CN105567703A

CN105567703A - LRK1 gene endowing plants with greensickness resistance and application of LRK1 gene

Info

Publication number: CN105567703A
Application number: CN201610111963.8A
Authority: CN
Inventors: 刘廷利; 顾周航; 周雪平; 张保龙; 杨郁文; 陈天子; 凌溪铁; 王金彦
Original assignee: Zhejiang University of Technology ZJUT; Jiangsu Yanjiang Agricultural Science Research Institute
Current assignee: Zhejiang University of Technology ZJUT; Jiangsu Yanjiang Agricultural Science Research Institute
Priority date: 2016-02-29
Filing date: 2016-02-29
Publication date: 2016-05-11

Abstract

The invention relates to LRK1 gene and its application for endowing plant with Verticillium wilt resistance, and belongs to the field of biotechnology. The LRK1 gene is a surface receptor protein gene obtained from the upland cotton variety Ao 3503. The complete coding frame length of LRK1 is 1860 bases, and the encoded protein contains 619 An amino acid with a molecular weight of 67.3KD and an isoelectric point of 5.4. The disease resistance of LRK1 transgenic Arabidopsis to deciduous Verticillium dahliae V991 and non-deciduous Verticillium dahliae BP2 was significantly enhanced. The incidence rate is more than 50%.

Description

Give LRK1 gene and the application thereof of plant verticillium wilt resistance

One, technical field

The invention provides LRK1 gene and application thereof that one is given plant verticillium wilt resistance, relate to Cloning Plant Genes and functional analysis, belong to plant genetic engineering field.For being improved disease resistance of plant and other useful production traitss by plant gene engineering technology.

Two, background technology

At present, in production, verticillium harm is very serious, is the Major Fungus Diseases of the plants such as cotton, tomato, capsicum, eggplant, causes the underproduction even to have no harvest, there is no effectively preventing measure.The cause of disease of verticillium is mainly verticillium dahliae (Verticilliumdahliae), with Microsclerotia (microsclerotia) form long-term existence in soil.Verticillium wilt pathogen can show Difference in Pathogenicity on host plant, interacting with host, under the impact of coevolution and ecotope difference, often produces Physiological Differentiation, occurs new Pathogenic Types.Cotton in China verticillium bacterial strain is divided into 8 classes by the RAPD finger printing cluster analysis such as Zhang Tianzhen, but may contain weak pathogenic bacterium by force in same class simultaneously, and verticillium wilt pathogen mechanism of causing a disease very complicated (Zhang Tianzhen, 2000) is described.In addition, the verticillium wilt pathogen heritable variation caused by transposon and DNA level transfer etc. also limit verticillium wilt resistance of cotton by same breeding (AmyotteSG, etal., 2012; DeJonge, etal., 2012).Therefore, clone resistant gene, to resolve disease resistance mechanisms significant and urgent to verticillium wilt resistance of cotton by same breeding.

But, to verticillium wilt pathogen, there is the gene of resistance at present less, especially report is had no to the gene that cotton verticillium wilt has a stable wide spectrum high resistance.Fradin etc. (2011) are by Ve1 arabidopsis thaliana transformation in tomato, obtain the resistance to verticillium wilt pathogen, and after this gene transformation tobacco and cotton, not there is resistance to verticillium wilt, show that the application of this gene on plant has species specificity (Liu Linlin etc., cotton and the anti-cotton verticillium wilt of tomato do not rely on Ve1, Chinese science: life science, 2014 (44) volume the 8th phase: 803-814).Clone from resisting verticillium sea island cotton kind H7124 and tomato Ve1 gene (Fradinetal., 2009) and cotton GbVe gene (Zhangetal., 2011) homeologous Gbve1 gene, this gene also has typical plant disease-resistant protein structure domain, utilizes the reticent Gbve1 gene of VIGS that sea island cotton H7124 can be made the resistant lose of verticillium; Further, the result of Gbve1 gene transformation Arabidopis thaliana and upland cotton all shows that the defoliation of Gbve1 gene pairs High pathogenicity and non-defoliation verticillium wilt pathogen all have resistance (Zhangetal., 2012a), but does not obtain high resisting verticillium kind.Except Ve gene, some downstream defense response genes are also for resisting verticillium study on prevention.The transgene cotton of overexpression AtNPR1 gene has resistance to non-defoliation verticillium, but does not have resistance (Parkhietal.2010) to defoliation verticillium.In addition, rice Xanthomonas harpin albumen, chitinase gene, lipid transferase gene, GastrodiaAntifungal Protein, antibacterial peptide, alexin (θ-Defensins) (Miaoetal., 2010; Munis, 2010; NiMetal., 2013) also certain resistance is shown to verticillium, but the overexpression on plant due to Analysis of Defence Genes Involved grows, band serves disadvantageous effect and resistance effect does not reach re-set target etc., thus limits the application of these genes on producing.

