CN109627303B

CN109627303B - Gene of pseudo-ginseng disease course related protein PnPR3 and application thereof

Info

Publication number: CN109627303B
Application number: CN201811509493.6A
Authority: CN
Inventors: 李昆志; 肖舒卉; 杨丹; 陈丽梅; 徐慧妮
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2018-12-11
Filing date: 2018-12-11
Publication date: 2021-11-12
Anticipated expiration: 2038-12-11
Also published as: CN109627303A

Abstract

The invention discloses a gene of a pseudo-ginseng disease course related protein PnPR3, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the gene codes a protein of an amino acid sequence shown as SEQ ID NO. 2; according to the invention, the exogenous gene PnPR3 is transferred into the tobacco to be expressed in the tobacco, and experiments show that the overexpression of the PnPR3 gene has a certain inhibiting effect on the typical fusarium solani of the root rot pathogen; the gene can be transferred into plants to improve the disease resistance of the plants, can improve the resistance of tobacco to fusarium solani which is a pathogenic bacterium of root rot, lays a good working foundation for disease resistance breeding of the root rot plants, and has higher economic benefit.

Description

Gene of pseudo-ginseng disease course related protein PnPR3 and application thereof

Technical Field

The invention relates to a coding gene of a pseudo-ginseng disease course related protein PnPR3 and application thereof, and the coding gene comprises a pseudo-ginseng disease course related protein genePnPR3And the coded protein, the carrier containing the gene and the plant cell line.

Background

After the panax notoginseng is infected by pathogenic bacteria, a series of genes in the plant respond to and up-regulate expression, wherein some genes are related to defense reaction and comprise a disease course related Protein (PR) and a bacteriostatic gene, and the disease resistance is improved by enhancing the protein activity of the genes; PR proteins are thought to be induced by pathogenic and abiotic stresses; based on the structure and function of PR proteins, PR proteins are currently found in monocotyledons and dicotyledons and 17 families have been identified. Among these PR proteins, PnPR3 protein is involved in biological processes such as carbohydrate metabolism, chitin catalysis, and cell wall macromolecule catalysis, and has molecular functions such as chitinase activity and chitin-binding activity.

Notoginseng (radix Notoginseng)Panax notoginseng) The panax notoginseng continuous cropping soil is a unique traditional rare traditional Chinese medicinal material in China, and the problem of continuous cropping obstacle mainly exists in large-scale planting for many years, and is severely limited by diseases such as root rot, powdery mildew and round spot, wherein the root rot is taken as a main disease, the annual incidence rate is 5% -20%, the panax notoginseng continuous cropping soil is planted more than 80% seriously, even the panax notoginseng continuous cropping soil is completely harvested, the root rot can be caused after the root rot is ill, the overground part is withered and yellow, and the yield and the medicinal material quality of panax notoginseng are seriously affected. At present, the control means of root rot mainly comprises the application of a large amount of antibacterial pesticides, and the harmless control of root rot becomes a hotspot and difficult problem of the research of the panax notoginseng cultivation technology. The pathogenic bacteria of the panax notoginseng root rot disease are complex and generally complex infection, the pathogenic bacteria include pseudomonas sp in bacteria, Fusarium sp in fungi, f.oxysporum schlecht, f.scirpi lamb, etc., and one of the pseudomonas sp, Fusarium sp or pseudomonas sp is generally mainly used, and the complex infection of several pathogenic bacteria accelerates root rot. The influence of root rot on the yield of panax notoginseng is one of the major bottlenecks of the current panax notoginseng planting industry.

The existing method for preventing and treating the root rot mainly applies a large amount of chemical pesticides, so that the quality and the safety of medicinal materials are influenced to a certain degree; no report related to the present invention is found at present.

Disclosure of Invention

The invention aims to provide a disease course related protein gene obtained by cloning pseudo-ginsengPnPR3The nucleotide sequence is shown as SEQ ID NO. 1, the full-length sequence of the gene cDNA is 918bp, and the gene cDNA encodes the protein of the amino acid sequence shown as SEQ ID NO. 2.

The pseudo-ginseng disease course related protein PnPR3 can also be a protein which is related to plant disease resistance and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid residue sequence in a sequence table.

The invention also aims to apply the gene of the pseudo-ginseng disease course related protein PnPR3 to the improvement of plant disease resistance.

