Application of MsNRAMP2 protein from alfalfa in inhibiting tobacco mosaic virus

Abstract

The invention discloses the application of alfalfa MsNRAMP2 protein in inhibiting tobacco mosaic virus, and belongs to the technical field of plant breeding. In order to effectively inhibit TMV disease of plants, the invention constructs alfalfa NRAMP2 gene as a plasmid of a plant gene expression vector, transforms the plasmid into Agrobacterium, and infects to obtain transgenic tobacco. Expressing MsNRAMP2 gene in wild-type tobacco can improve the disease resistance of tobacco, and the disease resistance is manifested in that the degree of leaf shrinkage and the degree of disease incidence are lighter than those of wild-type tobacco. MsNRAMP2 can improve the disease resistance of tobacco. The invention provides a new disease-resistant breeding gene resource.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to application of alfalfa MsNRAMP2 protein in inhibiting tobacco mosaic virus. Background Art

[0002] Tobacco mosaic virus (TMV) has been a focus of attention since it was discovered in 1883. TMV has a wide host range and can infect more than 30 families, including Cruciferae, Solanaceae, Leguminosae, Asteraceae, and more than 300 plant species. The diseases caused by it seriously affect the quality and yield of crops, causing significant economic losses. The incidence of TMV in tobacco fields is high. When tobacco is infected in the seedling stage, it will not only be dwarfed, but also affect its normal growth, resulting in economic losses of up to 50-70%. So far, very effective prevention and control methods have not been developed, so finding new environmentally friendly antiviral biopesticides is one of the prevention and control strategies. Antiviral proteins are also biopesticides, which can not only be directly developed and applied commercially, but also can be expressed in crops for a long time through transgenic technology to produce activity.

[0003] Leaf wrinkling is a typical feature of TMV disease. The current control method mainly includes chemical control. Chemical agents are quick, convenient, and effective. They have been widely used in production and have achieved good control effects in practice. However, chemical control pesticide residues are serious, which can easily cause poisoning in humans and animals, and can easily cause gene mutations and produce strong drug resistance, making control work increasingly difficult. Summary of the invention

[0004] The purpose of the present invention is to effectively inhibit TMV disease of plants.

[0005] The invention provides an alfalfa MsNRAMP2 protein, and the amino acid sequence of the alfalfa MsNRAMP2 protein is shown as SEQ ID NO.2.

[0006] The present invention provides an application of the above-mentioned alfalfa MsNRAMP2 protein or the gene of the above-mentioned alfalfa MsNRAMP2 protein in inhibiting tobacco mosaic virus.

[0007] It is further defined that the plant is tobacco.

[0008] It is further defined that the gene sequence encoding the above-mentioned alfalfa MsNRAMP2 protein is shown as SEQ ID NO.1.

[0009] The present invention provides a recombinant vector, which carries the gene sequence shown in SEQ ID NO.1.

[0010] It is further defined that the starting vector of the recombinant vector is pBI121.

[0011] The present invention provides a method for constructing the above-mentioned recombinant vector, characterized in that the specific steps of the construction method are as follows:

[0012] Step 1: The pBI121 vector was digested with Xba I and Sma I to obtain a linear vector, and the pMD18T-MsNRAMP2 vector was digested with Xba I and Sma I to obtain a gene fragment;

[0013] Step 2: Connect the linear vector obtained in step 1 to the gene fragment to obtain a recombinant vector.

[0014] The present invention provides a method for breeding plants that inhibit tobacco mosaic virus, and the specific steps of the breeding method are as follows:

[0015] Step 1: Connect the gene sequence shown in SEQ ID NO.1 with the pBI121 vector to obtain a recombinant vector;

[0016] Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0017] Step 3: Infect plants with the recombinant Agrobacterium obtained in step 2 to obtain transgenic plants.

[0018] It is further defined that the primers for cloning the gene sequence shown in SEQ ID NO.1 in step 1 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0019] It is further defined that in step 3, the transgenic plants are obtained by the Agrobacterium-mediated leaf disc method.

