CN110818783B - Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof - Google Patents

Abstract

The invention discloses a WRKY transcription factor gene LrWRKY2 of Lily of the Minjiang River. Studies have confirmed that the LrWRKY2 gene has the function of improving the anti-fungal function of plants. The anti-fungal LrWRKY2 gene of the present invention is constructed into a plant expression vector and transferred to tobacco for overexpression. The transgenic tobacco plants have strong anti-fungal infection ability. The experimental results show The transgenic tobacco overexpressing LrWRKY2 has a high level of resistance to infection by Nigella oryzae, Fusarium solani, Fusarium verticillium, Botrytis, and Alternaria ginseng.

Description

A Minjiang Lily WRKY transcription factor gene LrWRKY2 and its application

technical field

The invention relates to the research field of molecular biology and related technologies of genetic engineering, in particular to a Minjiang lily WRKY transcription factor gene LrWRKY2 with antifungal infection ability and application thereof.

Background technique

Plants undergo large-scale reprogramming of their transcriptomes in a highly variable and temporal manner in response to pathogen invasion or other stress, and this regulatory response that confers plastic adaptation to different environmental conditions is the result of multiple transcription factors The result of network action. WRKY transcription factors are a family of regulatory protein genes in the plant defense response-related transcription factor network, which are mainly involved in the plant immune system responding to a variety of different biotic and abiotic stresses (Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity . PlantPhysiol, 2009, 150(4): 1648-1655; Bakshi M, Oelmüller R. WRKY transcriptionfactor: Jack of many trades in plants. Plant Signal Behav, 2014, 9(2):e27700.). WRKY transcription factors are one of the largest types of transcription factors in plants. The N-terminus is generally a WRKY domain composed of the WRKYGQK heptapeptide sequence, and the C-terminus is composed of CX4-5CX22-23HXH (C2H2) or CX7CX23-HXC (C2HC). The zinc finger structure of (Rushton PJ, Somssich IE, Ringler P, et al. WRKY transcriptionfactors. Trends Plant Sci, 2010, 15(5): 247-258.).

Most of the promoter regions of target genes regulated by WRKY contain W-box (TTGACC/T), of which TGAC is the core sequence of W-box and is highly conserved. Once a nucleotide is changed, WRKY protein will bind to it. The ability to reduce or even lose (Xie Zhengwen, Wang Lianjun, Chen Jinyang, et al. Research progress on plant WRKY transcription factors and their biological functions. China Agricultural Science and Technology Review, 2016, 18(3): 46-54.). The WRKY domain is composed of four-strand β-sheets, and the conserved WRKYGQK residue corresponds to the N-terminal β-sheet (strand β-1), which can enter the DNA groove and interact with the W box on DNA (Ciolkowski I, Wanke D, Birkenbihl RP, et al. Studies on DNA-bindingselectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Mol Biol, 2008, 68(1-2): 81-92.).

WRKY transcription factors reprogram the plant transcriptome to respond to the invasion of different pathogens. The transcriptional regulation of genes related to disease resistance response is a key component of plant defense responses to pathogens and plays an important role in plant defense responses. The WRKY gene family in plants contains many members that regulate plant growth and development and various stress responses (Wu ZJ, Li XH, Liu ZW, et al. Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress. MolGenet Genomics, 2016, 291(1): 255-69.). Overexpression of PtrWRKY18 and PtrWRKY35 in Populus euphratica enhanced the resistance to rust fungus ( Melampsora ) and simultaneously increased PR gene expression in transgenic plants (Jiang Y, Guo L, Ma X, et al. The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promote Melampsora resistance in Populus . Tree Physiol, 2017, 37(5): 665-675.). The strawberry FaWRKY1 transcription factor negatively regulates resistance to Colletotrichum acutatum in fruit upon Infection. Front Plant Sci , 2019, 10: 480.). Overexpression of PtWRKY14 can enhance the resistance of tobacco ( Nicotiana tabacum ) to TMV infection (Wang Xing, Lin Shanzhi. PtWRKY14 gene transformed tobacco and its effect on TMV resistance. Guangdong Agricultural Science, 2014, 41(7): 130 -133.). Furthermore, salicylic acid (SA)-mediated reactive oxygen species (ROS) signaling enhanced the resistance of JcWRKY transgenic tobacco to alfalfa char rot pathogen (Agarwal P, Patel K, Agarwal PK. Ectopic expression of JcWRKY confers enhanced resistance in transgenic tobacco against Macrophomina phaseolina . DNA Cell Biol, 2018, 37(4): 298-307.).

