CN103937819B - A kind of lilium regale wilson glutathione S-transferase gene LrGSTL1 and application thereof - Google Patents

Abstract

The present invention discloses a Minjiang lily disease resistance gene <i>LrGSTL1</i> and its application. The nucleotide sequence of <i>LrGSTL1</i> gene is as SEQ? ID? NO: 1, encoding Lambda-like glutathione S-transferase, the present invention proves that the <i>LrGSTL1</i> gene of Minjiang lily has the function of improving the anti-fungal ability of plants through functional genomics technology, and the present invention The Minjiang lily glutathione S-transferase gene <i>LrGSTL1</i> was constructed on a plant expression vector and transferred to tobacco for overexpression. The transgenic tobacco has strong antifungal activity and expresses <i> LrGSTL1</i> transgenic tobacco leaves had obvious resistance to the infection of two pathogenic fungi, Fusarium oxysporum and Botrytis cinerea.

Description

A Minjiang lily glutathione S-transferase gene LrGSTL1 and its application

technical field

The invention relates to a Minjiang lily glutathione S-transferase gene LrGSTL1 with antifungal activity and application thereof, which belongs to the technical research field of molecular biology and genetic engineering.

technical background

With the continuous increase of world population, the demand for food is increasing day by day, so improving food production is an urgent problem to be solved. Crops are constantly attacked by various pathogenic microorganisms during their growth and development. The diseases caused by fungi account for more than 80% of the total plant diseases, and have seriously affected the yield of food. In addition, fungal diseases are the most common types of diseases, and fungi can invade any organ and tissue of plants and cause them to cause disease. The traditional methods of preventing and treating plant diseases are mainly the use of chemical pesticides and the cultivation of resistant varieties. Although both methods have achieved certain results, they also have serious drawbacks. Extensive use of chemical pesticides has caused serious environmental pollution and has had adverse effects on human and animal health; however, conventional breeding has disadvantages such as long cycle, time-consuming and labor-intensive, and less beneficial variation of plant resources, making them unable to fundamentally solve the problem of plant diseases . With the establishment and development of recombinant DNA technology, it is a new method to improve plant disease resistance by using molecular biology methods to obtain genes with antifungal activity and cultivating disease-resistant crops in a short period of time through transgenic technology .

When pathogens invade plants, plants mainly resist the invasion of pathogenic microorganisms through the expression of defense genes, hypersensitivity response (hypersensitivity response, HR) and systemic acquired resistance (Sytemic acquired resistance, SAR). Oxygen burst is one of the early responses of pathogens to infect plants, followed by hypersensitivity responses for self-defense. After the oxygen burst, the production of reactive oxygen species (reactive oxygen species, ROS) in plants increased significantly, such as superoxide anion (O ^2- ), hydroxide ion (HO ^- ), hydroxyl radical (-OH), Hydrogen peroxide, etc. (H ₂ O ₂ ). These reactive oxygen species can damage proteins, membrane lipids and other cellular components, and cause oxidative damage to plants. In order to prevent the damage of reactive oxygen species, plants use glutathione S-transferase (glutathioneS-transferase, GSTs), superoxide dismutase (superoxide dismutase, SOD), catalase (catalase, CAT), glutathione Reductase (glutathionereductase, GR), glutathione peroxidase (glutathioneperoxidase, GPX), etc. to remove free radicals in plants, thereby alleviating the stress effect.

GSTs widely exist in animals, plants, bacteria and fungi, and constitute supergene families with various physiological functions. GST monomer is a soluble protein with a molecular weight of 22-27kDa, mainly in the form of homologous or heterodimer. According to the similarity of amino acid sequence, gene composition and amino acid residues in the active site, plant GSTs can be divided into 6 types: phi, tau, theta, zeta, lambda and dehydroascorbate reductase. Among them, phi class and tau class are unique to plants. Glutathione usually exists in the form of reduced glutathione (GSH) under normal conditions, and a small amount exists in the form of oxidized glutathione (GSSH). GSTs can catalyze the conjugated combination of the sulfur atom on GSH and the electrophilic part of the second substrate, and produce a water-soluble complex, which is conducive to their excretion, thereby eliminating the accumulation of toxic substances in cells such as ROS and maintaining intracellular oxidation. The equilibrium of the reduced state (ArmstongRN, 1997. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chemical Research in Toxicology, 10:2-18). The research on plant GST started relatively late. In 1970, plant GST was first discovered in corn ( Zeamays ) ( EdwardsR , DixonDP, WalbotV. Functionally active GSTs have been found in crops such as Arabidopsisthaliana , wheat ( Triticum aestivum), and potato ( Solanum tuberosum ). Since GSTs are involved in the defense response of plants to biotic and abiotic stresses, the gene cloning and application of GSTs has important value in agricultural production and has been paid more and more attention by people.