[0020] Beneficial effects: The transgenic tobacco obtained by the Agrobacterium-mediated leaf disc method of the present invention has tall plants and large leaves. All tobaccos are grown under conditions of sufficient water and sufficient light. After inoculation with TMV, the degree of leaf shrinkage and disease severity is lighter than that of wild-type tobacco. Tobacco not inoculated with TMV under the same growth conditions has no leaf shrinkage, indicating that MsNRAMP2 can enhance the disease resistance of tobacco. Transgenic tobacco not inoculated with TMV under the same growth conditions has no leaf shrinkage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the pBI121 vector map.

[0022] Figure 2 This is an evolutionary tree diagram.

[0023] Figure 3The pBI121-MsNRAMP2 recombinant plasmid was identified by double restriction digestion with Xba I and Sma I.

[0024] Figure 4 This is a diagram of obtaining the resistant tobacco plants transferred with the MsNRAMP2 gene in Example 1 of the present invention; wherein A is callus tissue, B is Km-resistant buds, C is the rooting culture of resistant tobacco buds, and D is rooted tobacco.

[0025] Figure 5 This is a diagram of some PCR identification results in Example 1 of the present invention; wherein M is DL2000, + is the positive control, and 1-9 are the transgenic tobacco tested.

[0026] Figure 6 Leaf phenotypes of transgenic and wild-type tobacco after inoculation with TMV. DETAILED DESCRIPTION

[0027] Example 1. Construction of recombinant vector

[0028] 1. Use PCR technology to clone the MsNRAMP2 gene from alfalfa:

[0029] 1. Extraction of RNA from alfalfa:

[0030] (1) Place 0.1 g of whole alfalfa in a mortar pre-cooled with liquid nitrogen and grind into fine powder.

[0031] (2) Transfer the powder to a 1.5 mL sterile centrifuge tube and add 450 μL RL (confirm whether β-mercaptoethanol is added).

[0032] (3) Transfer all liquid to the filter column, centrifuge at 12000 rpm for 5 min, and carefully pipette the supernatant into a new RNase-Free centrifuge tube.

[0033] (4) Slowly add 0.5 times the volume of the supernatant anhydrous ethanol, mix well, transfer the resulting solution and precipitate into the adsorption column, centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0034] (5) Add 350 μL of deproteinized solution to the adsorption column, centrifuge at 12,000 rpm for 60 s, discard the filtrate, and return the adsorption column to the collection tube.

[0035] (6) Prepare DNase working solution: add 70 μL RDD solution to 10 μL DNase I reaction solution and mix gently.

[0036] (7) Add 80 μL of DNase working solution to the adsorption column filter and let it stand for 15 min.

[0037] (8) Add 350 μL of deproteinized solution to the centrifugal adsorption column, centrifuge at 12,000 rpm for 60 s, discard the filtrate, and return the adsorption column to the collection tube.

[0038] (9) Add 300 μL of rinse solution to the centrifugal adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 60 s, discard the filtrate, and return the adsorption column to the collection tube.

[0039] (10) Repeat step 9.

[0040] (11) Centrifuge at 12000 rpm for 2 min, discard the filtrate, and place the adsorption column at room temperature for 5 min to completely dry the rinsing solution remaining in the adsorption column.

[0041] (12) Transfer the centrifugal adsorption column to an RNase-free collection tube, add 30 μL RNA elution buffer and let stand for 1-2 min. Centrifuge at 12000 rpm for 2 min. The collection tube contains the RNA sample and is stored at -80°C.

[0042] 2. Synthesis of cDNA

[0043] Using the extracted RNA as a template, cDNA was obtained using the reverse transcription PCR method. The reaction process is as follows:

[0044] (1) Preparation of reaction solution I:

[0045]

[0046] (2) Mix the reaction solution I and place it in a 65°C water bath for 5 min. After the water bath, quickly place it in ice.

[0047] (3) Preparation of reaction solution II:

[0048]

[0049]

[0050] Reaction procedure:

[0051] 30℃10min

[0052] 42℃20min

[0053] 99℃5min

[0054] 4℃5min

[0055] Amplify the target gene.

[0056] MsNRAMP2 target gene fragment, gene CDS sequence: (SEQ ID NO.1)

[0057]

[0058]

[0059] The amino acid sequence of MsNRAMP2 protein is shown in SEQ ID NO.2.