This research is analyzed by the transcript profile of upland cotton height resisting verticillium kind Austria 3503, find a high resisting verticillium gene LRK1, this gene and Lei Mengdeshi cotton chitin receptoroid kinase gene 1 (Chitinelicitorreceptorkinase1-like, accession number XM_012617234) similarity 99%, with the sequence of this gene for template design primer is by LRK1 gene clone, and by this gene transformation Arabidopis thaliana, the transgenic Arabidopsis plants of acquisition all has higher resistance to defoliation and non-Strain of Defoliating Type.The separation of resistance gene of Verticillium wilt in cotton LRK1 can enrich resistant gene resource further, lays the foundation to the further utilization that its functional study is this genoid.

Three, summary of the invention

Technical problem

The object of the invention is: provide one and give plant verticillium wilt resistance LRK1 gene and application thereof, this genes encoding surface receptor protein, significantly increases by expression amount after verticillium pathogenic bacterium inducing.This genetic expression can significantly improve the resistance of recipient plant to fallen leaves verticillium pathogenic bacteria V991 and non-defoliation verticillium wilt pathogen BP2.The various plant expression vector of the gene constructed one-tenth of the present invention can be utilized, be applied to Agricultural biotechnologies breeding to improve crop disease-resistant proterties.

Technical scheme

The present invention relates to the LRK1 gene that is given plant verticillium wilt resistance, its sequence is SEQIDNO.1.This gene source is in Upland Cotton Austria 3503.The complete encoder block length of LRK1 gene is 1860 bases, and proteins encoded is containing 619 amino acid, and molecular weight is 67.3KD, and iso-electric point is 5.4.The functional study of LRK1 discloses its expression regulation mechanism and mechanism of action, can be applicable in the genetically engineered improvement of genetic engineering of plant for disease resistance and resistance.

Present invention also offers the primer of arbitrary fragment of expression vector containing gene of the present invention and Host Strains and this gene that increases.

Described LRK1 gene can give the application in plant verticillium wilt resistance.Especially the application in arabidopsis thaliana resistance to verticillium wilt is being given.

The present invention gives the LRK1 gene of plant verticillium wilt resistance, this gene increases by expression amount after the induction of cotton verticillium wilt High pathogenicity bacterial strain V991, LRK1 is building up to the upper arabidopsis thaliana transformation of plant expression vector pCambia2301 carrier (Bei Nuo bio tech ltd, Shanghai), result transfer-gen plant all has more resistance to cotton verticillium wilt High pathogenicity defoliation bacterial strain V991 and non-defoliation strain BP 2.The various plant expression vector of gene constructed one-tenth of the present invention can be utilized, be applied to Agricultural biotechnologies breeding with improve relevant host plant to the disease resistance of verticillium or be used in cotton verticillium wilt resistance improvement in.

Its application comprises:

1) clone of LRK1 gene and the structure of plant expression vector

With the cDNA of Upland Cotton Austria 3503 for template, use primer

P1:5’-TGCTCTAGAATGCTGAAATTAATCTCGATTC-3’，

P2:5’-CGCGGATCCTTATCTTCCGGACATAAGGT-3’

Amplification obtains 1860bp fragment, by this fragment called after LRK1, cuts, be connected to pCambia2301 carrier after PCR primer purifying with XbaI/BamHI enzyme, the plasmid called after pCambia2301:LRK1 that after order-checking, success builds;

2) acquisition of transfer-gen plant

By step 1) in the pCambia2301:LRK1 plasmid freeze-thaw method transformation Agrobacterium LBA4404 that builds, with dipping in colored method arabidopsis thaliana transformation, the Arabidopsis plant in the present age is T0 generation, and the seed of results is T1 generation;