GSTs play an important role in plant resistance to fungal stress. After inoculating potatoes with Phytophthorainfestans , the expression of GST protein encoded by prp1-1 was significantly increased, and the protein reacted with indole-3-acetic acid, indicating that indole-3-acetic acid can be used as a regulator or substrate of GST Drugs involved in disease resistance response (HahnK, StrittmatterG. Pathogen-defensegeneprp1-1frompotatoencodesanauxinresponsiveglutathioneS-transferase.EurJBiochem.1994,226:619-626). Ten days after maize was inoculated with Piriformosporaindica , the activities of catalase (CAT), GST and superoxide dismutase (SOD) in the roots of the plants increased by 44 times, 92 times and 48 times, respectively, compared with the control group. Thereby enhancing the antioxidant capacity of the plant, thereby improving the disease resistance of corn (KumarM, YadavV, TutejaN, JohriAK.AntioxidantenzymeactivitiesinmaizeplantscolonizedwithPiriformosporaindica.Microbiology.2009,155 :780-790). Treating Arabidopsis thaliana with plant hormones, herbicides, oxidative stress and downy mildew ( Peronospora parasitica ), it was found that AtGSTF16 was up-regulated under these treatments, indicating that AtGSTF16 has a certain role in Arabidopsis resistance to biotic stress and abiotic stress ( Wagner U, Edwards R, Dixon DP, Mauch F. Probing the diversity of the Arabidopsis glutathione S-transferase gene family. Plant Mol Biol. 2002, 49:515-532). Four GST genes were cloned from a tobacco cDNA library infected with anthracnose bacteria ( Colletotrichum destructivum ), and the expressions of NbGSTU1 and NbGSTU3 increased after the tobacco was inoculated with anthracnose bacteria . The sensitivity of silenced tobacco plants to the pathogen was significantly increased, indicating that GST genes play an important role in tobacco resistance to C. destructivum infection ( DeanJD , GoodwinPH , HsiangT . , 56:1525-1533).

Contents of the invention

The object of the present invention is to clone the lambda-like glutathione-sulfur transferase gene LrGSTL1 with antifungal activity from Lilium regale Wilson. The nucleotide sequence of LrGSTL1 is shown in SEQ ID NO: 1, and the cDNA of the gene is complete The long sequence is 1076bp, including a 717bp open reading frame (openreading frame, ORF), 22bp 5'untranslated region and 337bp 3'untranslated region, encoding a protein containing 238 amino acids, and the encoded amino acid sequence is as SEQ ID NO: 2.

The coding region of the Lilium Minjiang River glutathione S-transferase gene LrGSTL1 in the present invention is the nucleotide sequence shown in the 23rd-739th position in the sequence table SEQ ID NO: 1.

The LrGSTL1 gene of the present invention comes from Lily Minjiang. Minjiang Lily, also known as Wang Lily, is a perennial herbaceous bulbous plant of the Liliaceae Lily family. It is a unique wild lily in my country and is distributed in the Minjiang River Basin in Sichuan. Minjiang lily has strong resistance to fungi, viruses and drought stress, and is a rare genetic resource for lily disease-resistant breeding.

The present invention isolates and clones a complete cDNA fragment of an antifungal-related gene carried in Lily of Minjiang River, uses Agrobacterium tumefaciens to transfer the target gene into recipient plants and overexpresses it, and then verifies whether the gene has antifungal properties through experiments Activity, laying the foundation for the later use of the gene to improve the ability of tobacco and other plants to resist fungal diseases. The inventors named this gene LrGSTL1 .

When plants are stressed by pathogenic fungi, active oxygen in plants explodes, and GSTs catalyze the combination of GSH and electrophilic compounds to produce complexes that are easily soluble in water, which facilitates the excretion of electrophilic compounds, thereby reducing the level of toxic substances such as ROS in cells , maintaining the balance of intracellular redox state. In addition, GSTs also have glutathione peroxidase activity, so they play an important role in plant disease resistance.