[0060]

[0061] 3. Primer sequence:

[0062] MsNRAMP2 F:5'-GCTCTAGAATGAGTGGTCCATCGC-3' (SEQ ID NO.3);

[0063] MsNRAMP2 R:5'-CCCCCGGGTTAATTCTCAGAGGAACAAAAT-3' (SEQ ID NO.4);

[0064] PCR amplification program: 94°C for 5 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min 30 s, and finally 72°C for 10 min; 1% agarose gel electrophoresis detection;

[0065] 4. Gel recovery of target gene bands: Use a DNA gel recovery kit to recover the target gene fragment MsNRAMP2 (Alfalfa NRAMP2) amplified by PCR;

[0066] 5. Construction of cloning vector pMD18T-MsNRAMP2: Connect the target gene fragment of MsNRAMP2 to the cloning vector pMD18T, and transfer the ligation product into competent E. coli using the heat shock method:

[0067] After taking out the competent E. coli from the -80°C refrigerator, quickly put it on ice, add 10 μL of the ligation product after it melts, and place it on ice for 30 minutes; the reaction system is: 1 μL of pMD18-T vector, 4 μL of target gene fragment solution, and 5 μL of Solution I. The reaction conditions are: reaction temperature 16°C, reaction time more than 12 hours (overnight).

[0068] After taking out, quickly place in a 42℃ water bath for 1min 30s, then quickly insert into ice and place in ice bath for 2min. Add 450μLLB liquid culture medium, place in a shaker at 200rpm, 37℃, and shake for 1h;

[0069] Take out the centrifuge tube, centrifuge at 4000 rpm for 3 min, discard 350 μL of the supernatant, resuspend the remaining bacteria, and spread them on a plate of solid LB medium containing Amp;

[0070] Incubate the cells upside down in a 37°C incubator overnight; pick a single colony on the culture medium for identification;

[0071] 6. Sequencing and bioinformatics analysis of MsNRAMP2 gene:

[0072] (1) Use the software ORF Finder to predict the open reading frame of the MsNRAMP2 gene.

[0073] (2) The theoretical isoelectric point of MsNRAMP2 protein was analyzed using ProtParam (http: / / www.expasy.org / tools / protparam.html) software.

[0074] (3) The hydrophilicity and hydrophobicity of MsNRAMP2 protein were analyzed using the online analysis software Protscale.

[0075] (4) Use SOPMA software to predict and analyze the secondary structure domain of MsNRAMP2 protein.

[0076] (http: / / nhjy.hzau.edu.cn / kech / swxxx / jakj / dianzi / Bioinf7 / Expasy / Expasy8.htm).

[0077] (5) Prediction and analysis of the transmembrane structure of MsNRAMP2 protein The transmembrane domain of MsNRAMP2 protein was analyzed using TMHMM 2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / TMHMM2.0b.guide.php)(http: / / swissmodel.expasy.org / ) and the tertiary structure of MsNRAMP2 protein was analyzed.

[0078] (5) Comparison of protein sequences by NCBI blast: The protein sequences of NRAMPs from other species such as Medicagotruncatula, Cicer arietinum, Arabidopsis thaliana, and Abrus precatorius were obtained from NCBI and compared using ClustalX2 and MEGA6 software to construct a phylogenetic tree. The results are shown in Figure 2. Figure 2 shown.

[0079] 2. Construction of plant gene expression vector pBI121-MsNRAMP2 plasmid:

[0080] 1. Extract the plasmids of pMD18T-MsNRAMP2 and pBI121: Extract the plasmids of pMD18T-MsNRAMP2 and pBI121 using the AXYGEN kit;

[0081] 2. Enzyme digestion of pMD18T-MsNRAMP2 and pBI121 plasmids:

[0082] (1) Xba I and Sma I were used to double-digest the plasmids of pBI121 and pMD18T-MsNRAMP2. The reaction system of double-digestion was: 70 μL of pBI121 and pMD18T-MsNRAMP2 plasmids, 10 μL of 10×T Buffer, 3 μL of Xba I, 3 μL of Sma I, 10 μL of BSA and 4 μL of ddH2O;

[0083] (2) Recover the enzyme digestion products of the plasmids pMD18T-MsNRAMP2 and pBI121 using a DNA gel recovery kit;