Lily is the general name of the Liliaceae ( Liliaceae ) genus Lilium ( Lilium ) plants, is a perennial bulbous flowers. During the propagation of bulbs and the production of fresh cut flowers, lilies are susceptible to the harm of fungi, viruses, bacteria and other pathogens. There are dozens of lily diseases found so far. Among them, fusarium wilt (also known as base rot and stem rot) caused by fungi of the genus Fusarium spp. is the most serious disease in lily production. Fusarium infects lily bulbs, causing basal necrosis and scales to rot and fall off, resulting in a decline in the quality of bulbs. After plants are infected with Fusarium, the leaves turn yellow, wilt and droop, and the plants die prematurely, which seriously affects the yield and quality of lily cut flowers. Among them, F. oxysporum has the strongest pathogenicity and the highest isolation frequency, and is the main pathogen of lily fusarium wilt. Minjiang lily ( L. regale Wilson) is an endemic species in China. It is only distributed in the valleys of the Minjiang River at an altitude of 800-2700 m to the rock crevices on the mountainside. It has strong resistance to fusarium wilt and is an important germplasm resource for modern lily breeding. WRKYs are involved in responding to a variety of biotic and abiotic stresses and are an important part of plant defense systems. Therefore, the discovery and functional analysis of WRKY transcription factor genes in Minjiang lily has important research and application value.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Minjiang lily WRKY transcription factor gene LrWRKY2 and its application, that is, in improving tobacco resistance to Nigrospora oryzae , Fusarium solani , Fusarium verticillioides , Botryosphaeria dothidea and Alternaria panax resistance.

The present invention clones the full-length gene of WRKY transcription factor with antifungal activity from Lily of the Minjiang River. The nucleotide sequence of the WRKY transcription factor gene LrWRKY2 is shown in SEQ ID NO: 1, and the full-length cDNA sequence of the gene is 1302bp, including An open reading frame of 1032 bp, a 5' untranslated region of 45 bp, and a 3' untranslated region of 225 bp encodes a protein with the amino acid sequence shown in SEQID NO:2.

The coding region of the LrWRKY2 gene in the present invention is the nucleotide sequence shown in positions 46-1077 in SEQ ID NO: 1 of the sequence listing.

The present invention isolates and clones a complete cDNA fragment of an antifungal related gene of Lily of the Minjiang River, utilizes Agrobacterium tumefaciens to mediate the transfer of the target gene into the recipient plant and overexpresses it, and further experiments to verify whether the gene has antifungal properties The fungal activity lays the foundation for the later use of this gene to improve the ability of tobacco and other plants to resist fungal diseases. The inventor named this gene LrWRKY2 .

The above-mentioned LrWRKY2 gene can be applied to improve the antifungal properties of tobacco, and the specific operations are as follows:

(1) Using specific primers for amplifying LrWRKY2 , total RNA was extracted from the roots of Lily Minjiang after inoculation with Fusarium oxysporum, and amplified by reverse transcription-polymerase chain reaction (RT-PCR). The full-length coding region of LrWRKY2 was then ligated into the pGEM-T vector and sequenced to obtain a clone with the target gene;

(2) The pGEM-T- LrWRKY2 vector was digested with restriction enzymes Bam HI and Xba I, and the target gene fragment was obtained by gel recovery. The plant expression vector pCAMBIA2300s was digested with the same endonuclease, and the desired vector was obtained by gel recovery. Large fragment, then the obtained LrWRKY2 gene fragment was connected with the pCAMBIA2300s fragment to construct a plant overexpression vector, and then the constructed recombinant vector was transferred into tobacco through Agrobacterium tumefaciens for expression;

(3) Screen the transformants with the resistance markers on the recombinant vector T-DNA, and obtain the real transgenic plants by PCR and RT-PCR detection, analyze the ability of the leaves of the transgenic plants to resist fungal infection, and finally screen out the resistance to fungi. Transgenic plants with significantly enhanced sex.

