CN111850012A - Soybean bacillus subtilis protease gene GmSub and application thereof - Google Patents

Abstract

The invention discloses a soybean subtilisin gene GmSub, the base sequence of which is shown in a sequence table SEQ ID NO. 1; a plant expression vector, which is inserted with a gene shown in a sequence table SEQ ID NO.1 to replace a GUS gene; the soybean subtilisin GmSub1.5-2 is applied to the aspect of improving the disease resistance of plants; the results show that the resistance of positive plants TP1, TP2 and TP3 of the over-expressed GmSub1.5-2 gene to the soybean phytophthora root rot is obviously improved, and the fluorescent quantitative PCR analysis results show that the soybean bacillus subtilis protease gene GmSub1.5-2 is infected and induced by the soybean phytophthora root rot pathogenic bacteria to express, which indicates that the gene plays a positive role in the soybean reaction for resisting the phytophthora root rot.

Description

Soybean bacillus subtilis protease gene GmSub and application thereof

Technical Field

The invention belongs to the technical field of biological gene engineering, and particularly relates to a soybean bacillus subtilis protease geneGmSub1.5-2And application thereof.

Background

Serine proteases, a family of proteases centered on serine as the catalytically active site, can be roughly divided into 6 major classes, of which the subtilisin-like class consists of more than 200 different family members. Most subtilisins belong to the Kexins subfamily, while plants differ and are mostly classified in the Pyrolysins subfamily. Previous researches on serine protease find that the serine protease is widely involved in various physiological and biochemical processes in organisms, such as cell division, related tissue differentiation, seedling growth to mature fruit development, plant senescence, apoptosis and the like. A large number of studies have been reported on subtilisin-like genes in plants such as Arabidopsis, tomato, soybean and nightshade. Two mutant plants are obtained from arabidopsis thaliana by means of EMS chemical mutagenesis, such as Liangka, and the like, and subsequent researches show that the geneSDD1Can make serine of subtilisin-like produce mutation, the mutated gene can be fully expressed in epidermal cell for controlling stomata, and can make stomatal density of transgenic Arabidopsis plant and related index obviously increase, so that it can reasonably speculate geneSDD1Plays an important role in the plant morphogenesis and stomata development process. Seed coat specific gene cloned from soybean by Batchelor AK and the likeSCS1This gene belongs to a small class of genes with sequence similarity to subtilisin-like enzymes (serine proteases) and can regulate the differentiation of sclerenchymal cells by means of specific subtilisin-like genes. Beilinson V et al identified two subtilisins in soybeanSLP-1AndSLP-2the coding regions are homologous, but their promoters are very different and function differently.SLP-1The expression is mainly related in the seed coat of the seeds in the development stage, andSLP-2expression is mainly carried out in the cotyledons of the germinated seeds. GUS staining results of genetically transformed solanum nigrum plants show that,SaSBT1the promoter drives GUS gene to specifically express in tissues such as epidermis, vein and stem epidermal cells of the tender stem part of the transgenic solanum nigrum.

Subtilisin-like proteases (Subtilases) belong to one of serine proteolytic enzymes, and are found to exist in eukaryotes such as bacteria, fungi and parasites. The active site of the gene is a catalytic triad consisting of aspartic acid (Asp), histidine (His) and serine (Ser). The subtilisin-like enzyme is related to pathogenicity of pathogenic bacteria and fungi, and a plurality of researches report that the subtilisin-like enzyme can induce allergic necrosis reaction (HR) and Systemic Acquired Resistance (SAR) of a host in a plant-pathogen interaction process and plays an important role in plant disease resistance. Gindro et al, which use serine inhibitors on grapes, have found that they can effectively block the activation of the defense mechanism of xanthomonas vinifera, and cause part of the cells to die, thereby changing the sensitivity of grapes to germs. The cloned Subtilases protein StSBTc-3 in potato plays a role in inducing host cell death during interaction with Phytophthora infestans. The Chinese chestnut blight bacteria parasitized by virus is the main factor causing the Chinese chestnut blight, and plum blossom and the like are transferred into the subtilism-like protease geneprb1The chestnut and the wild chestnutEP155Compared with the prior art, the number of aerial hyphae is suddenly reduced, the spore production capability is greatly hindered, and the pathogenicity is obviously reduced. The research finds thatprb1Can block the autophagy pathway and influence the growth and development of the bacterial strain, so that the gene plays an important role in regulating and controlling the pathogenicity of the chestnut blight bacteria. Jord a L et al cloned four different genes from tomato,P69AandP69Dmainly takes part in the morphological expression in the tomato development process, and researches show thatP69BAndP69Cup-regulation of salicylic acid expression in tomato following Pseudomonas syringae infection, indicating69BAndP69Cplays a key role in the pathogenesis of plant exposure to pathogens. In recent literature studies Liu Xinjiang et al cloned two coding subtilisins in Arabidopsis thalianaMycoproteases, each containing the I9 domainBcser1And comprising the S8 domainBcser2The research result shows that the subtilisins-like geneBcser2Is an important factor for the generation of sclerotia of the bacillus polii and controls the generation quantity of conidia.