[0084] (3) Ligation of the expression vector pBI121 and the target gene fragment MsNRAMP2. The reaction system during ligation is: 5 μL of pBI121 plasmid digestion product, 3 μL of MsNRAMP2 digestion product, 1 μL of 10×T4 DNA Ligase Buffer and 1 μL of T4 DNA Ligase; the reaction conditions are: reaction temperature 16°C, reaction time more than 12 h (overnight);

[0085] (4) Ligation product: pBI121-MsNRAMP2 is transformed into competent E. coli;

[0086] (5) Identification of recombinant plasmids:

[0087] 1) Colony PCR identification: PCR identification was performed using primers containing the MsNRAMP2 gene with Xba I and Sma I restriction sites. The reaction system was:

[0088] ddH2O 7.35μL, 10×PCR Buffer 1μL, dNTP Mix (2.5mmol / L) 0.8μL, MsNRAMP2-XbaI (10μmol / L) 0.4μL, MsNRAMP2-Sma I (10μmol / L) 0.4μL, rTaq DNA polymerase (5U / μL) 0.05μL, Template DNA single colony

[0089] Reaction procedure: (30 cycles)

[0090]

[0091] 2) Extract the recombinant pBI121-MsNRAMP2 plasmid for enzyme digestion verification. The reaction system for verification is: 30 μL of pBI121-MsNRAMP2 recombinant plasmid, 5 μL of 10×T Buffer, 5 μL of BSA, 2.5 μL of Xba I, 2.5 μL of Sma I, and 5 μL of ddH2O. The identification results are as follows: Figure 3 shown.

[0092] Example 2. A method for controlling leaf shrinkage in plant breeding

[0093] 1. Transform Agrobacterium using the pBI121-MsNRAMP2 plasmid obtained in Example 1:

[0094] 1) The recombinant plasmid pBI121-MsNRAMP2 was transformed by freeze-thaw method as follows:

[0095] ①Take the competent cells stored at -80℃ and place them on ice to melt.

[0096] ② Add 10 μL of pBI121-MsNRAMP2 recombinant plasmid to 100 μL of Agrobacterium competent cells GV3101 and mix by hand at the bottom of the tube.

[0097] ③ Place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0098] ④ Add 700 μL of YEB liquid culture medium without antibiotics and culture at 28°C with shaking for 2-3 hours.

[0099] ⑤ Collect the bacteria by centrifugation at 6000 rpm for 1 min, take 100 μL of supernatant to resuspend the bacterial mass, spread the bacterial solution on YEB solid culture medium containing Km (50 mg / mL) + Rif (50 mg / mL), and place it upside down in a 28°C incubator for dark culture for 2-3 days.

[0100] pBI121-MsNRAMP2 plasmid was transformed into Agrobacterium;

[0101] The plant seedlings are co-cultured with the pBI121-MsNRAMP2 plasmid, and after the culture, bud induction, root induction and transplanting and domestication are performed to finally obtain transgenic plants with strong disease resistance.

[0102] The TMV-infected tobacco leaves were ground into juice in a mortar and then inoculated with transgenic MsNRAMP2 tobacco and wild-type tobacco for reproduction. The leaf phenotypes were observed after 15 days.

[0103] Identify the transformed Agrobacterium; the identification method is: pick a single colony with relatively uniform growth, and use gene-specific primers to perform colony PCR amplification; the reaction system for identification is: Template DNA single colony, 1μL of 10×Buffer, 0.8μL of dNTP Mix, 0.4μL of MsNRAMP2-F, 0.4μL of MsNRAMP2-R, 0.05μL of r Taq and 7.35μL of ddH2O; the reaction conditions are: ①94℃, 5min; ②94℃, 30s; ③55℃, 30s; ④72℃, 2min; ⑤72℃, 10min; ⑥4℃, 1h; ① to ⑥ are cycled 30 times;

[0104] 2. Co-cultivating plant seedlings with the pBI121-MsNRAMP2 plasmid, and then performing bud induction, root induction and transplanting and domestication to finally obtain transgenic plants with strong disease resistance.

[0105] 1) Aseptic culture of plant seedlings:

[0106] (1) Place wild-type tobacco seeds in a sterilized centrifuge tube, disinfect with 75% alcohol for 3 min, and rinse with sterile water 6-8 times.

[0107] (2) Treat with 1 mL of 10% sodium hypochlorite for 5 min and rinse with sterile water 6-8 times.