The invention provides a new method for improving the resistance of plants to fungal diseases, and cultivating disease-resistant plants by means of genetic engineering can overcome the shortcomings of traditional breeding, not only shortens the breeding cycle, but also is simple to operate and easy to obtain high-resistance materials. The LrWRKY2 gene from Minjiang lily of the invention can enhance the resistance of plants to fungi, and the gene can be introduced into tobacco to produce new varieties and new materials with fungal resistance. The use of genetic engineering technology to cultivate resistant plant varieties and materials has obvious advantages and irreplaceable importance; it can not only provide convenience for the large-scale production of crops, medicinal materials, horticultural plants, etc., greatly reduce the use of chemical pesticides, but also provide agricultural Production costs are saved and environmental pollution is reduced, so the invention has broad market application prospects.

Description of drawings

Fig. 1 is the PCR detection result diagram of LrWRKY2 transgenic tobacco genomic DNA of the present invention, among the figure: Marker is DL2000 DNA Marker (Dalian treasure biology); Positive control is the PCR result that plasmid pGEM-T- LrWRKY2 is template; WT is non-transgenic Tobacco (wild type) total DNA is the product of template PCR;

Fig. 2 is the expression analysis result chart of LrWRKY2 transcription level in positive LrWRKY2 transgenic tobacco of the present invention; among the figure: Marker is DL2000 DNA Marker (Dalian Bao Bio); WT is the PCR product of non-transgenic tobacco total RNA reverse transcription cDNA as template; positive control It is the PCR product of plasmid pGEM-T- LrWRKY2 as template;

Fig. 3 is the result picture of the LrWRKY2 transgenic tobacco resistance identification of the present invention; in the figure a, b, c, d, e are respectively inoculated with Nigella oryzae, Fusarium verticillium, Botrytis, Fusarium solani, Panax ginseng Alternaria LrWRKY2 transgenic tobacco leaves; WT is the leaves of wild-type tobacco, 1, 8, 17, 27 are LrWRKY2 transgenic tobacco leaves.

Detailed ways

The present invention will be described in further detail below through the accompanying drawings and examples, but the protection scope of the present invention is not limited to the content. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial methods unless otherwise specified. commercially available reagents or reagents prepared by conventional methods.

Example 1: LrWRKY2 full-length gene cloning and sequence analysis

Minjiang lily was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 h after inoculation. The inoculated roots of Minjiang lily were ground into powder with liquid nitrogen, and then transferred to a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method. RNA, using reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA with total RNA as template, the reaction system and operation process are: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM) in turn. each), DEPC water was added to the reaction volume of 14.5 μL; after mixing, heat denaturation at 70°C for 5 min, then quickly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U) , 1 μL M-MLV (200U), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take out and heat at 70°C for 10 min to terminate the reaction; store at -20°C after synthesis of the first strand of cDNA.

The target gene LrWRKY2 was amplified using the synthesized first-strand cDNA as a template. The upstream and downstream primer sequences used were 5' CTACAAGGTTCATCCTGCTAGACAT 3' and 5'TTTGCTTTCCATCCACTAGATAACA3', respectively. The target gene was amplified by Advantage ^TM 2 PCR Enzyme (Clontech); PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, 32 cycles; 72°C for 7 min; reaction system (20 μL) for 0.5 μL cDNA, 2 μL 10× Advantage 2 PCR Buffer, 0.4 μL 50× dNTP Mix (10 mM each), 0.4 μL Forward Primer (10 μM), 0.4 μL Reverse Primer (10 μM), 0.4 μL Advantage 2 PCR Polymerase Mix, 15.9 μL PCR-Grade water; after PCR, take 5 μL for agarose gel electrophoresis to test the specificity and size of the amplified product.

