阅读说明：本技术 一种岷江百合WRKY转录因子基因LrWRKY3及应用 (Lilium regale WRKY transcription factor gene LrWRKY3 and application thereof ) 是由 刘迪秋 普丽梅 王自娥 郑锂蕾 陈虹均 李珊 葛锋 于 2019-11-13 设计创作，主要内容包括：本发明公开了一种岷江百合WRKY转录因子基因<I>LrWRKY3</I>,其核苷酸序列如SEQ ID NO：1所述,编码如SEQ ID NO：2所示氨基酸序列的蛋白质,本发明通过功能基因组学相关技术研究证实<I>LrWRKY3</I>基因具有提高植物抗真菌的功能,将本发明抗真菌<I>LrWRKY3</I>基因构建到植物表达载体上并转入烟草中过量表达,转基因烟草植株具有很强的抗真菌侵染能力,实验结果显示超表达<I>LrWRKY3</I>的转基因烟草叶片对稻黑孢霉、葡萄座腔菌、禾谷镰刀菌、轮枝镰刀菌、茄腐镰刀菌等五种病原真菌的侵染有很强的抗性。(The invention discloses a Lilium regale WRKY transcription factor gene LrWRKY3 The nucleotide sequence is shown as SEQ ID NO:1, encoding the polypeptide shown as SEQ ID NO:2, the invention is proved by related technical research of functional genomics LrWRKY3 The gene has the function of improving the plant antifungal property, and the invention is used for resisting the fungi LrWRKY3 The gene is constructed on a plant expression vector and is transferred into tobacco for over-expression, a transgenic tobacco plant has strong capability of resisting fungal infection, and the experimental result shows that the over-expression is realized LrWRKY3 The transgenic tobacco leaf is used for treating Nilaparhizoma oryzae and grapeThe five pathogenic fungi such as lochiobacter, fusarium graminearum, fusarium verticillium, fusarium solani and the like have strong resistance to infection.)

1. Lilium regale WRKY transcription factor geneLrWRKY3The method is characterized in that: the nucleotide sequence is shown as SEQ ID NO. 1, and the protein of the amino acid sequence shown as SEQ ID NO. 2 is coded.

2. Lilium regale WRKY transcription factor gene as claimed in claim 1LrWRKY3In improving tobacco to Nicotiana oryzae (Nigrospora oryzae) Staphylococus viticola (A. vinifera)Botryosphaeria dothidea) Fusarium graminearum (F.graminearum)Fusarium graminearum) Fusarium verticillatum (A)Fusarium verticillioides) Fusarium solani (F.solani) (II)Fusarium solani) Use in resistance.

Technical Field

The present invention relates to molecular biology and genesEngineering related technical research field, in particular to Lilium regale WRKY transcription factor gene with antifungal infection capacityLrWRKY3And application thereof.

Background

Plant diseases are a very troublesome problem in agricultural production, especially fungal diseases, accounting for about 80% of the total plant diseases, and seriously affecting the yield and quality of crops. Plants have an innate immune system to avoid pathogenic invasion, including a complex network that tightly regulates defense responses, transcription factors play an important role as signal pathway regulatory genes in plant disease resistance responses, particularly the WRKY family of transcription factors, involved in regulation of biological and abiotic defense (Agarwal P, Patel K, Agarwal PK. echopic expression ofJcWRKYconfers enhanced resistancein transgenic tobacco againstMacrophomina phaseolina. DNA Cell Biol, 2018,37(4): 298-307.)。

The WRKY transcription factor is one of the largest transcription factors in plants, and has a WRKY structural domain at the N-terminal of the protein. The WRKY domain has a conserved WRKYGQK amino acid sequence, usually associated with a cis-acting element known as the W box (W box, C/TTGACT/C). The core structure of the cis-acting element of the WRKY protein and the promoter region of the target gene is the W-box specific binding of TTGAC (C/T), i.e. all the genes containing W-box in the promoter can be the target gene of WRKY, including WRKY itself (Dong J, Chen C, Chen ZArabidopsisWRKY genetic engineering Plant destination response. Plant Mol Biol, 2003, 51(1): 21-37.). WRKY domain is composed of four folds of β, with conserved WRKYGQK residues corresponding to the N-terminal β fold (strand β -1) capable of entering the major groove of the DNA duplex and interacting with the W-box on the DNA (Ciolkowski I, Wanke D, Birkenbihl RP, et al. study DNA-binding selection of transcription factors and WR structures bound. Plant Mol, 2008, 68(1-2): 81-92.).

