is one of the most important food crops in the world, and lives nearly half of the world population, and particularly, in Asia, rice is an absolute life food for Asian people. However, the cultivation of paddy rice faces serious challenges for weedy rice. Current rice varieties resistant to imidazolinone (acetolactate synthase, ALS) and acetyl-CoA carboxylase (ACCase) inhibitors, while capable of controlling weedy rice, are herbicide resistant as rice after 3-4 years of use. The herbicide resistance traits of rice can be hybridized with rice and weedy rice, namely pollen of the resistant rice is pollinated to the weedy rice with the herbicide resistance genes, so that the weedy rice also generates drug resistance, the resistant weedy rice is further developed, the problem becomes more and more serious, and the resistant weedy rice is widely distributed in south-east Asia at present.
Disclosure of Invention
In order to solve the problems, the invention creatively adopts the mutation of rice seeds to breed the rice variety of the PSII-resistant inhibition herbicide. Surprisingly, the bred mutant rice pollen pollinates other rice and weedy rice, and the generated progeny can not inherit the resistance character to herbicide. The existing weedy rice control technology is greatly changed, and a brand-new revolutionary solution is provided for the control of the weedy rice and the resistant weeds widely accepted in China, southeast Asia and the like.
In one aspect, the invention provides a mutant PSII D1 or psbA D1 protein, or a biologically active fragment thereof, wherein the amino acid sequence of the mutant PSII D1 or psbA D1 protein has one or more mutations selected from the group consisting of: 219I, 249A, 251V, 255Y, 256A, 264G, 264A, 264T, 266T, and 275F. Preferably, the amino acid sequence of the mutant PSII D1 or psbA D1 protein further has at least 90%, at least 95%, at least 98% and at least 99% sequence identity to the amino acid sequence set forth in SEQ id.04. More preferably, the mutant PSIID1 or psbA D1 protein has the amino acid sequence shown in SEQ id.04, except having one or more amino acid mutations as defined in claim 1.
In one embodiment, the amino acid sequence of the mutant PSII D1 or psbA D1 protein of the invention has one or more mutations selected from the group consisting of at positions corresponding to one or more of positions 219, 249, 251, 255, 256, 264, 266, and 275 in the amino acid sequence of the wild type rice PSII D1 or psbA D1 protein as set forth in SEQ id No. 04: V219I, V249A, a251V, F255Y, G256A, S264G, S264A, S264T, N266T and L275F.
In a further embodiment, the amino acid sequence of the mutant PSII D1 or psbA D1 protein of the invention has one or more mutations selected from the group consisting of at positions 219, 251 and 264 in the amino acid sequence corresponding to the wild-type rice PSII D1 or psbA D1 protein set forth in SEQ id No. 04: V219I, a251V and S264G, preferably the amino acid sequence of said mutant PSII D1 or psbA D1 protein has the following amino acid mutations: S264G/A251V or S264G/V219I.
In a still further embodiment, the mutant PSII D1 or psbA D1 protein of the invention has the amino acid sequence shown in SEQ id.06 or SEQ id.08 or SEQ id.10 or SEQ id.12 or SEQ id.14 or SEQ id.16 or SEQ id.18 or SEQ id.20 or SEQ id.22 or SEQ id.24 or SEQ id.26 or SEQ id.28.
The invention also provides a fusion protein, which comprises the mutant PSII D1 or psbA D1 protein or the bioactive fragment thereof and other components fused with the fusion protein. In a preferred embodiment, the additional component is a plastid targeting peptide, e.g., a peptide that targets the mutated PSII D1 or psbA D1 protein to the chloroplast. In another embodiment, the additional component is a tag peptide, such as 6 × His. In yet another embodiment, the additional component is a peptide, such as a NusA peptide, that contributes to increasing the solubility of the mutant PSII D1 or psbA D1 protein.
In another aspect, the invention also provides an isolated polynucleotide comprising a nucleic acid sequence encoding the PSII D1 or psbA D1 protein or biologically active fragment thereof, or the fusion protein, or a complement thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, whether double-stranded or single-stranded. The term "isolated" polynucleotide means that the polynucleotide contains substantially no components that normally accompany it in a naturally occurring environment.