The PCR products were cloned by TA using the pGEM-T easy Vector System I (Promega, USA). ) and 2.5 μL of 2×Ligation solution I, mixed well and placed at 16 °C for overnight reaction. The ligated product was transformed into E. coli DH5α by heat shock transformation. Use LB solid medium containing ampicillin (ampicillin, Amp) to screen positive clones, select several single colonies, and use specific primers for amplifying LrWRKY2 to identify clones with multiple cloning sites inserted into LrWRKY2 . Sequencing was performed, and the final LrWRKY2 full-length cDNA was 1302 bp, which was analyzed by NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and found that it contained an open reading frame of 1032 bp Box (see Sequence Listing), LrWRKY2 encodes a 343 amino acid protein with a molecular weight of about 37.91 KDa, an isoelectric point of about 5.88, and 4 cysteine residues. LrWRKY2 has a WRKYGQK conserved domain followed by a C2H2 (C-X4-C-X22/23-H-X1-H) type zinc finger motif immediately after each WRKY domain. Apparently, the protein encoded by LrWRKY2 belongs to class II WRKY transcription factor.

Example 2: Plant overexpression vector construction

The E. coli plasmid pGEM-T- LrWRKY2 inserted into LrWRKY2 and the plasmid of the plant expression vector pCAMBIA2300s were extracted using the SanPrep column-type plasmid DNA mini-extraction kit (Shanghai Shenggong), and 1 μL was taken for agarose gel electrophoresis to detect all the The integrity and concentration of the extracted plasmids; double-enzyme digestion of plasmids pGEM-T- LrWRKY2 and pCAMBIA2300s with restriction enzymes Bam HI (TaKaRa) and Xba I (TaKaRa) respectively (100 μL system), reaction system and operation The process is: take 20 μL pGEM-T- LrWRKY2 and pCAMBIA2300s plasmids, add 10 μL 10×Kbuffer, 4 μL Bam HI, 6 μL Xba I, 60 μL ddH ₂ O in turn, mix well, centrifuge for a short time, and place at 37°C overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then gel recovery of LrWRKY2 fragment and pCAMBIA2300s vector large fragment, using SanPrep column DNA gel recovery kit (Shanghai Sangong); 1 μL of the recovered product was detected by agarose gel electrophoresis for the size and concentration of the recovered fragments, and stored at -20°C for later use.

Using T4 DNA Ligase (TaKaRa), the recovered LrWRKY2 DNA fragment and pCAMBIA2300s vector fragment were ligated. The reaction system (20 μL) and the operation process were as follows: 10 μL LrWRKY2 DNA fragment was added to 2 μL pCAMBIA2300s vector DNA, 2 μL 10×T4 DNA Ligase Buffer, 1 μL T4 DNA Ligase, 5 μL ddH ₂ O, mixed well, centrifuged for a short time, and then reacted in a water bath at 16°C overnight. Then, the ligated product was transformed into E. coli DH5α by heat shock transformation, and positive clones were screened with solid medium containing 50 mg/L kanamycin (Km). A single colony was selected and shaken, and PCR was performed using the bacterial solution as a template with specific primers for amplifying LrWRKY2 to select clones that successfully linked LrWRKY2 to pCAMBIA2300s. If the detected strain was positive, add glycerol and store at -80°C for later use.