T1 containing that antibiotic 1/2MS substratum of 25mg/mL card is screening, obtains the positive T1 plant of candidate for seed, and on DNA and rna level, whether testing goal gene proceeds to and whether successful expression respectively;

Use primer

P3:5’-CTGGGAATGTTTTTGGAAG-3’

P4:5’-ACTCGGTCGTAGCTGAGGA-3’

The T1 plant of amplification acquisition 920bp size fragment is then the positive transgenic plant of successful conversion LRK1, and the seed of results is T2 generation.T2 is growing containing on that antibiotic 1/2MS substratum of 25mg/mL card for positive transgenic plant, and has resistance to verticillium wilt.

Beneficial effect

This research is analyzed by the transcript profile after the yellow bacterium that withers of upland cotton height resisting verticillium kind 3503 inoculation difficult to understand, find that a gene to wither bacterium abduction delivering by Huang, this gene and Lei Mengdeshi cotton chitin receptoroid kinase gene 1 (Chitinelicitorreceptorkinase1-like, accession number XM_012617234) similarity 99%, with the sequence of this gene for template design primer is by this gene clone, called after LRK1, and by this gene transformation Arabidopis thaliana, the transgenic Arabidopsis plants of acquisition all has higher resistance to defoliation and non-Strain of Defoliating Type.The separation of resistance gene of Verticillium wilt in cotton LRK1 can enrich resistant gene resource further, lays the foundation to the further utilization that its functional study is this genoid.There is following advantage and effect:

1. present invention obtains the LRK1 gene of a brand-new imparting plant verticillium wilt resistance.The LRK1 gene source that the present invention obtains is in Upland Cotton Austria 3503, and this gene obviously rises by expression amount after cotton verticillium wilt pathogenic bacterium inducing.By LRK1 and structure plant expression vector arabidopsis thaliana transformation, this gene of result all has stronger resistance to defoliation High pathogenicity verticillium wilt pathogen V991 and non-defoliation High pathogenicity verticillium wilt pathogen BP2 after expressing in transfer-gen plant, sickness rate is respectively 9.9%, 8.2%, and contrast wild-type sickness rate and be respectively 61.2%, 56.3%.The separation of LRK1 can enrich resistant gene resource further, and its functional study is that the further utilization of this genoid lays the foundation.

2. the present invention contributes to the mechanism of action understanding disease-resistant gene better.The clone of LRK1 is that understanding pathogenic bacteria and disease-resistant gene are done mutually further, and defense signaling path lays the foundation.Can utilize this gene transgenic plant be further analyzed LRK1 be how to transmit resistance signal and with which intracellular signaling process, thus obtain resistance signal transduction pathway.The separation of LRK1 and Function Identification are that the mechanism of action of Effect of Anti ospc gene lays the foundation.

3. the present invention is applied to resistance to verticillium wilt breeding.LRK1 resistance is respond well, to defoliation verticillium fungus strain V991 and the non-defoliation verticillium fungus strain BP2 of test, sickness rate all lower than 10%, significantly lower than the contrast wild-type sickness rate that is greater than 50%, significantly improve the resistance to verticillium, in breeding, have larger using value.

Four, accompanying drawing explanation

The expression of Fig. 1 defoliation verticillium fungus strain V991 and the rear LRK1 of non-Strain of Defoliating Type BP2 process Upland Cotton difficult to understand 3503.

The DNA level Molecular Detection of Fig. 2 LRK1 transfer-gen plant.M,Marker。WT is unconverted plant; LRK1 is the resistant transformants that kantlex filters out.

The rna level of Fig. 3 LRK1 transfer-gen plant detects.WT is unconverted plant; LRK2 is the resistant transformants that kantlex filters out.Upper row take cDNA as template, LRK1 primer amplification, and lower row is Actin primer amplification.

Fig. 4 defoliation verticillium fungus strain V991 and non-Strain of Defoliating Type BP2 inoculates the sickness rate of LRK1 transformed plant and contrast strain.WT is unconverted plant, and LRK1 is transformant.

Fig. 5 defoliation verticillium fungus strain V991 and non-Strain of Defoliating Type BP2 inoculates the phenotype of LRK1 transformed plant and contrast strain.WT is unconverted plant, and LRK1 is transformant.