Disclosure of Invention

The object of the present invention is to provide a soybean-derived subtilisin geneGmSub1.5-2And application thereof.

Soybean subtilisin GmSub1.5-2, the base sequence of which is shown in sequence table SEQ ID NO. 1.

A plant expression vector is inserted with a gene shown in a sequence table SEQ ID NO. 1.

The soybean subtilisin GmSub1.5-2 is applied to the aspect of improving the disease resistance of plants;

the plant is soybean;

the disease resistance is the resistance to phytophthora root rot.

The invention provides a soybean subtilisin gene GmSub1.5-2, the base sequence of which is shown in a sequence table SEQ ID NO. 1; a plant expression vector, which is inserted with a gene shown in a sequence table SEQ ID NO.1 to replace a GUS gene; the soybean subtilisin GmSub1.5-2 is applied to the aspect of improving the disease resistance of plants; the results show that the resistance of positive plants TP1, TP2 and TP3 of the over-expressed GmSub1.5-2 gene to the soybean phytophthora root rot is obviously improved, and the fluorescent quantitative PCR analysis results show that the soybean bacillus subtilis protease gene GmSub1.5-2 is infected and induced by the soybean phytophthora root rot pathogenic bacteria to express, which indicates that the gene plays a positive role in the soybean reaction for resisting the phytophthora root rot.

Drawings

FIG. 1 is a PCR amplification electrophoretogram of a core fragment of GmSub1.5-2 gene; m is DNA standard molecular weight; 1: PCR products;

FIG. 2 construction of a plant overexpression vector; A. double enzyme digestion of plant expression vector pCAMBIA 3301; B. PCR amplification of target fragments;

FIG. 3 construction of plant overexpression vector and verification of double enzyme digestion; B. PCR verification;

FIG. 4T-DNA region structure of plant overexpression vector pCAMBIA 3301-GmSub1.5-2; FIG. 4T-DNAstructual region of plant over-expression vector;

FIG. 5 PCR detection of transformed plants at T0 generation; A. screening a marker gene bar; the B.35S promoter; a NOS terminator;

m: DNA molecular weight standard; p: a plasmid positive control; n: water negative control; CK: non-transgenic plants (negative control); 1-3: transgenic positive plants TP1, TP2 and TP 3;

FIG. 6 PCR assay of T1, T2 transformed plants; A. screening a marker gene bar; the B.35S promoter; a NOS terminator; m: DNA molecular weight standard; p: a plasmid positive control; n: water negative control; CK: non-transgenic plants (negative control); 1-3: transgenic positive plants TP1, TP2 and TP3 of the T1 generation; 4-6: transgenic positive plants TP1, TP2 and TP3 of the T2 generation;

FIG. 7 shows the relative expression level of GmSub1.5-2 gene in the plant of transgenic plant overexpression vector pCAMBIA 3301-GmSub1.5-2.

Detailed Description

Example 1 GeneGmSub1.5-2Cloning and sequence analysis of

Extracting total RNA from root system tissues of soybean Jinong 74 seedling stage by using an RNAasso kit produced by TAKARA company, designing a specific primer Sub1.5-2 by using reverse transcription cDNA as a template according to an RNA-seq sequencing result sequence, amplifying a target fragment by adopting an RT-PCR method and sequencing, wherein PCR amplification primers required by a test are shown in a table 1;

connecting a GmSub1.5-2 gene complete sequence into a cloning Vector pMD18-T Vector, sequencing a recombinant Vector, and then performing bioinformatics analysis, wherein the sequencing is completed by Beijing Sanbo polygala tenuifolia biotechnology limited; the new gene ORF was translated into an amino acid sequence by DNAMAN software using the on-line analysis program ORFfinder (https:// www.ncbi.nlm.nih.gov/ORFfinder /) for possible open reading frame analysis of the new gene sequence.