The pCAMBIA2300s -LrWRKY2 plasmid in the above E. coli was extracted and purified using the SanPrep column plasmid extraction kit (Shanghai Shenggong). Subsequently, the above-constructed plant expression vector pCAMBIA2300s -LrWRKY2 was transformed into Agrobacterium tumefaciens LBA4404 competent cells by liquid nitrogen freeze-thaw method. The operation steps are as follows: add 2 μg pCAMBIA2300s- LrWRKY2 plasmid to a centrifuge tube containing 200 μL competent cells, mix gently, ice bath for 5 min, then transfer to liquid nitrogen to freeze for 1 min, then quickly place in a 37°C water bath for 5 min, and then Immediately ice-bath for 2 min, add 800 μL of LB liquid culture for 4 h at 28°C with shaking. The activated Agrobacterium was spread on LB solid medium containing 50 mg/L Km, and cultured at 28°C statically. A single colony was selected and shaken, and PCR was performed with specific primers for amplifying LrWRKY2 to detect whether pCAMBIA2300s -LrWRKY2 was transformed into Agrobacterium. For positive clones, add glycerol and store at -80°C for later use.

Example 3: Agrobacterium-mediated plant genetic transformation and screening of transgenic plants

The transgenic receptor in this experiment was tobacco. Tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water, soaked in 0.1% HgCl for ₈ min, then washed with sterile water for several times, and sown in 1/1/2 2 MS medium, cultivated in the dark at 28 °C for 6 d, transferred to a light incubator (25 °C, 16 h/d light) after germination, and then subcultured with 1/2 MS medium once a month.

Take out the preserved Agrobacterium LBA4404 strain containing pCAMBIA2300s -LrWRKY2 plasmid from the -80℃ refrigerator, inoculate it in 5 mL LB liquid medium containing 50 mg/L Km and 20 mg/L rifampicin, and cultivate it at 28℃ to The medium is cloudy. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg/L Km, and cultivate at 28 °C for 48 h; then scrape off an appropriate amount of Agrobacterium on the LB solid medium and inoculate it with 20 mg/L acetyl In the MGL liquid medium of syringone, shake cultured at 28°C for 2-3 h to activate Agrobacterium.

Take the leaves of tobacco sterile seedlings and cut them into leaf discs of about 1 cm ² , and completely immerse them in the above-mentioned MGL liquid medium containing activated Agrobacterium for 15 min. The leaf discs were cultured at room temperature on a co-culture medium. The co-culture medium for tobacco transformation was MS+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/Lsucrose+6 g/L agar, 22°C without light Cultivated for 2 days under the conditions.

The leaf discs after co-culture were transferred to MS screening medium with antibiotics and differentiated into shoots, and transgenic plants were screened at the same time. Tobacco screening medium is MS+0.5mg/L 6-BA+0.1mg/L NAA+30g/L sucrose+6g/L agar+50mg/L Km+200 mg/L cefotaxime sodium salt, Cef ; During the screening culture, transfer the culture flask to a light incubator (25°C, 16 h/d light, 8 h/d dark), and after the tobacco grows buds, culture it with MS containing 50 mg/L Km and 200 mg/L Cef The regenerated plants need to be further screened because of the high callus differentiation rate of tobacco. The tobacco regenerated seedlings are transferred to MS medium containing 50 mg/L Km to make them take root. Finally, the better rooting plants are selected. The regenerated seedlings were further tested.

The genomic DNA of the leaves of transgenic tobacco plants was extracted by CTAB method, and 1 μL of the extracted genomic DNA was taken to detect its integrity and concentration by agarose gel electrophoresis. After PCR, 8 μL of the product was taken for agarose gel electrophoresis to detect positive transgenic plants. The amplification results of some tobacco transgenic plants are shown in Figure 1. A total of 51 positive transgenic plants were screened from LrWRKY2 transgenic tobacco.

Example 4: Expression analysis of LrWRKY2 in transgenic tobacco and functional analysis of transgenic plants against fungal infection

The positive transgenic individual plants and young leaves of non-transgenic tobacco (wild-type) were taken to extract total RNA, reverse-transcribed to generate the first strand of cDNA, and used as a template for PCR with specific primers for amplifying LrWRKY2 . The expression of the LrWRKY2 transcript level in the strain, the extraction of total RNA and the RT-PCR method were the same as those in Example 1. After PCR, 8 μL was taken for agarose gel electrophoresis. The detection results of some individual strains are shown in Figure 2. A total of 47 transgenic individual plants were detected to express a large amount of LrWRKY2 at the transcriptional level, and these individual plants were numbered from 1 to 47.