The present invention relates to isolating the full-length cDNA fragment of LrGSTL1 and identifying its function. Plants with the gene fragment have the phenotype of resisting specific fungal infection to a certain extent. Wherein the DNA fragment is shown as SEQ ID NO: 1 in the sequence table. Sequence analysis of the gene revealed that the full-length cDNA of LrGSTl1 was 1076bp, with an open reading frame of 717bp, a 5'untranslated region (UTR) of 22bp and a 3'UTR of 337bp, encoding a protein of 238 amino acids. The protein encoded by LrGSTL1 has a conserved domain of the GSTs family, and has high homology with GSTs proteins from plants such as banana ( Musaacuminata ), corn, and rice. At the same time, cluster analysis classified LrGSTL1 as a plant lambda GSTs (GSTL). The above analysis results It indicated that LrGSTL1 belonged to the lambda-like GST gene in Lilium Minjiang. Overexpression of the amino acid sequence protein shown in SEQ ID NO: 2 can enhance the resistance of tobacco to two pathogenic fungi, Botrytiscinerea and Fusarium oxysporum.

Another object of the present invention is to apply the Minjiang lily glutathione S-transferase gene LrGSTL1 in improving the resistance of tobacco to Fusarium oxysporum and Botrytis cinerea. The specific operations are as follows:

(1) Gene acquisition: Total RNA was extracted from the roots of Lily of the Minjiang River after being inoculated with Fusarium oxysporum using specific primers for amplifying LrGSTL1 , and amplified by reverse transcription-polymerase chain reaction (RT-PCR). The full-length coding region of LrGSTL1 was added, then connected to the pMD-18T vector, and the clone with the target gene was obtained through sequencing verification.

(2) Plant expression vector construction and genetic transformation: digest the pMD-18T- LrGSTL1 plasmid with restriction endonucleases Eco RI and Pst Ⅰ, recover the target gene fragment through gel recovery, and use the same endonucleases to digest and digest the plant expression Carrier pCAMBIA2300S, gel recovery to obtain the large fragment of the required carrier; then the target gene fragment was connected with the large fragment of the pCAMBIA2300S vector to construct the plant overexpression vector pCAMBIA2300s- LrGSTL1 ; the pCAMBIA2300S -LrGSTL1 plasmid was introduced into the Agrobacterium strain LBA4404 by the liquid nitrogen freeze-thaw method ; LrGSTL1 was introduced into tobacco and expressed by Agrobacterium tumefaciens-mediated genetic transformation; positive transgenic plants were screened by antibiotic selection, genomic DNA PCR and RT-PCR.

(3) Analysis of antifungal activity of transgenic plants: Take the leaves of transgenic plants and wild-type plants (non-transgenic control) and inoculate them with spore suspensions of different fungi, detect the incidence of transgenic leaves after inoculation, and finally screen out those with obvious resistance to fungi. Enhanced transgenic plants.

The invention provides a new method for enhancing the resistance of plants to fungal diseases, and the cultivation of disease-resistant plants by means of genetic engineering can make up for the shortcomings of traditional breeding. The breeding cycle is short, the operation is simple, and high-resistant plants are easy to obtain. Introducing the LrGSTL1 gene from Minjiang Lily into tobacco can produce new materials and new varieties with antifungal properties, and the transgenic tobacco has strong antifungal activity. The transgenic tobacco overexpressing LrGSTL1 had obvious resistance to the infection of Fusarium oxysporum and Botrytis cinerea. The method of using genetic engineering technology to breed resistant plant varieties to prevent and treat plant diseases has significant advantages and irreplaceable importance. It can provide convenience for large-scale production of crops, flowers, etc., greatly reduces the use of chemical pesticides, reduces environmental pollution, and can also save costs for agricultural production, so the invention has broad market application prospects.

Description of drawings

Fig. 1 is the PCR detection result of partial LrGSTL1 transgenic tobacco genome DNA in the present invention, wherein Marker: DL2000DNAMarker (Dalian treasure biology), is made up of 2,000bp, 1,000bp, 750bp, 500bp, 250bp and 100bp six DNA fragments, positive control: with PCR product of plasmid pMD-18T- LrGSTL1 as template; WT: PCR product of non-transgenic tobacco (wild type) total DNA as template;

Fig. 2 is the expression analysis result of LrGSTL1 transcript level in positive LrGSTL1 transgenic tobacco in the present invention, wherein Marker: DL2000DNAMarker (Dalian treasure biology); WT: non-transgenic tobacco total RNA reverse transcription cDNA is the PCR product of template; Positive control: plasmid pMD -18T- LrGSTL1 is the PCR product of the template; the rest of the lanes are different positive LrGSTL1 transgenic tobacco individual plants;

