CN110862995B

CN110862995B - Construction and application of soybean sclerotiniose resistant gene GmPR5 and GmPR5 transgenic plants

Info

Publication number: CN110862995B
Application number: CN201911309597.7A
Authority: CN
Inventors: 赵雪; 姜海鹏; 韩英鹏; 战宇航; 李文滨; 李海燕; 滕卫丽
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2022-06-14
Anticipated expiration: 2039-12-18
Also published as: CN110862995A

Abstract

Construction and application of soybean sclerotinia sclerotiorum resistant gene GmPR5 and GmPR5 transgenic plants belong to the technical field of genetic breeding. In order to research a soybean sclerotinia sclerotiorum disease resisting mechanism and accelerate the soybean sclerotinia sclerotiorum disease resisting breeding process, the invention provides a soybean sclerotiorum disease resisting gene GmPR5, wherein the nucleotide sequence of the soybean sclerotiorum disease resisting gene GmPR5 is shown as SEQ ID No. 1; and a recombinant vector and a recombinant bacterium containing the gene; on the basis of obtaining a transgenic soybean plant by over-expressing a disease-resistant gene GmPR5, the invention determines that the GmPR5 can participate in a soybean sclerotinia sclerotiorum disease resistant reaction through progeny phenotype identification, provides a new thought for obtaining a soybean variety with complete resistance to sclerotinia sclerotiorum, provides a basis for further researching the function and the disease-resistant mechanism of the gene, and provides effective molecular markers and gene resources for soybean sclerotinia sclerotiorum disease resistant molecular design breeding.

Description

Construction and application of soybean sclerotiniose resistant gene GmPR5 and GmPR5 transgenic plants

Technical Field

The invention belongs to the technical field of genetic breeding, and particularly relates to construction and application of soybean sclerotinia sclerotiorum resistant genes GmPR5 and GmPR5 transgenic plants.

Background

Soybean (Glycine max) is an important food crop in China and even the world, and is also one of important sources of edible vegetable protein and edible oil and fat. Soybean sclerotiniose is a high-incidence disease with serious harm degree, and is one of three major diseases causing the most serious yield loss of soybeans worldwide. Soybean Sclerotinia sclerotiorum is a fungal disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, directly influences the quality and yield of soybeans, and has a disease rate of 50-90% when the disease is serious, even the soybeans are out of production. Due to the limitation of the traditional breeding method, the screening and identification of effective disease-resistant genes by using a molecular means are very important for breeding disease-resistant varieties, so that the efficiency and the accuracy of breeding the disease-resistant varieties are improved, and the process of breeding the disease-resistant varieties is accelerated. The mechanism of soybean sclerotinia rot resistance is complex, and a large number of loci and key genes are to be excavated and utilized. As sclerotinia sclerotiorum is a complex quantitative character controlled by multiple genes, soybean genome is huge, polyploidization phenomenon exists in soybean, low transformation rate and the like, so far, people do not excavate genes which play a main effect on sclerotinia sclerotiorum in plants, but some genes which play a relevant role in sclerotinia sclerotiorum resistance are reported, and the genes provide a molecular basis for researching physiological and biochemical processes of sclerotinia sclerotiorum resistance and cultivating excellent sclerotinia sclerotiorum resistance varieties. Jiang et al transformed a nonspecific antibacterial protein gene LJAMP2 in motherwort into rape, and inoculated the transgenic rape with Sclerotinia sclerotiorum, which proved that the LJAMP2 gene is related to the resistance of Sclerotinia sclerotiorum. Donaldson et al (2001) transformed soybean with the wheat Germin gene and increased tolerance of the stem and cotyledon of the transgenic soybean to Sclerotinia sclerotiorum. Zhang et al (2008) cloned the soybean Germin-like gene GmGLP10, and found that the resistance of transgenic tobacco over-expressing the gene to sclerotinia sclerotiorum is improved. Cunha (2010) and the like transform an oxalate decarboxylase gene in a desmodium mori (Flammulina) into soybeans, and the result shows that the lesion extension speed of a transgenic soybean plant is remarkably lower than the lesion area of wild soybeans. Ranjan et al (2017) found that gene silencing of the gene in the GmRBOH-VI group improves the resistance of soybeans to sclerotinia sclerotiorum and reduces the ROS level.