Several pathogenic fungi preserved in the laboratory were inoculated on PDA solid medium (200 g/L potato, 15 g/L agar, 20 g/L glucose), and cultivated in the dark at 28°C for 7 days. Wild-type tobacco leaves and LrWRKY2 transgenic tobacco leaves that grow well, uniform in size and fully expanded in the greenhouse were selected and cut from petioles with surgical scissors. Use a sterile plastic pipette tip to form wounds of the same size on the leaves at about the same position, and inoculate the fungal mycelium blocks of equal size respectively. The treated leaves were placed in a plate covered with filter paper soaked in sterile water, and cultivated in a light incubator at 28°C, and water was added every day for moisturizing. After 7 days of culture, the leaves were collected and the disease status of the leaves of each line was observed. The results are shown in Figure 3. After inoculation with five types of pathogenic fungi, including Nigella oryzae, Fusarium verticillium, Botrytis sp., Fusarium solani, and Alternaria ginseng, the leaves of the wild-type tobacco formed larger disease. The leaves appeared yellowing and rotting, while the symptoms of transgenic tobacco leaves were very mild, and the area of lesions formed was much smaller than that of wild-type tobacco. Obviously, LrWRKY2 transgenic tobacco has strong resistance to pathogenic fungi such as Nigella oryzae, Fusarium verticillium, Botrytis, Fusarium solani, and Alternaria ginseng.