Fig. 6 defoliation verticillium fungus strain V991 and non-Strain of Defoliating Type BP2 inoculates LRK1 transformed plant and the relative hyphae content contrasting strain.WT is unconverted plant, and LRK1 is transformant.

Five, embodiment

Method used in following embodiment is ordinary method if no special instructions, and primer sequence used is by the synthesis of the handsome Bioisystech Co., Ltd in Shanghai.In this experiment, gene source is in Upland Cotton Austria 3503 (Gossypiumhirsutum) (Liu little Shuan, Liu Tingli, Yuan Hongbo, Zhang Baolong, Wang Rongfu. the drought-enduring functional analysis of cotton natriuretic peptide gene GhPNP1. Scientia Agricultura Sinica, 2015,48 (12): 2306-2316).

(1) cotton LRK1 clones and sequential analysis

Primer is designed according to Lei Mengdeshi cotton (Gossypiumraimondii) chitin receptoroid kinase gene 1 (Chitinelicitorreceptorkinase1-like, accession number XM_012617234)

P1:5’-TGCTCTAGAATGCTGAAATTAATCTCGATTC-3’，

P2:5’-CGCGGATCCTTATCTTCCGGACATAAGGT-3’

With the cDNA template amplification of Upland Cotton Austria 3503, in 25 μ l reaction systems, add cDNA template 1 μ l, each 5nmol of primer, 5 μ l5 × primeSTARbuffer (Mg ²⁺plus) PCR damping fluid, 0.2mMdNTP, 1UprimeSTARHSDNAPolymerase (TaKaRa company) carry out pcr amplification.Pcr amplification condition is: after 94 DEG C of 3' 94 DEG C 45 ", 56 DEG C 45 ", 72 DEG C of 3', circulate 36 times, then 72 DEG C extend 10'.PCR primer detects on the sepharose of 1%, and obtaining fragment is 1860bp, and by this fragment called after LRK1 gene, its sequence is SEQIDNO.1.

This fragment is carried out end add A process (sky, Beijing bounties Gene Tech. Company Limited) and be connected with pGEM-Teasy carrier (Promega company).Connecting product JM109 (Beijing Quanshijin Biotechnology Co., Ltd) competent cell carries out heat-shock transformed.Order-checking is completed by the handsome bio-engineering corporation in Shanghai, the plasmid called after pGEM-Teasy:LRK1 that success builds.Carry out the determination of gene open reading frame with DNAclub, carry out sequence comparing analysis with DNAMAN, utilize internet database (http://www.ncbi.nlm.nih.gov/) to carry out BLAST analysis simultaneously.Database ExPASy (http://cn.expasy.org/) carries out the correlation analysis of albumen iso-electric point and molecular weight.This gene coded protein is containing 619 amino acid, and molecular weight is 67.3KD, and iso-electric point is 5.40.

(2) the abduction delivering analysis of cotton LRK1

Cotton used is 3 week seedling age, Bacteria culturing condition is: bacterial strain is defoliation High pathogenicity pathogenic bacteria V991 (Xu Rongqi, Wang Jiani, Chen Jieyin etc. verticillium dahliae T-DNA insertion mutation body surface type characteristic sum flanking sequence is analyzed. Scientia Agricultura Sinica, 2010, 43 (3): 489-496) and non-defoliation strain BP 2 (BaolongZhang, YuwenYang, TianziChenetal.IslandCottonGbve1GeneEncodingAReceptor-Li keProteinConfersResistancetoBothDefoliatingandNon-Defoli atingIsolatesofVerticilliumdahliae, PloSONE, 7 (12): e51091).Pathogenic bacteria is after the activation of PDA flat board, PDB nutrient solution (potato 200g is put into from colony edge picking bacterium block, boiling water boiling 40 minutes, double gauze filters, and adds 20g glucose, be settled to 1L, 121 DEG C of autoclave sterilization 20min), 25 DEG C, 120rmin cultivates 5-6d, use filtered through gauze nutrient solution, microscopy also counts with blood counting chamber.Inoculation method is for dipping in root method (FradinF., Abd-El-HaliemA., MasiniL.etal, InterfamilyTransferofTomatoVe1MediatesVerticilliumResist anceinArabidopsis, PlantPhysiology, 2011,156:2255 – 226), conidium concentration is adjusted to 1 × 10 ⁷/ mL, cleans soak 5min in conidium after by cotton root system, plant in burying.Gather respectively and do not induce and induce rear cotton root system, extract total serum IgE and synthesize cDNA, and carry out follow-up RT-PCR analysis (RNA extraction method and cDNA synthetic method are shown in annex), primer sequence is:

P5:5’-TTGTCAAGGCAAATGGTTCTCTC-3’

P6:5’-CGTCTAGTGGGTAATTATCTCCGA-3’。

After expression analysis result display V991 and BP2 process 2d, in root system, LRK1 gene expression amount obviously increases, up-regulated expression, reaches the highest to 8 days, and 12d expression amount reduces (Fig. 3).

(4) structure of LRK1 gene plant expression vector and Plant Transformation

With the above-mentioned pGEM-Teasy:LRK1 plasmid built for template, cut with XbaI/BamHI enzyme, and reclaim 1860bp fragment, be connected with pCambia2301 carrier, mate through sequence verification 100%, the positive plasmid called after pCambia2301:LRK1 of acquisition.After extracting plasmid, with freeze-thaw method by transfer vector plasmid transformation Agrobacterium LBA4404 (Bei Nuo bio tech ltd, Shanghai).With dipping in colored method arabidopsis thaliana transformation, T1 is obtained for plant in that resistance of 1/2MS card (25 μ g/mL) screening, whether PCR and RT-PCR testing goal gene on DNA and rna level proceeds to and whether successful expression respectively, that all can increase acquisition fragment at DNA and rna level is then considered as positive transformants, and primer is

P3:5’-CTGGGAATGTTTTTGGAAG-3’

P4:5’-ACTCGGTCGTAGCTGAGGA-3’

Expand DNA and expand PCR qualification 920bp size fragment (Fig. 5).By PCR identify can the T1 of amplified fragments for plant for candidate, recycling RT-PCR detects, and can amplify the positive strain (Fig. 6) then for expressing of 920bp fragment.Results T1 is T2 generation for transfer-gen plant seed.

In transfer-gen plant seed T2 generation, is seeded in 1/2MS substratum containing 25mg/L kantlex, selects green plant and transplant growth to Nutrition Soil and for Disease Resistance Identification.

(5) T2 is for the Disease Resistance Identification of transformant

The transformation plant of normal expression Disease Resistance Identification can be carried out to LRK1 gene.Green seedling by the 1/2MS Screening of Media containing kantlex is proceeded in Nutrition Soil, every pot transplanting 1 strain seedling.Disease Resistance Identification is carried out after Arabidopis thaliana grows 2 weeks.Bacterial strain uses therefor is respectively Verticillium Dahliae defoliation High pathogenicity bacterial strain V991 and non-defoliation strain BP 2.Pathogenic bacteria is after the activation of PDA flat board, and put into PDB nutrient solution, 25 DEG C from colony edge picking bacterium block, 120rmin cultivates 5-6d, uses filtered through gauze nutrient solution, and microscopy also counts with blood counting chamber, and adjustment spore concentration is 1 × 10 ⁷individual/mL, qualification strain number 40 strain of often kind of pathogenic bacteria, inoculation observes disease afterwards every day, and a situation arises, and just obviously can see disease symptom after 15 days, main manifestations is yellowing leaf, wilts, delayed growth.Disease Resistance Identification result shows, and for defoliation verticillium V991, contrast wild-type disease refers to reach 61.2%, and the average disease of transgenic line refers to be only 9.9%; For non-defoliation verticillium BP2, contrast wild-type disease refers to reach 56.3%, and the average disease of transgenic line refers to be only 8.2% (Fig. 4, Fig. 5).Further extraction morbidity Arabidopsis plant DNA, and by PCR, the mycelia of the Arabidopis thaliana of morbidity is carried out quantitatively.Data presentation quantitative result is consistent with phenotype, and the hyphae content in the transfer-gen plant of inoculation V991 is only wildtype Arabidopsis thaliana 2%, and inoculates 8% (Fig. 6) that hyphae content in the transfer-gen plant of BP2 is only wild-type content.Show that LRK1 significantly can strengthen the resistance of Arabidopis thaliana to Strain of Defoliating Type V991 and non-Strain of Defoliating Type BP2.