The panax notoginseng disease course related protein PnPR3 gene can be constructed into the existing prokaryotic expression vector or plant expression vector by the existing method, any one of promoters including a constitutive promoter, an enhanced promoter, an inducible promoter, a tissue specific promoter and a development stage special promoter can be added in front of the transcription initiation nucleotide, and in order to facilitate the identification and screening of the PnPR3 gene cells or plants, the used vector can be processed, such as adding an antibiotic marker with resistance (ampicillin, gentamycin, kanamycin and the like) or adding a plant selectable marker (GUS gene, luciferase gene and the like). The transformed plant host can be monocotyledons or dicotyledons, such as rice, wheat, corn, cucumber, tobacco and the like; the expression vector carrying the PnPR3 gene of the present invention can be used to transform plant cells or tissues by applying a plant viral vector, direct DNA transformation, Agrobacterium mediation or other conventional biological methods, and the transformed plants can be cultured into plants through tissues, thereby obtaining plants with improved disease resistance.

The invention transfers exogenous gene PnPR3 into tobacco to express the gene in the tobacco, and experiments show that the overexpression of the PnPR3 gene is to typical fusarium solani (F.) (Fusarium solani) Has certain inhibiting effect; the gene of the invention can be transferred into plants to improve the disease resistance of the plants and can improve the resistance of tobacco to fusarium solani (F) which is a pathogenic bacterium of root rotFusarium solani) The resistance of the strain lays a good working foundation for disease-resistant breeding of the root rot plants, and has higher economic benefit.

Drawings

FIG. 1 is an electrophoresis diagram of PnPR3 gene fragment amplified from a Panax notoginseng root induced by Rhizopus solani;

FIG. 2 is the PnPR3 protein conserved domain prediction;

FIG. 3 is a diagram of PnPR3 gene evolutionary tree analysis;

FIG. 4 shows the plant expression vector pK2GW7-35S-PnPR3Verifying a construction result; a is as shown inPnPR3Recovering fragment glue; FIG. 1 shows pENTRTM2B plasmid with no load; 2. double enzyme digestion of the entry clone plasmid pENTRTM 2B; the C diagram is pK2GW7-35S-PnPR3 PCR detection of bacterial liquid; graph D is pK2GW7-35S-PnPR3 Extracting plasmids;

FIG. 5 is a schematic diagram of PCR electrophoresis detection of Agrobacterium colonies;

FIG. 6 is a screening diagram of PCR detection of transgenic tobacco;

FIG. 7 is a PR-PCR validation of the PnPR3 gene in transgenic plants; 1: wild type tobacco genomic DNA PCR electrophoresis (negative control); 2: water PCR electrophoresis (blank); 3: plasmid electrophoresis of pMD-18TPnPR3 (positive control); 4-7: DNA PCR electrophoresis of No. 4, No. 6, No. 9 and No. 11 plants respectively;

FIG. 8 is a graph showing the resistance of transgenic tobacco overexpressing PnPR3 to Fusarium solani;

FIG. 9 is a graph showing the chitinase activity of PnPR3 transgenic tobacco.

Detailed Description

The test methods in the examples described below were all conventional methods unless otherwise specified, and the reagents used were all conventional commercially available reagents and reagents prepared according to conventional methods unless otherwise specified.

Example 1: panax notoginseng disease course related protein PnPR3 conserved fragment and cDNA full length acquisition thereof

And (3) screening a disease course related protein PR3 with obvious expression difference by transcriptome sequencing, obtaining a splicing sequence of PnPR3 from a CDS file, analyzing the fragment by using MEGA software and the full-length PR3 sequence of the ginseng of the species similar to the pseudo-ginseng, and finding that the fragment has a 3 'end and a 5' end. Therefore, the full-length sequence of the gene can be obtained only by carrying out high-fidelity PCR amplification on the cDNA sequence in the experiment; designing specific primers PR3-F (5'-GGATCCatgagattttggacagtaaccatattc-3', GGATCC is a BamHI enzyme cutting site) and PR3-R (5'-CAGCTGtagacctaaacctaaaccattaaatatgg-3', GTCGAC is a SalI enzyme cutting site), amplifying an open reading frame of the gene by adopting high-fidelity PCR reaction enzyme provided by Dalianbao bio-corporation to obtain a strip with the size of about 1000bp, and the strip conforms to the size of a target gene; recovering DNA fragments from agarose gel, connecting with a pMD-18T cloning vector, transforming into E.coli DH5 alpha, carrying out colony PCR detection, extracting plasmids, carrying out double enzyme digestion detection, determining that the plasmids can be cut open, and sending the plasmids to Kunming Optimak biology Limited for sequencing; finally, the open reading frame of the PnPR3 gene was determined and a cDNA fragment containing an enzyme cleavage site was obtained (shown in SEQ ID NO: 1).