[0108] (3) Spread the treated seeds evenly on 1 / 2MS solid culture medium and culture them until seedlings grow.

[0109] (4) The seedlings were transferred into bottles containing MS solid culture medium and continued to be cultured to grow into large seedlings. The leaves were used for infection experiments.

[0110] 2) Cut tobacco leaves into squares of about 1 cm x 1 cm and pre-culture for 48 hours.

[0111] 3) Activating the Agrobacterium:

[0112] In a clean bench, pBI121-MsNRAMP2 positive bacteria were spread on YEB solid medium containing Km and Rif, inverted and cultured at 28°C for 48 hours, and then the bacterial suspension was resuspended in YEB liquid medium to obtain a bacterial solution, which was then diluted with YEB to an OD600 of about 0.4-0.6. The bacterial solution was transferred to a 50 mL conical flask for infecting tobacco.

[0113] Take out the pre-cultured leaves and infect them in the bacterial solution for 6 minutes, turning them upside down to ensure that the leaves are fully in contact with the bacterial solution. Take out the leaves and place them on sterile filter paper, absorb the attached bacterial solution, place the leaves with the back facing up on MS1 ​​culture medium, and culture them in the dark for 2 days.

[0114] 4) Bud induction:

[0115] The leaves were transferred to MS2 medium for bud induction, and the medium was changed every 3 days. After 3 consecutive times, the medium was changed every 2 weeks.

[0116] 5) Rooting induction:

[0117] When the differentiated buds grow to about 2 cm, they are cut off and transferred to MS3 medium for rooting culture.

[0118] 6) Transplantation and domestication:

[0119] The rooted tobacco seedlings were transferred to soil for cultivation.

[0120] The tobacco plants obtained after the above transformation are MsNRAMP2 gene-transformed tobacco resistant plants.

[0121] like Figure 4 As shown in the figure, pBI121-MsNRAMP2 positive bacteria were spread on YEB solid medium containing Km and Rif, and the bacterial solution was obtained after the colony was resuspended with liquid YEB, and then the bacterial solution was diluted with YEB to an OD600 of about 0.4-0.6. The MsNRAMP2 gene was genetically transformed into tobacco by the Agrobacterium-mediated leaf disc method, and resistant plants with the MsNRAMP2 gene were obtained. The infected leaves were first cultured in the dark on MS1 ​​for 2-3 days, and then moved to MS2 for screening. Callus tissue would grow around the leaves that were successfully infected, and then small buds would differentiate, while callus tissue and small buds would not grow around the leaves that were not successfully infected. The resistant buds were cut off and moved to MS3 medium for rooting culture. A total of 10 kanamycin-resistant plants were obtained, and 9 positive plants were found after PCR identification. The grown resistant plants were transplanted into pots containing soil: vermiculite at a ratio of 1:1.

[0122] PCR identification was performed on Km-resistant plants. Using pBI121-MsNRAMP2 plasmid as a positive control, 9 of the plants were able to amplify fragments of about 500 bp that were consistent with the positive control fragment, which preliminarily proved that MsNRAMP2 had been transferred into tobacco. Some PCR identification results are shown in the figure below. Figure 5 shown.

[0123] The leaves of TMV (Tobacco Mosaic Virus) were ground into juice and rubbed with infected tobacco to reproduce. The leaf phenotypes and disease severity of transgenic tobacco and wild-type tobacco after inoculation with TMV were observed. After inoculation with TMV, the leaves of MsNRAMP2-transgenic tobacco showed less wrinkling and disease severity than wild-type tobacco, which proved that MsNRAMP2 can improve tobacco's disease resistance. Figure 6The disease grade is shown in Table 1 (the disease grade is graded according to the tobacco disease grade standard (GB / T23222)).