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, the polynucleotide has a nucleic acid sequence selected from the group consisting of: (1) a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID.06 or SEQ ID.08 or SEQ ID.10 or SEQ ID.12 or SEQ ID.14 or SEQ ID.16 or SEQ ID.18 or SEQ ID.20 or SEQ ID.22 or SEQ ID.24 or SEQ ID.26 or SEQ ID.28 or a complementary sequence thereof; (2) any one of the nucleic acid sequences shown in SEQ ID.05 or SEQ ID.07 or SEQ ID.09 or SEQ ID.11 or SEQ ID.13 or SEQ ID.15 or SEQ ID.17 or SEQ ID.19 or SEQ ID.21 or SEQ ID.23 or SEQ ID.25 or SEQ ID.27, degenerate sequences thereof or complementary sequences 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, due to the degeneracy of the genetic code, encodes the same amino acid sequence as the sequence shown in (1) or (2), or a complementary sequence thereof. The stringent conditions may be 6M urea, 0.4% SDS, or 0.5 XSSC, or equivalent hybridization conditions, or may be more stringent conditions, such as 6M urea, 0.4% SDS, or 0.1 XSSC, or 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℃. In addition, the nucleic acid sequence is optimized for expression in a plant cell.
The invention also provides an expression cassette (cassette) comprising the polynucleotide. Preferably, the expression cassette comprises a promoter region, a nucleic acid and a termination region for transcription and translation in the 5 '-3' direction of transcription. More preferably, the expression cassette is suitable for expressing a nucleic acid in a plant. Exemplary promoters include those from the CaMV35S promoter or the rice actin I gene, maize alcohol dehydrogenase gene, maize dwarf I gene, or Tobacco Mosaic Virus (TMV) omega element; exemplary transcriptional and translational termination regions include transcription termination elements or polyadenylation signals, and the like. The expression cassette may also include an origin of replication required for replication in bacteria (e.g., the ORI region from pBR322 or P15 Aori), elements required for Agrobacterium T-DNA transfer (e.g., the left and/or right borders of the T-DNA), to facilitate transformation from bacteria into plants. In addition, the expression cassette may also comprise enhancers, introns, multiple cloning sites, operators, repressor binding sites, transcription factor binding sites, etc., such as, for example, leader sequences from TMV, Maize Chlorotic Mottle Virus (MCMV) or Alfalfa Mosaic Virus (AMV) and the like as enhancers, and may, for example, comprise introns from Adh1, bronze1, actin 1 or actin 2.
The invention also provides a vector comprising the polynucleotide. 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 the PSII D1 or psbA D1 gene endogenous to the host cell.
Vectors may be of the plasmid, virus, cosmid, phage, etc. type, 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(ATCC37017), 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 a host cell comprising said PSII D1 or psbA D1 protein or a biologically active fragment thereof, said fusion protein, said polynucleotide, said expression cassette or said vector.
The vector comprising a nucleic acid 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 the PSII D1 or psbA D1 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), Spodopterasf9, CHO, COS, etc. The choice of an appropriate host cell is within the ability of those skilled in the art. Preferably, the host cell is a plant cell.
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, expression cassettes 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., Battey, I., Basicmethods in Molecular Biology, 1986).
In a particular embodiment, a mutant PSII D1 or psbA D1 protein of the invention can be targeted to a plastid, e.g., a chloroplast, within a plant. This can be achieved by linking in frame a nucleic acid sequence encoding a mutant PSII D1 or psbA D1 protein of the invention to a nucleic acid sequence encoding a plastid leader peptide, e.g., a chloroplast transit peptide. Alternatively, the polynucleotide, expression cassette 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, a nucleic acid sequence encoding a mutant PSII D1 or psbA D1 protein of the invention can be integrated by bombarding leaves of a 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 an appropriate cell density, the selected promoter may be induced by appropriate means, such as temperature shift or chemical induction, and the cell may be additionally cultured for a period of time to allow production of the mutant PSII D1 or psbA D1 protein or biologically active fragment or fusion protein thereof of the invention.
The invention also provides a method for obtaining a plant resistant or tolerant to any one PSII class herbicide, comprising regenerating said plant cell into a plant. Preferably, this is done by means of transgenics, gene editing, crossing, backcrossing, selfing or asexual propagation.
In another aspect, the present invention also provides plants produced by the methods.
The present invention also provides a method of increasing resistance or tolerance to a PSII class herbicide in a plant cell, plant tissue, plant part, or plant comprising expressing in the plant cell, plant tissue, plant part, or plant the PSII D1 or psbA D1 protein or biologically active fragment thereof or the fusion protein; or comprising crossing a plant expressing said PSII D1 or psbA D1 protein or biologically active fragment thereof or said fusion protein with another plant and selecting plants or parts thereof having increased resistance or tolerance to a PSII class herbicide; or comprising genetically editing the PSII D1 or psbA D1 protein of said plant cell, plant tissue, plant part or plant to achieve expression therein of said PSII D1 or psbA D1 protein or biologically active fragment thereof or said fusion protein.