Protein gene related to the course of disease of panax notoginsengPnPR3The sequence is input into ORF (open Reading Frame Finder) (www.ncbi.nlm.nih.gov/gorf. html) in NCBI and DNAMAN software analysis sequence, the result shows that the full length of the PnPR3 gene is 918bp, 305 amino acids are coded (shown as SEQ ID NO: 1); meanwhile, the molecular weight of the protein coded by PnPR3 is 33393.25 daltons by using ExPASY (ProtParam) software, the theoretical pI value is 6.41, and the molecular formula is C₁₄₇₁H₂₂₀₀N₄₀₈O₄₄₉S₁₉(ii) a The 305 amino acids of the PnPR3 contain 26 basic amino acids (R, H, K), 23 acidic amino acids (D, E), 132 nonpolar, hydrophobic amino acids (G, A, V, L, I, F, P), and 124 nonionized polar, neutral amino acids (W, S, T, C, Y, M, N, Q); wherein the negatively charged amino acids include D and E for a total of 23 and the positively charged amino acids include R, H and K for a total of 26.

The PnPR3 protein predicted conserved domain is analyzed by using an online tool InterPro (http:// www.ebi.ac.uk/InterPro/search/sequence-search), and the PnPR3 protein full length is a conserved domain family, namely glycosyl hydrolase family19 (Glycoside hydrolase, family19(IPR 016283)). This family is believed to contain only chitinase activity. The family contains a Chitin-binding, a type 1 structure (IPR 001002) is located at 21-57 sites, and 1 Lysozyme-like domain (Lysozyme) (IPR 023346) structure is located at 66-305 sites. The protein contains four regions with chitinase catalytic activity, which are respectively located at 211-221, 69-298, 70-298 and 87-109, and also contains a conserved site Chitin-binding, type 1 at 31-50. The PnPR3 protein body (GO) prediction shows that the protein is involved in biological processes such as carbohydrate metabolism process, chitin catalysis process and cell wall macromolecule catalysis process; the protein has molecular functions such as chitinase activity and chitin binding activity.

Performing homology analysis by using DNAMAN software, and constructing phylogenetic tree by using the protein of Panax notoginseng PnPR3 and the protein of Ginseng radix, radix Dauci Sativae, Juglandis, herba Spinaciae, cacao, semen Cajani, etc., as shown in FIG. 3; the pseudo-ginseng PnPR3 protein and the ginseng PR3 protein (accession number: ACN 96317.1) are gathered into a group and show a closer relationship.

Experimental example 2: notoginseng course of disease related protein genePnPR3Plant expression vector pK₂GW₇Construction of-35S-PnPR 3

Carrying out double digestion on the pMD-18T-PnPR3 vector plasmid with correct sequencing detection by using restriction enzymes BamHI and SalI, and recovering a PnPR3 gene fragment; mixing pENTR^TMCarrying out double enzyme digestion on the 2B plasmid by using restriction enzymes BamHI and SalI, and recovering a linear vector fragment; connecting the recovered PnPR3 gene fragment with the recovered linear plasmid vector to successfully construct a pENTR-PnPR3 entry vector; carrying out enzyme digestion detection on the entry expression vector, and constructing the entry vector and a plant expression vector pK according to the operation instruction of a Gateway kit₂GW7.0 is subjected to LR reaction, and pK can be successfully constructed₂GW₇-35S-PnPR3 plant expression vector; the plant expression vector is subjected to PCR detection, enzyme digestion detection, plasmid sequencing, and the next experiment is carried out after the gene is verified to be free from mutation (figure 4).