At present, few reports about the PR5 gene exist, and the function of PR5 is not clear.

Disclosure of Invention

In order to research a soybean sclerotinia sclerotiorum disease resisting mechanism and accelerate the soybean sclerotinia sclerotiorum disease resisting breeding process, the invention provides a soybean sclerotiorum disease resisting gene GmPR5, wherein the nucleotide sequence of the soybean sclerotiorum disease resisting gene GmPR5 is shown as SEQ ID No. 1.

The invention also provides a primer for amplifying the soybean sclerotinia sclerotiorum resistant gene GmPR5, wherein the sequence of the primer is shown as SEQ ID No.2-SEQ ID No. 3.

The invention also provides a recombinant vector containing the soybean sclerotinia sclerotiorum resistant gene GmPR 5.

The invention also provides a recombinant bacterium containing the soybean sclerotinia sclerotiorum resistant gene GmPR 5.

The invention also provides a construction method of the soybean sclerotinia sclerotiorum resistant transgenic plant, which comprises the following steps:

1) constructing a recombinant vector for over-expressing the soybean sclerotinia sclerotiorum resistant gene GmPR 5;

2) transforming the recombinant vector into agrobacterium to obtain a recombinant strain;

3) then, the recombinant bacteria are transformed into plants to obtain the soybean sclerotinia sclerotiorum resistant transgenic plants.

Further limited, the intermediate vector used for constructing the recombinant vector in the step 1) is pCambia 3300.

Further limited, the agrobacterium of step 2) is agrobacterium tumefaciens EHA 105.

Further defined, the plant of step 3) is soybean, preferably soybean variety Maple Arrow, Hefeng 25 or Williams 82.

The invention also provides application of the soybean sclerotinia sclerotiorum resistant gene GmPR5 in sclerotinia sclerotiorum resistant crop breeding.

Advantageous effects

The GmPR5 is a newly discovered gene with the function of resisting the soybean sclerotinia rot, and the research determines that the GmPR5 can participate in the soybean sclerotinia rot reaction through the phenotype identification of descendants on the basis of obtaining a transgenic soybean plant by over-expressing a disease-resistant gene GmPR5, provides a new idea for obtaining a soybean variety with complete resistance to the sclerotinia rot, provides a basis for further researching the function and the disease-resistant mechanism of the gene, and provides effective molecular markers and gene resources for the molecular design and breeding of the soybean sclerotinia rot.

Drawings

Fig. 1 cloning of the GmPR5 gene, wherein M: DL 2000; 1: PCR products; 2: water control;

FIG. 2 tissue/organ specific expression of the soybean GmPR5 gene, R-root; s-stem; l-leaf; h-hypocotyl; c-cotyledon; f-flower; p-petiole; s-seeds;

FIG. 3 is a graph showing the change in the relative expression level of GmPR5 within 24 hours after sclerotinia sclerotiorum inoculation, wherein MC: disease resistant variety "Maple Arrow" control; MT: treating a disease-resistant variety 'mapleArrow'; HC: the susceptible variety 'Hefeng 25' is compared; HT: treating susceptible varieties 'Hefeng 25';

FIG. 4 is the increase amplitude of the relative expression amount of GmPR5 within 24h after sclerotinia sclerotiorum inoculation, wherein MA: disease resistant variety "Maple Arrow"; HF: susceptible variety "confluent 25": p <0.05, p < 0.01;

fig. 5 expression amount of GmPR5 in soybean leaves under different hormone treatment conditions, a: salicylic acid-treated susceptible variety "Hefeng 25" and water control; b: treating a disease-susceptible variety 'Hefeng 25' with hydrogen peroxide and comparing with water; c: jasmonic acid treatment susceptible variety 'Hefeng 25' and water control; d: ethylene treatment susceptible variety "Hefeng 25" and water control;