WRKY transcription factors are reprogrammed to cope with the invasion of different pathogens by regulating the plant transcriptome. Positively or negatively regulating the expression of downstream target genes by participating in different regulatory pathways, including participationHormone signaling pathways, interaction with other members of the WRKY transcription factor family, and the like. Grape WRKY transcription factor geneVqWRKY52Ectopic expression in Arabidopsis enhances the control of powdery mildew and Pseudomonas syringae ((R))Pseudomonas syringaepv.tomatoDC 3000) (Wang X, Guo R, Tu M, et al, Ectopic expression of the wire vane WRKY transformation factor VqWRKY52 inArabidopsis thalianaenhances resistance tothe biotrophic pathogen powdery mildew But Not to the necrotrophic pathogenBotrytis cinereaFront Plant Sci, 2017, 8: 97.). Withania somnifera (Withania somnifera）WsWRKY1Overexpression in tobacco improves tolerance to biotic stress (Singh AK, Kumar SR, Dwivedi V, et al. A WRKY transcription factor from)Withania somniferaregulated depend-ment with lipid accumulation and biological stress tolerance of phytosterol and defect pathways, New Phytol, 2017, 215(3):1115 and 1131.). In addition, chickpeasCaWRKY40Up-regulating the expression of defense-related genes to enhance resistance to Fusarium oxysporum (F.) (Fusarium oxysporum) Resistance (Chakraborty J, Ghosh P, Sen S, et al, CamPK9 inputs the stability of CaWRKY40 transformation factor which trigger sensitivity in chickpea uponFusariumoxysporumf. sp.ciceriRace1infection. Plant Mol Biol, 2019, 100(4-5): 411-431.）。

Flax (2)Linum usitatissimum) The transcription factor LuWRKY36 plays a key regulation role in the defense reaction of resisting fusarium oxysporum through the mediation of hormone and calcium signal pathways, and LuWRKY36 is used for up-regulation and control under the condition of biological stressLuPLR1Biosynthesis of genes and Lignin (Markulin L, Corbin C, Renouard S, et al, Characterisation of LuWRKY36, a Flax transfer factor promoting growth of a lignin in a lignin to a lignin resinFusarium oxysporumelicitorsinLinum usitatissimumL, hairpin roots, Planta, 2019, 250(1): 347-. Strawberry (A)Fragaria vesca) WRKY transcription factor FvWRKY42 interacting with various stress-related proteins and over-expressing in Arabidopsis thalianaFvWRKY42Resulting in cell death, sporulation and slow hyphal growthSlow to increase resistance to powdery mildew and up-regulate in Arabidopsis thalianaPR1Expression of the Gene (Wei W, Cui MY, Hu Y, et al, Ectopic expression ofFvWRKY42, a WRKY transcription factor from the diploid woodland strawberry(Fragaria vesca), enhances resistance to powdery mildew, improves osmoticstress resistance, and increases abscisic acid sensitivity inArabidopsisPlant Sci, 2018, 275: 60-74.). Arabidopsis thaliana (Arabidopsis) Transcription factor AtWRKY50 andPR1co-expression of the two promoter elements TGA2 or TGA5 enhances the Salicylic Acid (SA) -inducible marker genePR1Expression level of (HussainRMF, Sheikh AH, Haider I, et al. Arabidopsis WRKY50 and TGA TranscriptionnActivities synergy Expression ofPR1Front Plant Sci, 2018, 9: 930). In addition, WRKY transcription factors including pepper CaWRKY22, CaWRKY6, CaWRKY27, CaWRKY40 and CaWRKY58 are integrated to form a sub-regulation network, and the signal pathway mediated by salicylic acid, jasmonic acid and ethylene is used for positively regulating ralstonia solanacearum (A), (B) and (C) in a synergetic wayRalstonia Solanacearum) Resistance of (Hussain A, Li X, Weng Y, et al. CaWRKY22 Acts as a Positive Regulator in Pepper Response toRalstonia Solanacearumby conforming Networks with CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58. Int J Mol Sci, 2018, 19(5): E1426.). WhileCaWRKY40bThe immune related gene is regulated to play a negative regulation role in resisting ralstonia solanacearum of the hot pepper (Ifnan Khan M, Zhang Y, Liu Z, et al).CaWRKY40bin pepper acts as a negative regulator in response toRalstonia solanacearumby directly modulating defense genes includingCaWRKY40.Int JMol Sci, 2018, 19(5): E1403.)。