Experimental example 3: agrobacterium transformation with pK2GW7-35S-PnPR3 plant expression vector

Agrobacterium-infected cells were prepared, the above-constructed plant expression vector pK2GW7-35S-PnPR3 was transferred to Agrobacterium (C58C 1(pPMP 90)) by electroporation, and transformants were selected on LB plates supplemented with spectinomycin. Adding a small amount of plasmid into agrobacterium tumefaciens competent cells, slightly and uniformly mixing, adding the mixture into a precooled electric transformation cup, slightly knocking the cup body to enable the mixed solution to fall to the bottom of the cup, placing the electric transformation cup into a chute of an electric transformation instrument (BIO-RAD), electrically shocking by using a 1 mm click cup and a parameter of 200 ohm and 2.5 Kv/0.2cm, immediately taking out the electric transformation cup after clicking, quickly adding 0.5mL of LB culture medium, uniformly mixing, and transferring into a 1.5mL centrifuge tube; shaking-culturing at 28 deg.C and 200 rpm for 3-5 hr; centrifuging at 7500 rpm for 1 min at room temperature, discarding most of the supernatant, and retaining 50 μ l to resuspend the cells; the resuspended agrobacteria are spread on LB solid medium added with spectinomycin (Spe, 50. mu.g/mL), single colonies are obtained after 2 days of culture at 28 ℃, single colonies are picked up on 20mL liquid LB medium added with spectinomycin (Spe, 50. mu.g/mL) and after 36 hours of shake culture at 28 ℃ and 180rmp, PCR detection is carried out by using the upstream and downstream primers of PnPR3 gene, a 1.0kp band can be amplified by successfully transformed colonies (figure 5), and transformant colonies confirmed by colony PCR are used for transforming plants.

Experimental example 4: agrobacterium-mediated genetic transformation of plants and screening of transgenic plants

The receptor tobacco seeds are sterilized on the surface by NaClO, sowed on 1/2 MS solid culture medium (pH5.8), placed in a tissue culture room with constant temperature of 25 ℃ and continuous illumination (100 mu mol/m 2. s) for culturing for 4 weeks to obtain aseptic seedlings, and cultured in the tissue culture room. Selecting tobacco leaves in an ideal state as explants, dip-dyeing the tobacco leaves in the prepared agrobacterium liquid for 20min, sucking off surface water, paving the leaves on an MS1 culture medium for 2d (the time required when the first leaf visually shows the thallus), carrying out dark co-culture on the tobacco leaf explants, transferring the tobacco leaf explants to a bud induction culture medium containing screening factors such as MS4 for screening (with the front face facing downwards), and carrying out subculture for about 15 d. Culturing the transformed tobacco leaf explant in a subculture medium under illumination, observing callus or cluster buds growing on the edge of the leaf after 20-25 days, transferring the leaf explant into a rooting medium MS (containing antibiotic Cef + Km), and inducing rooting under illumination of 25 ℃. Selecting transgenic tobacco containing resistance.

Extracting the genome of the tobacco by adopting a CTAB method: 0.1 g of plant leaves are weighed into a 1.5mL centrifuge tube, and ground into powder by adding liquid nitrogen. 900. mu.l of 2 XCTAB buffer (Tris-HCl pH 7.5100 mM, EDTA 20 mM, NaCl 1.4M, CTAB 2%) pre-warmed to 65 ℃ was added, and the mixture was stirred evenly every 2 min in a water bath at 65 ℃ for 20min, taken out and cooled to room temperature, and 500. mu.l of chloroform was added: rotating and shaking the isoamyl alcohol (24: 1) mixed solution uniformly at 4 ℃, 7500 rpm, centrifuging for 10 min, transferring the supernatant to a new centrifuge tube, and repeating the steps; adding 1/10 volumes of 3M NaOAc with pH5.2 and equal volume of isopropanol, shaking uniformly, centrifuging for 20min at 4 ℃, 12000 rpm, discarding the supernatant, washing twice with 75% ethanol, drying, dissolving and degrading RNA with 1 XTE buffer solution containing RNase to obtain genome DNA of transgenic tobacco, performing PCR amplification by using the DNA as a template and specific primers of PnPR3 gene, and detecting 4 transgenic plants (T4, T6, T9 and T11) in the obtained 12 suspected PnPR3 transgenic seedlings after antibiotic screening, wherein the PCR product band is single and the size of the PCR product band accords with the length of the PnPR3 sequence segment (figure 6). The PCR confirmed transgenic plants were used for RT-PCR analysis.