[0124] Table 1

[0125] Tobacco plants Disease grade (GB / T23222) WT 4 MsNRAMP2#1 2 MsNRAMP2#2 2 SEQUENCE LISTING <110> Harbin Normal University <120> Application of MsNRAMP2 protein from alfalfa in inhibiting tobacco mosaic virus <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1593 <212> DNA <213> Synthesis <400> 1 atgagtggtc catcgcgaga agataattca aaggaagagg taaaagaaga agatgaaggt 60 aaccggttac tggaagttga cggtgaaggt gaaggtgatt ctacatcgga agaagaagca 120 gcgtatgaat ccggtgagaa aatcgtggtg gtggatttcg aattcggcac cgtcgacgat 180 tcaaccgttc cacctttttc atggaagaag ctatggatgt tcacaggacc aggattcttg 240 atgagtatag catttttgga tccagggaat ttggaaggtg atctacaagc aggagcgatt 300 gcagggtatt cattgctatg gttgttgatg tgggctactt tgatgggatt gttgattcaa 360 tcgctatcgg cgagggttgg tgttgctact ggtcgtcatc ttgctgagct ttgtagagat 420 gagtatccga attgggcgag atatgtgctt tggtttatgg ctgaacttgc tttgattggt 480 gctgatattc aagaggttat tggtagtgct attgctattc agattcttag tcgtggtgtt 540 ttgcctcttt gggctggtgt tcttattact gcttctgatt gctttttctt tctatttctt 600 gagaactacg gagtgagaa gttggaagct gcttttgcag ttctcatagc taccatggct 660 ctttcgtttg cctggatgtt tggtgacgca caacctagtg gaaaagagct tgtgaggggt 720 attttgattc caagacttag ctcaaaaacg attcatcagg ctgtgggcgt tgtgggttgt 780 gtaattatgc ctcacaatgt atttctacat tctgctttag tgcagtcaag gaagattgat 840 ccccataaga aaggtcgggt gcaagaggct cttaattact samaattga gtccacagtt 900 gcactttcag tcaccttcat gatcaatctg tttgtcacaa cagtttttgc taagggtttt 960 1020 aaatatggag gaggactctt ccctattctt tatatatggg gaattgggtt actggcggcc 1080 ggaaagga gcactatcac tggcacatat gctgggcaat tcataatggg gggtttccta 1140 aaccttcgat taaagaaatg gttgagagca ttgatcactc ggagttgtgc aattgtgcca 1200 actataattg tagctattgt ttataacagg tcagaagcct cattggatgt tttgaatgaa 1260 tggctcaatg tgcttcagtc catacaaatt ccctttgcat tgattcccct tctcacatta 1320 gtctccaaag agcgtatcat ggggtccttt aaggttgggc ctgttcttga gagggtggca 1380 tggacagttg ctgcactgat aatagtaatt aatgggtatc tcttggtaga tttcttcctt 1440 tctgaggtga acagcgtatt gtttggcttt gtagcctgct cttgcacagt tgcatacatt 1500 gcattcattg tatatttaat ttcacaaagt ggtgctcttc cttccgcatt ggtggatcga 1560 cttccaaagg gattttgttc ctctgagaat taa 1593 <210> 2 <211> 530 <212> PRT <213> Synthetic <400> 2 Met Ser Gly Pro Ser Arg Glu Asp Asn Ser Lys Glu Glu Val Lys Glu 1 5 10 15 Glu Asp Glu Gly Asn Arg Leu Leu Glu Val Asp Gly Glu Gly Glu Gly 20 25 30 Asp Ser Thr Ser Glu Glu Glu Ala Ala Tyr Glu Ser Gly Glu Lys Ile 35 40 45 Val Val Val Asp Phe Glu Phe Gly Thr Val Asp Asp Ser Thr Val Pro 50 55 60 Pro Phe Ser Trp Lys Lys Leu Trp Met Phe Thr Gly Pro Gly Phe Leu 65 70 75 80 Met Ser Ile Ala Phe Leu Asp Pro Gly Asn Leu Glu Gly Asp Leu Gln 85 90 95 Ala Gly Ala Ile Ala Gly Tyr Ser Leu Leu Trp Leu Leu Met Trp Ala 100 105 110 Thr Leu Met Gly Leu Leu Ile Gln Ser Leu Ser Ala Arg Val Gly Val 115 120 125 Ala Thr Gly Arg His Leu Ala Glu Leu Cys Arg Asp Glu Tyr Pro Asn 130 135 140 Trp Ala Arg Tyr Val Leu Trp Phe Met Ala Glu Leu Ala Leu Ile Gly 145 150 155 160 Ala Asp Ile Gln Glu Val Ile Gly Ser Ala Ile Ala Ile Gln Ile Leu 165 170 175 Ser Arg Gly Val Leu