As a result: designing a specific primer Sub1.5-2 with the amplification length of 1841bp according to the sequence fragment of the GmSub1.5-2 obtained by early screening, obtaining a nucleic acid band (figure 1) with the same size as the expected size after PCR amplification, cloning the PCR product to a PMD18-T vector, and sequencing; comparing the sequencing result with data in NCBI (national center for information and Biotechnology information) to find out a known sequence consistent with a target gene, and preliminarily judging that the cloned gene is an unknown new gene; the comparison shows that the target gene has the highest homology compared with the sequence number XM-028363170.1, the consistency on the 100% coverage level reaches 99%, and the comparison similarity with other Glycine soja subtilisin-like protease SBT1.5 gene sequences is higher (https:// blast.ncbi.n.m.nih.gov/blast.cgi), the target gene is presumed to be one of the members of the Glycine soja subtilisin-like protease SBT1.5 family, and therefore the target gene is named GmSub1.5-2;

the nucleic acid sequence of the new gene GmSub1.5-2 was analyzed by the on-line analysis program ORF FINDER and the position of the theoretical coding region was predicted. The analysis result shows that the GmSub1.5-2 gene has a theoretical open reading frame of 1524 and can code 507 amino acid residues.

Example 2 construction of plant expression vectors and genetic transformation

The plant expression vector pCAMBIA3301 was digested simultaneously with restriction enzymes BglII and BstElI, and the original GUS gene on 3301 was replaced with the cloned GmSub1.5-2 full-length gene using the Seamless Assembly cloning Kit. Constructing a pCAMBIA3301-GmSub1.5-2 plant overexpression vector which takes Bar as a screening marker and uses a CaMV35S promoter to start a target gene GmSub1.5-2. The constructed plant over-expression vector pCAMBIA3301-GmSub1.5-2 is used for transforming soybean receptor material by adopting an agrobacterium-mediated method.

As a result: carrying out double enzyme digestion on a plant expression vector pCAMBIA3301 by using BglII and BstElI restriction endonucleases to obtain a3301 vector large fragment with the size of 10kb and a GUS gene fragment with the size of 2029 bp; carrying out PCR amplification on a cloning vector Pmd 18-T-GmSub1.5-2 by using a primer Sub1.5-2 to obtain a GmSub1.5-2 gene ORF full-length fragment (shown in figure 2) with the size of 1841bp, and connecting the GmSub1.5-2 gene ORF full-length fragment into a plant expression vector pCAMBIA3301 by using T4 ligase;

PCR and double enzyme digestion identification are carried out on the successfully constructed plant over-expression vector pCAMBIA3301-GmSub1.5-2, and the full-length fragment 1841bp of the ORF of the GmSub1.5-2 gene (shown in figure 3) which is consistent with the expected size is obtained, and the sequence is carried out, so that the sequencing result is correct, and the successful construction of the plant over-expression vector pCAMBIA3301-GmSub1.5-2 is shown (shown in figure 4).

The constructed plant overexpression vector pCAMBIA3301-GmSub1.5-2 is transformed into soybean cotyledon nodes by an agrobacterium-mediated method by taking a soybean material 'Jinong 74' as a receptor, and resistant transformed plants are obtained by germination culture, recovery culture, co-culture, selective culture, elongation culture, rooting, seedling hardening and transplanting culture and are cultured and generation adding in a greenhouse.

Example 3 molecular testing of transformed plants

By adopting an improved CTAB method, genome DNA is extracted from soybean leaves, and is used as a template, a vector plasmid is used as a positive control, an untransformed plant is used as a negative control, and PCR amplification is carried out by respectively using specific primers (table 1) for screening marker genes bar and 35S promoters and NOS terminators.

As a result: through genetic transformation, 7 resistant seedlings are obtained together, and 3 positive strains are detected by PCR detection of three exogenous fragments of a selection marker Bar gene, a 35S promoter and an NOS terminator (figure 5) and are respectively marked with TP1, TP2 and TP3 (TP, Transgenic Plants); and (3) culturing the plants which are detected to be positive in an artificial climate chamber until the plants are mature, harvesting seeds, carrying out generation addition in the greenhouse, carrying out PCR detection on plants of T1 and T2 generations, and detecting screening marker genes bar and 35S promoters and NOS terminators of plants TP1, TP2 and TP3 of GmSub1.5-2 gene overexpression plants (figure 6).