In order to examine the transcription of PnPR3 gene in the transgenic tobacco line containing the gene, total RNA is extracted from the transgenic plant, and is subjected to reverse transcription into cDNA for RT-PCR analysis, and the transcription level of AtKUP1 gene in the transgenic plant is detected. After RNA was extracted using TRIZOL Reagent (Invitrogen), cDNA was synthesized using RevertAIdTM-M μ l V Reverse Transcriptase Kit (Reverse transcription Kit, Fermentas) with a total plant RNA of about 0.1-0.5 ug, oligo (dT) 50 ng, 10 mM dNTP mix 1 μ l, made up to 9 μ l with DEPC treated water, mixed well, collected in the bottom of a tube by brief centrifugation, heated at 65 ℃ for 5 min in an ice bath for 10 min, added to reaction mixture of 11 μ l (5 × reaction buffer 4 μ l, 25 MgCl 24 μ l, 0.1M DTT 2 μ l, RNase inhibitor 1 μ l), mixed well, collected in the bottom of a tube by brief centrifugation, incubated at 25 ℃ for 2 min, then incubated at 42 ℃ for 70 min, and cDNA was synthesized. Using cDNA as template and PnPR3 gene upstream and downstream specific primers as PCR, 1.0 kb band can be amplified from the plants successfully transferred into PnPR3 (FIG. 7), which proves that PnPR3 gene can be successfully transcribed in transgenic plants. The transgenic plants confirmed by RT-PCR were used for further physiological and biochemical analysis.

Experimental example 4: overexpression of PnPR3 Gene on Fusarium solani (Fusarium solani) Resistance testAnd (6) testing.

To evaluate the level of resistance of the PnPR3 transgenic tobacco, tobacco lines (T4, T6, T9, T11) into which the PnPR3 gene had been successfully transferred were selected and healthy young leaves of the transgenic tobacco and the control group were cut. Wounds of uniform size were formed on the leaves, and 200. mu.L of F.solani spore suspension (spore concentration 1X 10) was inoculated with a sterile tip⁵Conidia/ml). The treated leaves were placed on sterile and moist filter paper and co-cultured in glass dishes at 28 ℃ and the development of the leaves was observed. After 7 days, the control tobacco leaves developed severe yellowing and slight decay, while the leaves of the transgenic tobacco lines showed only slight yellowing; the leaves were collected, and the chitinase activity in the leaves was determined by a chitinase kit purchased from myza organism ltd, Yunnan using a spectrophotometric method. Overexpression of PnPR3 in tobacco can enhance the transgenic tobacco pairs by increasing chitinase activityF. solani Resistance of (2) (fig. 8);

analytical inoculationF.solaniChitinase activity in transgenic tobacco and control leaves of fungal spores; in the initial phase, the level of chitinase activity in tobacco leaves was maintained at a low level, whereas transgenic tobacco leaves obtained higher chitinase activity than the control due to overexpression of PnPR 3. By using F. solani After 2 days of spore inoculation, the chitinase activity in the tobacco leaves is obviously increased, and the chitinase activity in the leaves is accumulated along with time. Related studies have shown that higher plants do not normally contain chitin, but when infected with bacteria, fungi or viruses, plant chitinase activity increases rapidly, enhancing the resistance of the plant to pathogenic microorganisms; thus, PnPR3 is an important defense gene involved in the resistance of notoginseng to root rot infection (fig. 9).

Sequence listing

<110> university of Kunming science

<120> gene of pseudo-ginseng disease course related protein PnPR3 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 918

<212> DNA

<213> Panax notoginseng (Panax notogeng)