Annex:

1.RNA extracting method

(1) draw materials and add liquid nitrogen and be fully ground into powder and be transported in centrifuge tube;

(2) add the RNA Extraction buffer of 10 times of volumes, concussion mixing, 50 DEG C of water-baths are about 20min, and midway can mix 2-3 time;

(3) add the chloroform of 0.6 times of volume, mixing, leave standstill ice bath 20min;

(4) the centrifugal 20min of 12000rpm, is transported in a new centrifuge tube by supernatant;

(5) add the 8MLiCl solution of 1/2 volume, ice bath spends the night (more than 12h);

(6) the centrifugal 20min of 12000rpm, abandons supernatant, washes precipitation 1 time and be transported in a new centrifuge tube by precipitation with 70% alcohol;

(7) the centrifugal 20min of 12000rpm, abandons ethanolic soln, and precipitation drains 20min;

(8) add 200ul to precipitate without the water dissolution of RNase, the water-saturated phenol/chloroform=1:1 adding 1 times of volume fully mixes, and leaves standstill 5min;

(9) the centrifugal 20min of 12000rpm, is transported to supernatant in another new centrifuge tube, then the water-saturated phenol/chloroform=1:1 adding 1 times of volume repeats extracting once;

(10) the centrifugal 20min of 12000rpm, above resets and add the chloroform of 1 times of volume once;

(11) the centrifugal 20min of 12000rpm, above reset and add 1/2 volume 8MLiCl solution, ice bath spends the night (more than 12h);

(12) the centrifugal 20min of 12000rpm, precipitation is washed once with 70% alcohol.100-200ul is dissolved in without in the water of RNase after draining.Get 2ul Detection job.

The synthesis of 2.cDNA

By for subsequent use after the RNA DNaseI37 DEG C purification process 30min of extraction.The first chain cDNA is synthesized with TransScriptReverseTranscriptase test kit (Transgen company).System is as follows:

42℃45min，85℃5min。In-20 DEG C of preservations.

LRK1 gene, its sequence is SEQIDNO.1:

ATGCTGAAATTAATCTCGATTCTGTTGCTATTAATCTCGATTTCATCAATTTCAGTTGAATCAAGGTGCAGCAGAGGCTGTGACTTGGCTTTAGCATCGTACTACGTTTGGCAAGGATCAAACTTGACCTTCATATCTCAGATTCTTAACTCAAGCCTCGTACCGTACTCAACTACTAATTTCGATTCGATCCTTGCTTATAACCCACAAGTTGCGAACAAAGACAGTGTCGAAGCTTTTAGCAGGCTTAACATCCCTTTCCCTTGTGATTGCATCAATAACGATTTCCTTGGCCATGTTTTCACCTACTCGGTTCAACCGGGCGATACTTACGATAAAATCGCTGGTTATTATTCCGATTTGACGACGGTTGCTTGGTTGCGGCCGTTTAATAGCTATCCTGAGACCAATATACCCGATAGTGGGGTGGTTAATGTGGTGGTCAATTGTTCGTGTGGAGATTCTGCTATTTCCAAGGATTACGGTTTGTTTGTTACGTATCCGCTCCGGGAAAATGAGACTTTGGATACTGTGTTGACTCAGGCGAATTTGTCATCGGATTTATCGGGGTTGGTGCGGCTTTACAACCCGGGGGCAAATTTCAGCTCAGGGTCTGGCTTGGTGTATATTCCGGGACGAGATGCTGAAAATAGTTTTCGTCCCCTCAAATCAAGCAAAGGAATTTCAGGTGGAGTTATCGCCGGGATAGCTATAGCAGCCGTAGTGGTATCACTATTGCTGGCATTTGGTATTTATGCTAAATTTTTCCGAAAGAAGTCAAAGACAACATCATTGCTTTCGACGGTTTCCCATGACGTATCGGCTGAAGCTGGGAATGTTTTTGGAAGCAAAGCAGCAGAATCAACTGGAACTGTTGCTGCTTCTCCCGGCCTTACCGGCATTACTGTAGACAAATCAGTTGAGTTCTCATATGAAGAACTTGCCCAAGCGACTGATTATTTCAGTATGGCTAACAAGATTGGTGAAGGTGGCTTCGGGGCTGTTTACTATGCAAATTTGAGAGGCGAGGAAGCTGCAATCAAGAAGATGGATATGCAGGCATCCAAAGAATTTCTGGCTGAATTGAAGGTTTTGACACGTGTTCATCACCTGAACCTGGTGCGTTTAATTGGATACTGTGTTGAAGGCTCTCTTTTCCTAGTTTACGAATACATCGAGAATGGCAATCTAAGCCAGCATTTGCGAGGATCAAGCAGGGAGCCACTTCCTTGGTCTACTCGCGTGCAAATTGCCCTTGACTCAGCCAGAGGTCTTGAATATATCCATGAGCATACGGTCCCTGTTTACATTCACCGTGACATTAAATCAGCTAATATATTGATAGACAAAAAGTTCCGTGCGAAGGTTGCCGATTTTGGATTAACGAAACTGACAGAAGTCGGAAGTGCATCACTACCCACACGTCTTGTTGGGACATTTGGATACATGCCACCAGAATATGCTCAATATGGTGATGTTTCTCCTAAAATAGATGTATATGCCTTCGGGGTTGTGCTCTATGAGCTTATTTCCGCTAAAGAAGCTATTGTCAAGGCAAATGGTTCTCTCGCTGAATCAAAAGGTCTTGTTGCCCTGTTTGACGATGCTCTTGATGAGCCTGATCCTAAAGAAGGCCTTTGCAGACTTATCGATGCAAGGCTCGGAGATAATTACCCACTAGACGCTGTATTCAAGATGGCTCAGCTTGCGAAAGCATGCACAAAAGAAAATCCTCAGCTACGACCGAGTATGAGATCCATAGTAGTTGCTTTAATGACCCTTTCATCGTCAACAGAGGATTGGGATGTTGGTTCTTTCTATGAAAATCAAGCTCTCGTCAACCTTATGTCCGGAAGATAA。