The Bulbus Lilii is of Liliaceae (Liliaceae) genus Lilium (Liliaceae)Lilium) The plant is a general term of perennial root flower. During the processes of seed ball propagation and fresh cut flower production, lily is vulnerable to various pathogenic bacteria such as fungi, viruses and bacteria. The lily diseases found at present are more than dozens, among which, the lily is from the genus Fusarium (A), (B), (CFusarium) Blight (also called basal rot and stem rot) caused by fungi is the most serious disease in lily production. FusariumAfter the lily seed balls are infected, the basal disc is necrotic, the scales are rotten and fall off, and the quality of the seed balls is reduced; after the plants are infected by fusarium, the leaves turn yellow and droop wilting, and the plants die in advance, so that the yield and the quality of the cut lily flowers are seriously influenced. The pathogen of the lily wilt is mainly fusarium oxysporum, China is an important origin of lily and has rich lily germplasm resources, such as wild grown Lilium regale (R) ((R))L. regale) High resistance to Fusarium oxysporum. The Lilium regale is a unique species in China, is only distributed in rock cracks from valleys with the altitude of 800-2700 m in the Lilium regale watershed to the waist of a mountain, has extremely strong antifungal and antiviral properties, and is an important germplasm resource for modern lily breeding. Due to the influence of factors such as frequent geological disasters, artificial mining, wasteland exploitation, tree planting and afforestation, the distribution area and the number of the Lilium regale wild resources are obviously reduced, the Lilium regale wild resources are listed as endangered species by the world natural protection alliance, and the protection of the rare Lilium regale wild resources is urgent. Therefore, the full development and utilization of the disease-resistant gene of the Lilium regale is imperative. The WRKY transcription factor is a key regulating factor for plant disease-resistant defense reaction, and the WRKY transcription factor gene related to disease resistance is discovered from the Lilium regale, so that the disease-resistant regulation mechanism of the Lilium regale can be deeply known, and a candidate disease-resistant gene can be provided for disease-resistant genetic engineering.

Disclosure of Invention

The invention aims to provide a Lilium regale WRKY transcription factor geneLrWRKY3And application thereof, namely improving the effect of tobacco on the black sporotrichum oryzae (B)Nigrospora oryzae) Staphylococus viticola (A. vinifera)Botryosphaeria dothidea) Fusarium graminearum (F.graminearum)F. graminearum) Fusarium verticillatum (A)F. verticillioides) Fusarium solani (F.solani) (II)F. solani) Use in resistance.

The invention relates to a full-length gene of a WRKY transcription factor with antifungal activity, which is cloned from Lilium regaleLrWRKY3The nucleotide sequence is shown as SEQ ID NO. 1, the gene cDNA full length sequence is 722bp, comprises an open reading frame of 537 bp, a5 'untranslated region of 19 bp and a 3' untranslated region of 166 bp, and encodes the protein of the amino acid sequence shown as SEQ ID NO. 2.