The invention also provides the use of the PSII D1 or psbA D1 protein or a biologically active fragment thereof, the fusion protein, the polynucleotide, the expression cassette or the vector for improving the resistance or tolerance of plant cells, plant tissues or the whole plant to any PSII herbicide.
The invention also provides a method of controlling weeds at a locus for growing plants, comprising applying to the locus a weed controlling effective amount of one or more PSII-type 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 identifying a plant, comprising the steps of: a) determining whether said plant comprises said polynucleotide; or, b) determining whether said plant expresses said PSII D1 or psbA D1 protein or a biologically active fragment thereof or said fusion protein.
In the present invention, the "PSII-type herbicide" is a substance having herbicidal activity by itself or in combination with other herbicides and/or additives capable of changing the effect thereof, and can act by inhibiting photosynthesis of plants by targeting D1 protein to the action of the photosynthesis system ii (PS ii). PSII-type herbicides are well known in the art and include many types, (1) amides: chlorantraniliprole/paraquat (pentaochlor, 2307-68-8), propanil (propanil, 709-98-8); (2) benzopyrazoles: bentazone/bentazone (25057-89-0); (3) oximes/hydroxybenzonitriles: bromophenol oxime/desmofenoxim (bromofenoxim, 13181-17-4); bromoxynil (bromoxynil, 1689-84-5); ioxynil (ioxynil, 1689-83-4); (4) carbamates: desmedipham/diphenoxylate (13684-56-5), phenmedipham/diphenoxylate (13684-63-4), phenmedipham-ethyl (13684-44-1); (5) pyridazinones: chlorpyrifos (Chlorazone/Pyrazon, 1698-60-8); (6) triazolinones: amicarbazone/amicarbazone (amicarbazone, 129909-90-6); (7) uracils: bromacil (bromoacil, 314-40-9), lenacil (2164-08-1), terfenadine (terbacil, 5902-51-2); (8) phenylpyridazines: pyridazinol (pyridafol, 40020-01-7), pyridate/pyridate (pyridate, 55512-33-9), QYP02410 (structural formula:
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: gramineae such as rice, wheat, corn, millet or barley.
The term "wild-type" refers to a nucleic acid molecule or protein that can be found in nature. In the present invention, the wild-type protein may be derived from any plant, in particular from the aforementioned monocotyledonous or dicotyledonous plants. Several sources of wild-type protein sequences and coding sequences have been disclosed in the prior art documents, which are incorporated herein by reference. Preferably, the wild-type protein of the invention is derived from the genus oryza, in particular rice. More preferably, the wild type PSIID1 or psbA D1 protein has the amino acid sequence shown in SEQ id.04, or an amino acid sequence having 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.04. The mutant PSII D1 or psbA D1 protein or biologically active fragment thereof is derived from a rice PSII D1 or psbA D1 protein and has one or more amino acid substitutions selected from the group consisting of those described above.
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.
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.04, and the second letter represents a different amino acid substituted for the natural amino acid. For example V219I shows that valine at position 219 is replaced by isoleucine with respect to the amino acid sequence of SEQ id.04. 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.04, relative to the amino acid sequence of its wild-type protein. For double or multiple mutations, each mutation is separated by a "/". For example, S264G/a251V indicates that the serine at position 264 is replaced by glycine and the alanine at position 251 is replaced by valine, both mutations being present within the particular mutant PSII D1 or psbA D1 protein, relative to the amino acid sequence of SEQ id.04.
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.04 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.04 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, etc., by computerized operations algorithms (GAP, BESTFIT, FASTA, and TFASTA, Genetics computer Group in the Wisconsin Genetics package). The National center for Biotechnology Information available from the United states of America can 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.
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 mutant PSII D1 or psbA D1 proteins of the invention may comprise, in addition to the above mutations, one or more further mutations in the amino acid sequence, such as conservative substitutions. In addition, mutant PSII D1 or psbA D1 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 another aspect, the present invention also relates to fragments lacking one or more amino acid residues from the N-and/or C-terminus of the mutant PSII D1 or psbA D1 protein, while retaining its desired functional activity, which are also within the scope of the present invention and are referred to as biologically active fragments. In the present invention, a "biologically active fragment" refers to a portion of the mutant PSII D1 or psbA D1 protein of the invention that retains the biological activity of the mutant PSII D1 or psbA D1 protein of the invention while increasing tolerance or resistance to a PSII class herbicide. For example, a biologically active fragment of a mutant PSII D1 or psbA D1 protein may be a portion of the protein lacking one or more (e.g., 1-50, 1-25, 1-10, or 1-5, such as 1, 2, 3, 4, or 5) amino acid residues at the N-and/or C-terminus of the protein, but which still retains the biological activity of the full-length protein.