Fig. 3 is the resistance analysis result after LrGSTL1 transgenic tobacco leaf and WT leaf inoculation fungus in the present invention, wherein the fungi inoculated in figure a, b are Botrytis cinerea and Fusarium oxysporum respectively; L-1, L-5, L-8, L-10, L-13 are five different LrGSTL1 transgenic tobacco individual plants, WT is a non-transgenic tobacco individual plant.

detailed description

The present invention will be described in further detail below by the examples, but the content of the present invention is not limited thereto, the methods in the present embodiment are all operated according to conventional methods if there is no special instructions, and the reagents used are conventional reagents if there are no special instructions or according to Reagents configured by conventional methods.

Example 1: Cloning and sequence analysis of the full-length LrGSTL1 gene

Lilium Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 hours after inoculation. Grind the treated root of Lilium Minjiang into powder with liquid nitrogen, transfer it to a centrifuge tube, extract total RNA by using guanidine isothiocyanate method, and use reverse transcriptase M-MLV (promega) to synthesize cDNA using total RNA as a template For the first strand, the reaction system and operation process are as follows: take 5 μg TotalRNA, add 50ngoligo(dT)15, 2 μL dNTP (2.5mMeach), and DEPC water in sequence until the reaction volume is 13.5 μL; Cool down for 5 minutes, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U) in sequence, mix well and centrifuge for a short time, incubate at 42 °C for 1.5 h, take it out and heat at 70 °C for 10 min to terminate the reaction, cDNA Store at -20°C after first-strand synthesis for future use.

Using the synthesized first-strand cDNA as a template, the target gene LrGSTL1 was amplified, and the upstream and downstream primer sequences used were 5'CAAGCAGTGGTATCAACGCAGAGT3' and 5'CACCACCTGTGAGACGATGAAGA3'. The target gene was amplified by Advantage ^TM 2PCEnzyme (Clontech). PCR reaction conditions: 94°C for 2min; 94°C for 30s, 62°C for 30s, 72°C for 1min, 30cycles; 72°C for 5min. The reaction system (20 μL) was 1 μL of first-strand cDNA, 2 μL of 10×Buffer, 0.5 μL of dNTP (10 mMeach), 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 0.25 μL of Advantage ^TM 2PCEnzyme, and 15.65 μL of PCR-GradeWater. After PCR, 5 μL was used for agarose gel electrophoresis to detect the specificity and size of the amplified product.

Gel electrophoresis showed that the PCR product was a single specific band, so the PCR product was directly cloned by TA. The kit used was pMD18-Tvectorkit (Dalian Bao Biology). 1 μL of pMD-18Tvector (50ng/μL) and 2.5 μL of 2×LigationsolutionI were mixed and placed at 16°C for overnight reaction. The ligation product was transformed into Escherichia coli DH5α by heat shock transformation method. Use LB solid medium containing X-Gal, IPTG, and ampicillin (ampicillin, Amp) to screen positive clones, select several white colonies, and use specific primers for amplifying LrGSL1 after shaking to identify clones with multiple cloning sites inserted into LrGSTL1 . The identified clones were sequenced, and the final obtained LrGSTL1 full-length cDNA was 1076bp, which was found to contain a 717bp ORF (see sequence listing). LrGSTL1 encodes a protein containing 238 amino acids, the molecular weight of LrGSTL1 is about 27.28KDa, and the isoelectric point is 5.21. SignalP3.0 analysis results showed that there was no signal peptide in LrGSTL1 , and the protein localization prediction results showed that LrGSTL1 might be located in chloroplast.

Embodiment 2: plant expression vector construction

The Escherichia coli plasmid pMD-18T- LrGSTL1 inserted into LrGSTL1 and the plasmid of the plant expression vector pCAMBIA2300S were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong), and 1 μL was used for agarose gel electrophoresis to detect the extracted Plasmid integrity and concentration. The plasmids pMD-18T- LrGSTL1 and pCAMBIA2300S were digested with Eco RI (TaKaRa) and Pst I (TaKaRa) respectively (100 μL system) . Mix 10 μL 10×Hbuffer, 5 μL EcoR I, 5 μL Pst I, 60 μL ddH ₂ O, centrifuge for a short time, and place at 37°C overnight for reaction. All digested products were spotted on agarose gel for electrophoresis, and then the LrGSTL1 fragment and the pCAMBIA2300S large fragment were gel-recovered separately. The SanPrep column DNA gel recovery kit (Shanghai Sangong) was used for the whole process. Take 1 μL of the recovered product and detect the size and concentration of the recovered fragments by agarose gel electrophoresis.