<400> 1

atgagatttt ggacagtaac catattcatt ctagcctcat accttgcagt ttctgcagaa 60

caatgtggga aacaagctgg aatggctttg tgcccaaatg ggctctgttg cagccaattc 120

gggtggtgtg gcagcacccc tgagtactgc actaattgcc aaagccagtg cagcgaacct 180

tccccaggtg gaggtgttag ctccataatt actgagtctg tgtttaatca aatgcttaaa 240

tatcgaaacg acggaaggtg ccgtactaat ggattctaca catacaatgc ttttatcaat 300

gctgcaaaat ctttcaatgg ttttgggaca actggtaata ctgtccaaca gaaacaagag 360

ctcgccgctt tcttggctca gacctctcat gaaaccacag gtggatgggc aagtgcccca 420

gatggtcaat atgcatgggg atattgcttt ataagagaaa acaaccaggc tgcttactgc 480

acttccaaag actggccttg tcctcctggc aaactatact atggcagagg acctatccaa 540

ctcactcaca actacaacta tgggcaagct ggaaatgcaa ttggagtgga cctaataaac 600

aaccctgacc tagttgccac ggacgctacc atatcattca aaacagccat atggttctgg 660

atgactccac aggctaacaa gccatcgagc cacgatgtga ttacacaaag atggtcaccc 720

tctgcagcag ataccgcggc tggtcgcgtc cctggcttcg gtgtcatcac taacataatt 780

aacggcgggc tcgagtgtga tcacggcagg gatgataagg cggaggatag gattggattc 840

tacaagaggt attgtgacat tctgcaagtt ggctatggga atgaactcaa ttgcaacaat 900

caaaggcctt ttggctaa 918

<210> 2

<211> 305

<212> PRT

<213> Panax notoginseng (Panax notogeng)

<400> 2

Met Arg Phe Trp Thr Val Thr Ile Phe Ile Leu Ala Ser Tyr Leu Ala

1 5 10 15

Val Ser Ala Glu Gln Cys Gly Lys Gln Ala Gly Met Ala Leu Cys Pro

20 25 30

Asn Gly Leu Cys Cys Ser Gln Phe Gly Trp Cys Gly Ser Thr Pro Glu

35 40 45

Tyr Cys Thr Asn Cys Gln Ser Gln Cys Ser Glu Pro Ser Pro Gly Gly

50 55 60

Gly Val Ser Ser Ile Ile Thr Glu Ser Val Phe Asn Gln Met Leu Lys

65 70 75 80

Tyr Arg Asn Asp Gly Arg Cys Arg Thr Asn Gly Phe Tyr Thr Tyr Asn

85 90 95

Ala Phe Ile Asn Ala Ala Lys Ser Phe Asn Gly Phe Gly Thr Thr Gly

100 105 110

Asn Thr Val Gln Gln Lys Gln Glu Leu Ala Ala Phe Leu Ala Gln Thr

115 120 125

Ser His Glu Thr Thr Gly Gly Trp Ala Ser Ala Pro Asp Gly Gln Tyr

130 135 140

Ala Trp Gly Tyr Cys Phe Ile Arg Glu Asn Asn Gln Ala Ala Tyr Cys

145 150 155 160

Thr Ser Lys Asp Trp Pro Cys Pro Pro Gly Lys Leu Tyr Tyr Gly Arg

165 170 175

Gly Pro Ile Gln Leu Thr His Asn Tyr Asn Tyr Gly Gln Ala Gly Asn

180 185 190

Ala Ile Gly Val Asp Leu Ile Asn Asn Pro Asp Leu Val Ala Thr Asp

195 200 205

Ala Thr Ile Ser Phe Lys Thr Ala Ile Trp Phe Trp Met Thr Pro Gln

210 215 220

Ala Asn Lys Pro Ser Ser His Asp Val Ile Thr Gln Arg Trp Ser Pro

225 230 235 240

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

245 250 255

Thr Asn Ile Ile Asn Gly Gly Leu Glu Cys Asp His Gly Arg Asp Asp

260 265 270

Lys Ala Glu Asp Arg Ile Gly Phe Tyr Lys Arg Tyr Cys Asp Ile Leu

275 280 285

Gln Val Gly Tyr Gly Asn Glu Leu Asn Cys Asn Asn Gln Arg Pro Phe

290 295 300

Gly

305

<210> 3

<211> 33

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

ggatccatga gattttggac agtaaccata ttc 33

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

cagctgtaga cctaaaccta aaccattaaa tatgg 35

Claims

1. A gene of a pseudo-ginseng disease course related protein PnPR3 has a nucleotide sequence shown as SEQ ID NO. 1 and codes a protein with an amino acid sequence shown as SEQ ID NO. 2.

2. The gene of the pseudo-ginseng pathogenesis-related protein PnPR3 as claimed in claim 1 is used for improving the resistance of tobacco to fusarium solani (F.) (Fusarium solani) Use in resistance.