Example 4 identification of disease resistance of transformed plants

And (3) inoculating the phytophthora root rot to the transformation positive material by adopting a hypocotyl infection method, and performing identification standard according to the quality standard of soybean germplasm resource data. Selecting soybean plants at the initial growth stage, inoculating phytophthora sojae root rot pathogenic bacteria after the true leaves of the soybean plants grow to be completely flat, and moisturizing the wounds for 7 days after inoculation, wherein the temperature is controlled at 20-25 ℃, and the humidity is kept at more than 90%. Mortality of transgenic lines and control recipient varieties was investigated 5 days after inoculation. If 70% or more of plants in the strain or variety die, the plant is infected with disease (S); 70% or more of plants can be disease resistant (R) when growing normally; 31% -69% of plant dead lines or varieties are resistant (MR).

The hypothesis test is used for analyzing the data obtained by identifying the disease resistance of T2 generation transgenic positive strains TP1, TP2 and TP3 phytophthora root rot, and the statistical mortality identification result shows that the resistance level of the transgenic positive strains TP1, TP2 and TP3 to phytophthora 1 physiological race is obviously improved compared with that of a receptor control strain, wherein the inoculation mortality of the TP1 strain is the lowest and is 26.66 percent, and the resistance level is achieved; the resistance levels of TP2 and TP3 strains are same as that of a control CK and are all neutral, but the inoculation mortality rates are respectively 43.33 percent and 36.66 percent and are obviously lower than that of the control CK of 63.33 percent, which shows that the resistance of positive plants TP1, TP2 and TP3 of a transgenic plant overexpression vector pCAMBIA3301-GmSub1.5-2 to the phytophthora sojae root rot is obviously improved (Table 2).

Example 5 analysis of relative expression amount of target Gene

Extracting total RNA of root tissues after T2 generation transformed plants, of which marker genes bar and 35S promoters and NOS terminators are positive through PCR detection, are inoculated for 5 days, performing reverse transcription to obtain corresponding cDNA, and performing real-time fluorescence quantitative PCR detection on a target gene GmSub1.5-2; taking soybean beta-actin gene (GenBank accession number is TC 204137) as an internal reference gene, carrying out relative quantitative analysis on the expression quantity of GmSub1.5-2 gene in transgenic soybean plants, and setting 3 times of repetition; amplifying a target gene according to the operation of an SYBR Premix Ex TaqTM kit instruction; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 10s, reaction at 60 ℃ for 40s, 40 cycles. And analyzing the relative expression quantity of the endogenous target gene by adopting a 2-delta CT calculation method.

As a result: t2 generation transgenic positive strains TP1, TP2 and TP3 are planted in a greenhouse, and phytophthora sojae is inoculated through a hypocotyl infection method in a seedling stage. Carrying out moisture preservation treatment after inoculation, controlling the environmental temperature at 20-25 ℃ and keeping the humidity at more than 90%, extracting root tissue total RNA after 5 days of inoculation treatment, and carrying out Real-time PCR (polymerase chain reaction) determination on a target gene GmSub1.5-2. And calculating data by adopting a 2-delta CT method, and analyzing the relative expression quantity of the target gene.

In soybean roots, the expression level of GmSub1.5-2 of a control plant which is not infected with phytophthora root rot is set to be 1, and the expression levels of other transformation materials and other treatments are compared with the set value. As shown in FIG. 7, under the condition of no bacterium, the relative expression amounts of the positive plants TP1, TP2 and TP3 of the transgenic plant overexpression vector pCAMBIA3301-GmSub1.5-2, namely the positive plants TP1, TP2 and TP3, of the GmSub1.5-2 genes are respectively 3.22, 2.26 and 2.51 which are obviously higher than those of the control. After 5 days of inoculation of phytophthora sojae root rot pathogenic bacteria, the relative expression level of the GmSub1.5-2 gene of each plant is obviously increased, the relative expression level of the GmSub1.5-2 gene in a control plant is increased to 3.05, and the relative expression levels are respectively 7.88, 6.09 and 5.85 in positive plants TP1, TP2 and TP3 of a transgenic plant overexpression vector pCAMBIA 3301-GmSub1.5-2. The GmSub1.5-2 gene is infected by phytophthora sojae root rot pathogen to induce expression.