FIG. 6 expression level of GmPR5 in soybean leaves under different stress treatment conditions, A: salicylic acid treated disease resistant variety "Maple Arrow" and water control; b: jasmonic acid treatment of disease resistant variety "Maple Arrow" against water control; c: hydrogen peroxide treated disease resistant variety "Maple Arrow" and water control; d: ethylene treated disease resistant variety "Maple Arrow" and water control;

FIG. 7 transformation of E.coli with the plant expression vector, wherein M: DL 2000; 1-3: PCR products; 4, water control;

FIG. 8 transformation of Agrobacterium tumefaciens with recombinant vector, wherein M: DL 2000; 1, water control; 2-6: PCR products;

FIG. 9 PCR assay of transgenic soybean, wherein M: DL 2000; 1-5: the receptor is GmPR5 gene over-expression plant of 'mapleArrow'; 6-11: the receptor is a GmPR5 gene over-expression plant of 'Hefeng 25'; 12: negative control; 13: water; 14: a positive control;

FIG. 10 PCR assay of transgenic soybean, wherein M: DL 2000; 1: water; 2-4: the receptor is GmPR5 gene over-expression plant of 'mapleArrow'; 5-7: the receptor is a GmPR5 gene over-expression plant of 'Hefeng 25';

FIG. 11 disease-resistant variety "mapleArrow" T₁qRT-PCR detection of soybean plants transferred with GmPR5 gene, wherein CK: disease resistant variety "Maple Arrow"; 1-5: GmPR5 gene over-expression disease-resistant variety T₁Plant generation;

FIG. 12 the disease-susceptible varieties Hefeng 25T₁qRT-PCR detection of soybean plants transferred with GmPR5 gene, wherein CK: susceptible variety "Hefeng 25"; 1-5: disease-sensitive variety T with GmPR5 gene overexpression₁Plant generation;

FIG. 13T₁Carrying out Western blotting detection on transgenic soybean plants, wherein M: marker 10-170 kd; 1: positive control 2-4; a plant over-expressing GmPR5 gene; 5: a non-transgenic plant;

FIG. 14T₁The leaf change of a 'Hefeng 25' plant transformed with a GmPR5 gene after inoculation of soybean sclerotinia sclerotiorum is shown in the specification, wherein a: a 'hefeng 25' control; b-c: 'Hefeng 25' plant over expressing GmPR5 gene;

FIG. 15T₁Transformation of leaves of soybean inoculated with soybean sclerotinia sclerotiorum after transfer of GmPR5 gene 'Maple Arrow' plant, wherein a: "Maple Arrow" control; b-c: a "Maple Arrow" plant overexpressing the GmPR5 gene;

FIG. 16T₁The change of leaves of a 'Hefeng 25' plant transformed with a GmPR5 gene after being inoculated with phytophthora sojae for 3d is shown in the specification, wherein a: "hedeng 25" control; b-d: 'Hefeng 25' plant over expressing GmPR5 gene;

FIG. 17T₁And (3) leaf change after 3d of soybean phytophthora sojae is inoculated to a 'Maple Arrow' plant with a GmPR5 gene by transfer, wherein a: 'Maple Arrow' control; b-d: 'Maple Arrow' plant overexpressing the GmPR5 gene.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The pharmaceutical reagents, YEP solid plates, MS0 medium and the like used in the following examples were, unless otherwise specified, conventional reagents or purchased from conventional biochemical reagent stores.

The plant varieties used in the present invention: the world recognized sclerotinia-resistant varieties 'Maple Arrow' (partially resistant varieties), susceptible varieties 'coseng 25' and Williams 82 introduced in canada.

Pathogenic bacteria: sclerotinia sclerotiorum is collected from the onset plot of sclerotinia sclerotiorum of Daqing big beans in Heilongjiang province, and is separated, purified and stored in a laboratory, and the pathogenic bacteria are recorded in a document which is published in 2014 such as Zhao snow and the like and is entitled "disease tolerance evaluation and resource screening of soybean germplasm for sclerotinia sclerotiorum" (Anhui agricultural science, 2014, v.42, No.449(16): 5014-. Phytophthora: the method is described in a literature which is published in 2014 of Zhao Xue and the like and is entitled "excavation of excellent allelic variation of phytophthora root rot resistance of soybean germplasm resources" [ J ]. Soybean science 2014,33(4): 000488-.