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<220>

<221>P3

<222>(1)..(19)

<223>

<400>4

ctgggaatgtttttggaag19

<210>5

<211>19

<212>DNA

<213>

<220>

<221>P4

<222>(1)..(19)

<223>

<400>5

actcggtcgtagctgagga19

<210>6

<211>23

<212>DNA

<213>

<220>

<221>P5

<222>(1)..(23)

<223>

<400>6

ttgtcaaggcaaatggttctctc23

<210>7

<211>24

<212>DNA

<213>

<220>

<221>P6

<222>(1)..(24)

<223>

<400>7

cgtctagtgggtaattatctccga24

Claims

1. The LRK1 gene that confers resistance to Verticillium wilt in plants, the sequence of which is SEQ ID NO.1.

2. The application of the LRK1 gene according to claim 1.

3. The application of the LRK1 gene according to claim 1 in conferring resistance to Verticillium wilt in plants.

4. The application of the LRK1 gene of claim 1 in conferring resistance to Verticillium wilt in Arabidopsis plants.

5. The use according to claim 4, comprising:

1) Cloning of LRK1 gene and construction of plant expression vector

Using the cDNA of upland cotton variety Ao 3503 as a template, primers

P1: 5'-TGCTCTAGAATGCTGAAATTAATCTCGATTC-3',

P2:5'-CGCGGATCCTTATCTTCCGGACATAAGGT-3'

A 1860bp fragment was amplified, and the fragment was named LRK1. After the PCR product was purified, it was digested with XbaI/BamHI and connected to the pCambia2301 vector. The plasmid successfully constructed after sequencing was named pCambia2301:LRK1;

2) Obtaining of transgenic plants

Transform the pCambia2301:LRK1 plasmid constructed in step 1) into Agrobacterium LBA4404 by the freeze-thaw method, and transform Arabidopsis thaliana with the flower dipping method. The contemporary Arabidopsis plants are the T0 generation, and the harvested seeds are the T1 generation;

T1 generation seeds were screened on 1/2MS medium containing 25mg/mL kana antibiotics to obtain candidate positive T1 plants, and detected whether the target gene was transferred and successfully expressed at the DNA and RNA levels;

with primers

P3:5'-CTGGGAATGTTTTTGGAAG-3'

P4:5'-ACTCGGTCGTAGCTGAGGA-3'

The T1 plants that amplified the 920bp size fragment were the positive transgenic plants successfully transformed into LRK1.

6. The recombinant vector pCambia2301:LRK1 constructed in the application described in claim 5.