Pro Leu Trp Ala Gly Val Leu Ile Thr Ala Ser 180 185 190 Asp Cys Phe Phe Phe Leu Phe Leu Glu Asn Tyr Gly Val Arg Lys Leu 195 200 205 Glu Ala Ala Phe Ala Val Leu Ile Ala Thr Met Ala Leu Ser Phe Ala 210 215 220 Trp Met Phe Gly Asp Ala Gln Pro Ser Gly Lys Glu Leu Val Arg Gly 225 230 235 240 Ile Leu Ile Pro Arg Leu Ser Ser Lys Thr Ile His Gln Ala Val Gly 245 250 255 Val Val Gly Cys Val Ile Met Pro His Asn Val Phe Leu His Ser Ala 260 265 270 Leu Val Gln Ser Arg Lys Ile Asp Pro His Lys Lys Gly Arg Val Gln 275 280 285 Glu Ala Leu Asn Tyr Tyr Asn Ile Glu Ser Thr Val Ala Leu Ser Val 290 295 300 Thr Phe Met Ile Asn Leu Phe Val Thr Thr Val Phe Ala Lys Gly Phe 305 310 315 320 Tyr Gly Thr Lys Gln Ala Asn Ser Ile Gly Leu Val Asn Ala Gly Gln 325 330 335 Tyr Leu Glu Glu Lys Tyr Gly Gly Gly Leu Phe Pro Ile Leu Tyr Ile 340 345 350 Trp Gly Ile Gly Leu Leu Ala Ala Gly Gln Ser Ser Thr Ile Thr Gly 355 360 365 Thr Tyr Ala Gly Gln Phe Ile Met Gly Gly Phe Leu Asn Leu Arg Leu 370 375 380 Lys Lys Trp Leu Arg Ala Leu Ile Thr Arg Ser Cys Ala Ile Val Pro 385 390 395 400 Thr Ile Ile Val Ala Ile Val Tyr Asn Arg Ser Glu Ala Ser Leu Asp 405 410 415 Val Leu Asn Glu Trp Leu Asn Val Leu Gln Ser Ile Gln Ile Pro Phe 420 425 430 Ala Leu Ile Pro Leu Leu Thr Leu Val Ser Lys Glu Arg Ile Met Gly 435 440 445 Ser Phe Lys Val Gly Pro Val Leu Glu Arg Val Ala Trp Thr Val Ala 450 455 460 Ala Leu Ile Ile Val Ile Asn Gly Tyr Leu Leu Val Asp Phe Phe Leu 465 470 475 480 Ser Glu Val Asn Ser Val Leu Phe Gly Phe Val Ala Cys Ser Cys Thr 485 490 495 Val Ala Tyr Ile Ala Phe Ile Val Tyr Leu Ile Ser Gln Ser Gly Ala 500 505 510 Leu Pro Ser Ala Leu Val Asp Arg Leu Pro Lys Gly Phe Cys Ser Ser 515 520 525 Glu Asn 530 <210> 3 <211> 24 <212> DNA <213> Synthetic <400> 3 gctctagaat gagtggtcca tcgc 24 <210> 4 <211> 30 <212> DNA <213> Synthetic <400> 4 cccccgggtt aattctcaga ggaacaaaat 30

Claims

1. Use of alfalfa MsNRAMP2 protein or a gene encoding alfalfa MsNRAMP2 protein in improving tobacco mosaic virus resistance, characterized in that: The amino acid sequence of the alfalfa MsNRAMP2 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The gene sequence encoding the alfalfa MsNRAMP2 protein according to claim 1 is shown as SEQ ID NO.

1.

3. A method for breeding tobacco resistant to tobacco mosaic virus, characterized in that: The specific steps of the breeding method are as follows: Step 1: Connect the gene sequence shown in SEQ ID NO.1 with the pBI121 vector to obtain a recombinant vector; Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Infect tobacco with the recombinant Agrobacterium obtained in step 2 to obtain transgenic tobacco.

4. The breeding method according to claim 3, characterized in that The primers for cloning the gene sequence shown in SEQ ID NO.1 in step 1 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. The breeding method according to claim 3, characterized in that: In step 3, transgenic tobacco is obtained by the Agrobacterium-mediated leaf disc method.

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