The present invention also relates to methods of producing a mutant PSII D1 or psbA D1 protein or biologically active fragment or fusion protein thereof of the invention comprising: (a) culturing the host cell under conditions conducive to the production of the mutant PSII D1 or psbA D1 protein or biologically active fragment or fusion protein thereof; and (b) recovering the mutant PSII D1 or psbA D1 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 mutant PSII D1 or psbA D1 protein or biologically active fragment or fusion protein thereof of the invention can be lysed by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of a lytic agent. These methods are well known to those skilled in the art. The mutant PSII D1 or psbA D1 protein of the invention, or a biologically active fragment thereof, can be recovered and purified from a culture 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, phytohemagglutinin chromatography, and the like.
In a particular embodiment, the present invention 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 a PSII class herbicide, comprising transforming said host organism with a nucleic acid sequence encoding a mutant PSIID1 or psbA D1 protein of the present invention or a biologically active fragment thereof, an expression cassette 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 WO92/09696 for monocotyledonous plants, in particular rice transformation, etc. 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.
In another embodiment, the present invention provides a method of producing host organisms, particularly plant cells, plant tissues, plant parts or plants, which are tolerant or resistant to PSII herbicides, comprising integrating and expressing nucleic acids encoding the mutant PSII D1 or psbA D1 proteins or biologically active fragments thereof of the present invention into the genome of the host organism, suitable vectors and selectable markers are well known to those skilled in the art, for example, a method of integration into the tobacco genome is described in WO06/108830, the disclosure of which is incorporated herein by reference, the gene of interest is expressed in plant cells preferably from a constitutive or inducible promoter, mRNA, once expressed, is translated into a protein, thereby incorporating the amino acid of interest into the protein, the gene encoding the protein expressed in plant cells may be under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter, the promoters encoding the protein may be used, for example, promoters derived from bacteria, such as the octopine synthase promoter, nopaline synthase promoter, mannopine synthase promoter, promoters from viruses, such as cauliflower mosaic virus (cauliflower mosaic virus), and other promoters which may be used to regulate the activity of a plant cell-specific promoter, such as a constitutive promoter, e.g. a constitutive promoter, a promoter of a plant cell-specific promoter, a promoter which may be used to regulate the plant cell-specific promoter, such as a constitutive promoter, a promoter which may be used to regulate the plant cell-specific promoter, a plant cell-specific promoter, E promoter, e.g. a promoter, a promoter which may be used to regulate the plant cell-specific promoter, a promoter which may be used to regulate the plant cell-specific promoter, a constitutive promoter, a promoter which may be used in a promoter, a promoter which may be used in a promoter, a promoter which may be used in a promoter which may.
In a specific embodiment, the method of the invention for increasing PSII herbicide tolerance or resistance in a plant cell, plant tissue, plant part or plant comprises transforming the plant or part thereof with a nucleic acid molecule comprising a nucleic acid sequence encoding a mutant PSII D1 or psbA D1 protein or 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.
In another embodiment, the method of increasing PSII herbicide tolerance or resistance in a plant cell, plant tissue, plant part or plant of the invention comprises crossing a plant expressing a mutant PSII D1 or psbA D1 protein or biologically active fragment or fusion protein thereof of the invention with another plant and selecting a plant or part thereof having increased PSII herbicide tolerance or resistance.
In another embodiment, the method of increasing tolerance or resistance to a PSII class herbicide in a plant cell, plant tissue, plant part or plant of the invention comprises genetically editing the endogenous PSII D1 or psbA D1 protein of the plant cell, plant tissue, plant part or plant to achieve expression therein of a mutant PSII D1 or psbA D1 protein or a biologically active fragment or fusion protein thereof of the invention.
In a specific embodiment, the present invention provides a method of making a plant having increased herbicide tolerance or resistance by conventional breeding techniques comprising selfing or crossing a plant having integrated in its genome a nucleic acid sequence encoding a mutant PSII D1 or psbA D1 protein of the present invention, or a biologically active fragment thereof, and screening for progeny that comprise said encoding nucleic acid sequence heterozygous or homozygous for the progeny.
The invention further relates to plant cells, plant tissues, plant parts and plants, and progeny thereof, obtainable by the above process. 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.).
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.
The invention endows the rice with the PSII inhibition herbicide resistance through the mutation sites so as to solve the problem that severe weedy rice and other crops are phytotoxicity to the herbicide.
Detailed Description
The methods used in the following examples are those described in molecular biology, tissue culture techniques and agricultural manuals, unless otherwise specified.