Use T4DNALigase (TaKaRa) to connect the recovered LrGSTL1 DNA fragment and the pCAMBIA2300S carrier fragment. The reaction system (20 μL) and the operation process are as follows: take 10 μL LrGSTL1 gene fragment and add 2 μL pCAMBIA2300S carrier DNA, 2 μL 10×T4DNALigaseBuffer, 1 μL T4DNALigase, 5 μL ddH ₂ O, After mixing, centrifuge for a short time and place in a metal bath at 16°C for overnight reaction. Then, the ligation product was transformed into Escherichia coli DH5α by heat shock transformation method, and positive clones were screened with solid medium containing 50 mg/L kanamycin (Km). Select a single colony and shake the bacteria, and use the bacterial liquid as a template to carry out PCR with the specific primers for amplifying LrGSTL1 , and select the clone that successfully connects LrGSTL1 and pCAMBIA2300S. If the detected strain is positive, add 20% glycerol and mix well and place Store at ℃ for later use.

The pCAMBIA2300S -LrGSTL1 plasmid in the above Escherichia coli was extracted and purified with the SanPrep Column Plasmid Extraction Kit (Shanghai Sangong). Competent cells of Agrobacterium LBA4404 strain were prepared and divided into 1.5mL centrifuge tubes, 200μL per tube, and stored at -80°C after quick freezing in liquid nitrogen. The plant expression vector pCAMBIA2300S -LrGSTL1 constructed above was transformed into Agrobacterium LBA4404 competent cells by liquid nitrogen freeze-thaw method. The operation steps are as follows: take 200ngpCAMBIA2300S- LrGSTL1 plasmid and add it to a centrifuge tube containing 200μL competent cells, mix gently, then ice bath for 5min, then transfer to liquid nitrogen and freeze for 1min, then quickly place it in a water bath at 37°C for 5min, then immediately freeze After bathing for 2 minutes, add 800 μL LB liquid medium, and culture with shaking at 28°C for 4 hours. Spread the activated Agrobacterium on the LB solid medium containing 50mg/LKm, and culture it statically at 28°C. Select the monoclonal shaking bacteria, carry out PCR with specific primers for amplifying LrGSTL1 , and detect whether pCAMBIA2300S -LrGSTL1 is transferred into Agrobacterium. For positive clones, add glycerol and store at -80°C for later use.

Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

The transgenic recipient in this experiment is tobacco. Soak the tobacco seeds in 75% alcohol for 30 seconds, wash them with sterile water, disinfect them with 0.1% HgCl ₂ for 8 minutes, and then wash them several times with sterile water. On 2MS medium, culture in dark at 28°C for 5-8d, after germination, transfer to light incubator (25°C, 16h/d light), and subculture once a month with 1/2MS medium.

Take out the preserved Agrobacterium LBA4404 strain containing the pCAMBIA2300S -LrGSTL1 plasmid from the -80°C refrigerator, inoculate it in 5 mL of LB liquid medium containing 50 mg/L Km and 20 mg/L rifampicin, and culture it at 28°C until cloudy. Pipette 1mL of turbid bacterial solution onto LB solid medium containing 50mg/LKm, and incubate at 28°C for 48h. Scrape off an appropriate amount of Agrobacterium on the LB solid medium and inoculate it in MGL liquid medium containing 20 mg/L acetosyringone, shake and culture at 28°C for 2-3 hours to activate the Agrobacterium.

Take the young leaves of sterile tobacco seedlings and cut them into leaf discs of about 1 cm ² , soak them completely in the above-mentioned MGL liquid medium containing activated Agrobacterium, and dip for 15 minutes. Blot the bacterial solution on the surface of the leaf discs with sterile filter paper, arrange the leaf discs on the tobacco transformation co-culture medium and culture them in the dark at 22°C for 2 days. The co-culture medium is MS+0.02mg/L6-BA+2.1mg/LNAA+30g/L sucrose+6g/L agar.