sequence listing

<110> Kunming University of Science and Technology

<120> A Minjiang Lily WRKY transcription factor gene LrWRKY2 and its application

<160> 4

<170> SIPOSequenceListing 1.0

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<211> 1302

<212> DNA

<213> Lilium regale Wilson

<400> 1

ctcccctctc cctctctcac ccctacaagg ttcatcctgc tagacatgtg tgatctcttc 60

tggcaaagaa tggaaggcga gcttgccgac atcgtccgca ccagcggcct caccagccat 120

ccgcccatct ccgactttgc tgattgggat ctaccttccg accccataac cttctcccct 180

tcatcccaag acaacttcgg cgacccattc atcaacttgc cagatccatt gcttcatgaa 240

tcaccggcca ccaatatcgc tccagtctct gactccgaca accatgaaat aggtgtgcca 300

cttgctcaga ggatgatagc agcgagcgag gagctgaaga ggcctaacaa catattctca 360

cggatgctgc agatctcccc aagggagcta aaagctactc aaatgatatc gaccagtgat 420

ttgatgaaga cgagcaacag tggctcgaca ggcgccgtgc agatttcctc tccaaggggt 480

cttggaatca agaggaggaa aagccaggca aagaaggtgg tgtgcatccc agcaccagca 540

gctacaacca gcaggagcag tggagaggtt gttccagctg atctttgggc ttggaggaag 600

tatggacaga aacccatcaa aggttctcct catccaaggg gctactatag atgcagcagc 660

tcaaaaggat gctcagcaag gaagcaagtc gagaggagcc gaactgatcc aaacatgcta 720

gtcatcactt acacatccga gcacaaccat ccctggccga cacagaggaa cgctctcgct 780

ggatcgacaa gatcgtcaca cccggctaag aacatctctt ccgcggcatc caagatctcc 840

ccacctgcaa atttgaagga agaagaaccc aaggaggagg taatctcaac cattgtgcca 900

gtgaaggaag aaatggcagg aaatgatcat caagaattcc aagaccaagt gttgcagcat 960

acttacaagc caatgatacc agactcgaat caatccgatg atttctttga tgagttggga 1020

gaattgggga ctgattctat ggattcattt aacatgtttg aatgggccgg gaaataatct 1080

ggagtcatag atttcgaata aaaagttgtt atctagtgga tggaaagcaa aaactattga 1140

ggtttgaagc acatacattt tcacatactt gatttaacca aaatatgaat taaatcctta 1200

gtgtcctata agtgaaaatg tattgtgcat ggattctgtt ttatggattg ctacatatat 1260

ttgtgttttg gactatctga ccaatgactg gttaacttat aa 1302

<210> 2

<211> 343

<212> PRT

<213> Lilium regale Wilson

<400> 2

Met Cys Asp Leu Phe Trp Gln Arg Met Glu Gly Glu Leu Ala Asp Ile

1 5 10 15

Val Arg Thr Ser Gly Leu Thr Ser His Pro Pro Ile Ser Asp Phe Ala

20 25 30

Asp Trp Asp Leu Pro Ser Asp Pro Ile Thr Phe Ser Pro Ser Ser Gln

35 40 45

Asp Asn Phe Gly Asp Pro Phe Ile Asn Leu Pro Asp Pro Leu Leu His

50 55 60

Glu Ser Pro Ala Thr Asn Ile Ala Pro Val Ser Asp Ser Asp Asn His

65 70 75 80

Glu Ile Gly Val Pro Leu Ala Gln Arg Met Ile Ala Ala Ser Glu Glu

85 90 95

Leu Lys Arg Pro Asn Asn Ile Phe Ser Arg Met Leu Gln Ile Ser Pro

100 105 110

Arg Glu Leu Lys Ala Thr Gln Met Ile Ser Thr Ser Asp Leu Met Lys

115 120 125

Thr Ser Asn Ser Gly Ser Thr Gly Ala Val Gln Ile Ser Ser Pro Arg

130 135 140

Gly Leu Gly Ile Lys Arg Arg Lys Ser Gln Ala Lys Lys Val Val Cys

145 150 155 160

Ile Pro Ala Pro Ala Ala Thr Thr Ser Arg Ser Ser Gly Glu Val Val

165 170 175

Pro Ala Asp Leu Trp Ala Trp Arg Lys Tyr Gly Gln Lys Pro Ile Lys

180 185 190

Gly Ser Pro His Pro Arg Gly Tyr Tyr Arg Cys Ser Ser Ser Lys Gly

195 200 205

Cys Ser Ala Arg Lys Gln Val Glu Arg Ser Arg Thr Asp Pro Asn Met

210 215 220

Leu Val Ile Thr Tyr Thr Ser Glu His Asn His Pro Trp Pro Thr Gln

225 230 235 240

Arg Asn Ala Leu Ala Gly Ser Thr Arg Ser Ser His Pro Ala Lys Asn

245 250 255

Ile Ser Ser Ala Ala Ser Lys Ile Ser Pro Pro Ala Asn Leu Lys Glu

260 265 270

Glu Glu Pro Lys Glu Glu Val Ile Ser Thr Ile Val Pro Val Lys Glu

275 280 285

Glu Met Ala Gly Asn Asp His Gln Glu Phe Gln Asp Gln Val Leu Gln

290 295 300

His Thr Tyr Lys Pro Met Ile Pro Asp Ser Asn Gln Ser Asp Asp Phe

305 310 315 320

Phe Asp Glu Leu Gly Glu Leu Gly Thr Asp Ser Met Asp Ser Phe Asn

325 330 335

Met Phe Glu Trp Ala Gly Lys

340

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial)

<400> 3

ctacaaggtt catcctgcta gacat 25

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial)

<400> 4

tttgctttcc atccactaga taaca 25

Claims (1)

1. A Minjiang lily WRKY transcription factor gene LrWRKY2 can improve tobacco resistance to Nigrospora oryzae , Fusarium solani , Fusarium verticillioides , Alternaria panax For the application in resistance, the nucleotide sequence of the WRKY transcription factor gene LrWRKY2 of Lily of the Minjiang River is shown in SEQ ID NO: 1.

Images