Sequence listing

<110> Jilin university of agriculture

<120> soybean subtilisin gene GmSub and application thereof

<160>2

<170>SIPOSequenceListing 1.0

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<211>1841

<212>DNA

<213> Soybean (Glycine max Linn. Merr.)

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cccacgtcat aagcctcatt cccgaacagc ttcgccaact ccacaccacc cgctcccctc 60

agttcctggg cctcaacact gccgacaggg ctggccttct caaggagacc gatttcggct 120

ccgatctcgt catcggagtc atcgacaccg gcatttcccc cgagagccag agcttcaacg 180

accgtcatct cgctctccct ccccctaagt ggaagggcca ctgcgttgct gctaaagact 240

ttccccccac ctcctgcaac cggaaactca tcggagctcg ctacttctgc gctggctacg 300

aagccaccaa cggcaaaatg aacgacactt tggagtcccg ttccccgaga gactccgacg 360

gtcacggcac ccacacggct tccatcgccg ccggcaggta cgtcttcccg gcctccacca 420

tgggctacgc caaaggaatg gccgccggga tggctcccaa ggctcgcctc gccgtctaca 480

aggtctgctg gaacgccggc tgctacgact ccgacatcct cgccgcgttc gacgccgccg 540

tggccgacgg cgtcgacgtg gtctccctca gcgtcggggg cgtggtggtg ccctaccacc 600

tcgacgtcat cgccgtcggg gccttcggag cctccgaggc tggcgtcttc gtgtctgcct 660

cggcgggtaa cggcggtccc ggcggcctca cggtgaccaa tgtggcaccc tgggtcacca 720

ctgtgggcgc cggaaccata gacagagatt tcccggcgga tgttgtgctg gggaacggca 780

aggtgattgg cgggatgagt gtgtacggtg gaccgggtct gactccaggt cggctctatc 840

ctctggtgta tgcgggttcg gatggttact cttcctctct ttgcttggaa gattcgttgg 900

atcccaagtc tgtgaggggg aagattgttg tgtgtgagag aggcgtcaat tcaagagcag 960

ccaaaggcca agtggtgaag aaagccggag gggttggaat ggtgttgacg aatggaccct 1020

tggacgggga aggcttggtg gctgactgcc aagtcctccc ggcgacctcc gtgggtgctg 1080

aaggagggga cgagcttagg agatacatgg cattcgccgc acagctgcgg acaccagcaa 1140

cggcaaccat aatattcaag ggcacaaggc tcgggattaa accagcgccc aaggtggcat 1200

ctttttcggc cagagggcca aacccggagt cccctgagat tctgaagccg gatgtgatag 1260

ctcccggatt gaacattctg gcggcttggc ctagtacgct ttccccttcc gggcttccct 1320

ctgacgagcg caggagccag tttaacattc tgtctggcac ttcaatggct tgcccccacg 1380

tttctggttt ggctgctctt ctgaaagcgg ctcaccctga ctggagtcct gccgccatca 1440

gatctgccct catcaccact gcttacactc tagacaacgg aggaggccct ctgctggatg 1500

agtccaatgc caatgtttcc tccgtcttcg atcatggggc tggccatgtt catcccgaca 1560

aggccatcaa ccctggcctg gtctatgaca tctctaccta cgattatgtc gatttcctct 1620

gcaattccaa ctacacctcc cacaacatcc gagttatcac cagaaaggct gctgtctgca 1680

gtggggccag gagtgctggt cactctggga atctcaatta cccctcctta gccgccgtct 1740

ttcaacagta tggaaagcag cacatgtcca cccacttcat cagaactctc accaatgttg 1800

gagaccccaa ctcgctctac aaagtcactg ttgcaccccc t 1841

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<213> Soybean (Glycine max Linn. Merr.)