The co-cultured leaf discs were transferred to the MS selection medium added with antibiotics to differentiate into seedlings, and the transgenic plants were screened at the same time. The selection medium is MS+0.5mg/L6-BA+0.1mg/LNAA+30g/L sucrose+6g/L agar+50mg/LKm+200mg/L cephalosporin (cefotaximesodiumsalt, Cef); Transfer to light incubator (25°C, 16h/d light, 8h/d dark). After leaf disks germinated, they were subcultured with MS medium containing 50mg/LKm and 200mg/LCef. Because of the high callus differentiation rate of tobacco, further screening of regenerated plants was required, and regenerated tobacco seedlings were moved to medium containing 50mg/LKm. Make it root on the MS medium, and finally select the regenerated shoots with better rooting to carry out the following detection.

The genomic DNA of leaves of transgenic tobacco plants was extracted by CTAB method, and 1 μL of genomic DNA was tested for its integrity and concentration by agarose gel electrophoresis. Using the genomic DNA of the transgenic plants as a template, PCR was performed with specific primers for amplifying LrGSTL1 . After PCR, 8 μL of the product was used for agarose gel electrophoresis to detect positive transgenic plants. The amplification results of some transgenic tobacco plants are shown in Fig. 1, and a total of 62 positive transgenic plants were screened from LrGSTL1 transformed tobacco.

Example 4: Expression Analysis of LrGSTL1 in Transgenic Tobacco and Analysis of Antifungal Activity of Transgenic Plants

Randomly select 30 positive transgenic plants and one non-transgenic tobacco plant (wild type, WT), take young leaves and extract total RNA, reverse transcribe to generate first-strand cDNA, and use this as a template to amplify LrGSTL1 with specific primers Perform PCR, and analyze the expression of LrGSTL1 transcript level in each transgenic individual plant according to RT-PCR results. The method and steps of total RNA extraction and RT-PCR were the same as in Example 1. After PCR, 8 μL was used for agarose gel electrophoresis. The detection results of some individual plants are shown in FIG. 2 . A total of 16 transgenic individual plants were detected to express LrGSTL1 at the transcriptional level, and these plants were numbered 1-16 respectively.

Several pathogenic fungi preserved in the laboratory were inoculated on PDA solid medium (200g/L potato, 15g/L agar, 20g/L glucose), cultured in the dark at 28°C for 6 days, and the conidia were removed from the After being eluted from the culture medium, the conidia suspension was diluted to a concentration of 10 ⁶ spores·mL ^-1 after counting on a hemocytometer, and used to inoculate tobacco leaves to analyze the antifungal activity of transgenic plants. There are 7 species of fungi tested: Botrosphaeriadothidea , Fusarium oxysporum, Phomopsis sp., Alternaria sp., Botrytis cinerea ( Botrytiscinerea ), Colletorichum gloeosporioides , Gibberella moniliformis . Take young leaves of different transgenic lines and WT, inoculate 20 μL of spore suspension after injury treatment, spread the inoculated tobacco leaves on wet filter paper, place them in an incubator at 28°C for 3-5 days, and then observe the fungal inoculation Tobacco leaf disease degree, and based on this to evaluate the antifungal activity of LrGSTl1 transgenic tobacco. The results are shown in Figure 3, LrGSTL1 transgenic tobacco has obvious inhibitory effect on the infection of Botrytis cinerea and Fusarium oxysporum.

Sequence listing (SEQID)

<110> Kunming University of Science and Technology

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Claims (3)

1. a lilium regale wilson glutathione S-transferase gene lrGSTL1, it is characterized in that: its nucleotide sequence as shown in SEQIDNO:1, the protein of as shown in the SEQIDNO:2 aminoacid sequence of encoding.

2. lilium regale wilson glutathione S-transferase gene described in claim 1 lrGSTL1raising tobacco to the application in Fusarium oxysporum, Botrytis cinerea resistance.

3. lilium regale wilson glutathione S-transferase gene according to claim 2 lrGSTL1application, it is characterized in that the concrete operations of fungal resistance improving tobacco are as follows:

(1) by above-mentioned glutathione S-transferase gene lrGSTL1be connected with plant overexpression vector pCAMBIA2300S, build recombinant vectors;

(2) recombinant vectors of above-mentioned structure is proceeded in tobacco by Agrobacterium tumefaciens mediated;

(3) screen transformant by kantlex, and adopt amplification lrGSTL1special primer carry out polymerase chain reaction and obtain real transfer-gen plant, get the blade inoculation fungi of different transgenic line, and detect the occurring degree of transgenic tobacco leaf, finally filter out the transfer-gen plant that fungus resistant is obviously strengthened.