In the inventionLrWRKY3The coding region of the gene is a nucleotide sequence shown in 20 th to 556 th positions in a sequence table SEQ ID NO. 1.

The invention separates and clones a complete cDNA segment of an antifungal related gene of Lilium regale, and utilizes agrobacterium tumefaciens (A), (B) and (C)Agrobacterium tumefaciens) The target gene is transferred into a receptor plant and is overexpressed, whether the gene has antifungal activity is verified through further experiments, a foundation is laid for the capability of improving tobacco and other plants to resist fungal diseases by utilizing the gene in the later period, and the inventor names the gene asLrWRKY3。

As described aboveLrWRKY3The gene can be applied to improving the antifungal property of tobacco, and the specific operation is as follows:

(1) using amplificationLrWRKY3The specific primer is used for extracting total RNA from the root of Lilium regale after inoculating fusarium oxysporum and amplifying the total RNA by reverse transcription-polymerase chain reaction (RT-PCR)LrWRKY3Then connecting the full-length coding region to a pGEM-T vector, and obtaining a clone with a target gene through sequencing;

(2) using restriction endonucleasesXbaI andEcoRi enzyme digestion pGEM-T-LrWRKY3Recovering the carrier by glue to obtain target gene segment, using the same endonuclease to enzyme-cut plant expression carrier pCAMBIA2300s, recovering the glue to obtain the required carrier large segment, and recovering the obtained carrier large segmentLrWRKY3Connecting the gene fragment with the pCAMBIA2300s fragment to construct a plant overexpression vector, and then transferring the constructed recombinant vector into tobacco to express through the mediation of agrobacterium tumefaciens;

(3) screening transformants by using the resistance marker on the recombinant vector T-DNA, obtaining a real transgenic plant through PCR and RT-PCR detection, analyzing the resistance level of the transgenic plant to different pathogenic fungi, and finally screening the transgenic plant with obviously enhanced fungal resistance.

The invention provides a new method for improving the resistance of plants to fungal diseases, overcomes the defects of the traditional breeding by cultivating disease-resistant plants by a genetic engineering means, shortens the breeding period and operatesSimple and easily obtained high-resistance material. The invention is derived from Lilium regaleLrWRKY3The gene can enhance the resistance of plants to fungi, and can be introduced into tobacco to produce new varieties and new materials with fungal resistance. The cultivation of resistant plant varieties and materials by using genetic engineering technology has obvious advantages and irreplaceable importance. The invention not only can provide convenience for large-scale production of crops, medicinal materials, flowers, nursery stocks and the like, greatly reduces the use of chemical pesticides, but also can save the cost for agricultural production and reduce the environmental pollution, thereby having wide market application prospect.

Drawings

FIG. 1 is a drawing of the present inventionLrWRKY3PCR detection result graph of transgenic tobacco genome DNA, in the graph: the Marker is DL2000 DNA Marker (Dalibao biology); the positive control is plasmid pGEM-T-LrWRKY3PCR products as templates; WT is the product of PCR using total DNA of non-transgenic tobacco (wild type) as template;

FIG. 2 shows the positivity of the present inventionLrWRKY3In transgenic tobaccoLrWRKY3A graph of the results of expression analysis at the transcriptional level; in the figure: marker is DL2000 DNA Marker (Dalibao biology); WT is a PCR product with non-transgenic tobacco total RNA reverse transcription cDNA as a template; the positive control was plasmid pGEM-T-LrWRKY3A PCR product as a template;

FIG. 3 is a drawing of the present inventionLrWRKY3A result graph of the disease resistance identification of the transgenic tobacco; in the figure, a, b, c, d and e are tobacco leaves inoculated with fusarium graminearum, fusarium verticillium, botrytis cinerea and fusarium solani respectively; WT is leaf of wild type tobacco, 19, 20, 27, 34 areLrWRKY3Leaves of transgenic tobacco.

Detailed Description

The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited to the above description, and the examples are conventional methods unless otherwise specified, and reagents used are conventional commercially available reagents or reagents formulated according to conventional methods unless otherwise specified.