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Met Asn Asp Thr Leu Glu Ser Arg Ser Pro Arg Asp Ser Asp Gly His

1 5 10 15

Gly Thr His Thr Ala Ser Ile Ala Ala Gly Arg Tyr Val Phe Pro Ala

20 25 30

Ser Thr Met Gly Tyr Ala Lys Gly Met Ala Ala Gly Met Ala Pro Lys

35 40 45

Ala Arg Leu Ala Val Tyr Lys Val Cys Trp Asn Ala Gly Cys Tyr Asp

50 55 60

Ser Asp Ile Leu Ala Ala Phe Asp Ala Ala Val Ala Asp Gly Val Asp

65 70 75 80

Val Val Ser Leu Ser Val Gly Gly Val Val Val Pro Tyr His Leu Asp

85 90 95

Val Ile AlaVal Gly Ala Phe Gly Ala Ser Glu Ala Gly Val Phe Val

100 105 110

Ser Ala Ser Ala Gly Asn Gly Gly Pro Gly Gly Leu Thr Val Thr Asn

115 120 125

Val Ala Pro Trp Val Thr Thr Val Gly Ala Gly Thr Ile Asp Arg Asp

130 135 140

Phe Pro Ala Asp Val Val Leu Gly Asn Gly Lys Val Ile Gly Gly Met

145 150 155 160

Ser Val Tyr Gly Gly Pro Gly Leu Thr Pro Gly Arg Leu Tyr Pro Leu

165 170 175

Val Tyr Ala Gly Ser Asp Gly Tyr Ser Ser Ser Leu Cys Leu Glu Asp

180 185 190

Ser Leu Asp Pro Lys Ser Val Arg Gly Lys Ile Val Val Cys Glu Arg

195 200 205

Gly Val Asn Ser Arg Ala Ala Lys Gly Gln Val Val Lys Lys Ala Gly

210 215 220

Gly Val Gly Met Val Leu Thr Asn Gly Pro Leu Asp Gly Glu Gly Leu

225 230 235 240

Val Ala Asp Cys Gln Val Leu Pro Ala Thr Ser Val Gly Ala Glu Gly

245 250 255

Gly Asp Glu Leu ArgArg Tyr Met Ala Phe Ala Ala Gln Leu Arg Thr

260 265 270

Pro Ala Thr Ala Thr Ile Ile Phe Lys Gly Thr Arg Leu Gly Ile Lys

275 280 285

Pro Ala Pro Lys Val Ala Ser Phe Ser Ala Arg Gly Pro Asn Pro Glu

290 295 300

Ser Pro Glu Ile Leu Lys Pro Asp Val Ile Ala Pro Gly Leu Asn Ile

305 310 315 320

Leu Ala Ala Trp Pro Ser Thr Leu Ser Pro Ser Gly Leu Pro Ser Asp

325 330 335

Glu Arg Arg Ser Gln Phe Asn Ile Leu Ser Gly Thr Ser Met Ala Cys

340 345 350

Pro His Val Ser Gly Leu Ala Ala Leu Leu Lys Ala Ala His Pro Asp

355 360 365

Trp Ser Pro Ala Ala Ile Arg Ser Ala Leu Ile Thr Thr Ala Tyr Thr

370 375 380

Leu Asp Asn Gly Gly Gly Pro Leu Leu Asp Glu Ser Asn Ala Asn Val

385 390 395 400

Ser Ser Val Phe Asp His Gly Ala Gly His Val His Pro Asp Lys Ala

405 410 415

Ile Asn Pro Gly Leu Val TyrAsp Ile Ser Thr Tyr Asp Tyr Val Asp

420 425 430

Phe Leu Cys Asn Ser Asn Tyr Thr Ser His Asn Ile Arg Val Ile Thr

435 440 445

Arg Lys Ala Ala Val Cys Ser Gly Ala Arg Ser Ala Gly His Ser Gly

450 455 460

Asn Leu Asn Tyr Pro Ser Leu Ala Ala Val Phe Gln Gln Tyr Gly Lys

465 470 475 480

Gln His Met Ser Thr His Phe Ile Arg Thr Leu Thr Asn Val Gly Asp

485 490 495

Pro Asn Ser Leu Tyr Lys Val Thr Val Ala Pro Pro

500 505

Claims (5)

1. Soybean subtilisin GmSub1.5-2, the base sequence of which is shown in sequence table SEQ ID NO. 1.

2. A plant expression vector is inserted with a gene shown in a sequence table SEQ ID NO. 1.

3. Application of soybean subtilisin gene GmSub1.5-2 in improving plant disease resistance.

4. Use according to claim 3, characterized in that: the plant is soybean.

5. Use according to claim 4, characterized in that: the disease resistance is the resistance to phytophthora root rot.

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