The noun abbreviations are explained as follows:

str: streptomycin; kan: kanamycin; rif: rifampin; PPT: glufosinate-ammonium;

and SA: salicylic acid; JA: jasmonic acid; ET: ethylene.

The primer information related to the invention is as follows:

primer set 1

1S:5’-GGATCCATGTATGAAAGTATTGAAAACTATCT-3’,

1A:5’-ACTAGTTCATTCGATATTGTTTGGTGTTCT-3’；

Primer set 2

2S:5’-AGAACAAAACAACTTGATGCCTATTAG-3',

2A:5’-CGCAACTTTCCCTTGAACACAT-3’；

Primer set 3

3S:5’-TTTGGCGATGAGACTGAGGAA-3',

3A:5’-TCCCATAACCAACAGGAACCAG-3’；

Primer set 4

4S:5’-GTGTCAGCCATACTGTCCCCATTT-3’,

4A:5’-GTTTCAAGCTCTTGCTCGTAATCA-3’；

Primer set 5

5S:5’-CGGGGGACTCTTGACATGTATGAAAGTATTGAAAACT-3’,

5A:5’-GTCAGATCTACCATGCATTCGATATTGTTTGGTGTTC-3’；

Primer pair 6

6S:5’-GCGGTACCGGCAGGCTGAAG-3',

6A:5’-CCGCAGGAACCGCAGGAGTG-3’；

Primer set 7

7S:5’-ATGCCATCATTGCGATAAAGG-3’,

7A:5’-TCCTTTTACTTGCCATCTCCAT-3’；

Primer pair 8

8S:5’-GTTTCAAGCTCTTGCTCGTAATCA-3’,

8A:5’-GTGTCAGCCATACTGTCCCCATTT-3’

Example 1 cloning of the GmPR5 gene.

1) Taking a soybean anti-bacterial nuclear disease variety 'mapleArrow' as a material, taking the material when a first group of three compound leaves grow out, extracting total RNA and carrying out reverse transcription to synthesize a cDNA first chain.

2) According to the predicted GmPR5 gene sequence on the Phytozome, a Primer 5 software is utilized to design a gene cloning Primer (Primer pair 1), cDNA is taken as a template to carry out RT-PCR reaction, the reaction system is as follows, and the reaction program is as follows: 5min at 94 ℃; 37 cycles: 30s at 95 ℃, 30s at 60 ℃ and 80s at 72 ℃; 10min at 72 ℃. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis and the target fragment was recovered and purified by gel (FIG. 1).

3) According to the procedure of the PGM-T cloning kit of TIANGEN company, the gel recovery product obtained above was linked with a cloning vector, and Top10 large intestine competent cells were transformed, and a single clone was picked up and verified by PCR and sequencing. Finally obtaining the GmPR5 gene of which the target fragment size is 996 bp. The GmPR5 gene is connected with a PGM-T carrier and successfully transformed into escherichia coli, and sequencing verification confirms that the GmPR5 gene and the nucleotide sequence are shown as SEQ ID NO. 1.

Example 2 analysis of expression pattern of GmPR5 gene.

1. Pathogenic bacteria, sclerotinia sclerotiorum, are collected from the disease-causing plots of the soybean sclerotinia sclerotiorum in the Daqing of Heilongjiang province, are separated and purified by a laboratory, and inoculated bacteria are propagated on a PDA culture medium.

2. The materials are obtained.

(1) The seeds were sown with "Maple Arrow" and William82(William82 as experimental reference control group), and roots, stems, leaves, hypocotyls and cotyledons, flowers and seeds were taken in three replicates each during their growth to maturity and at the respective three generations and three generations.

(2) Seeding a disease-resistant variety 'Maple Arrow' and a susceptible variety 'Hefeng 25' in vermiculite, growing until true leaves are extracted, separating leaf edges until the true leaves are completely unfolded, cutting a wound with the depth of about 1 mm of one centimeter at the position 1/3 close to the surface of a lower hypocotyl, cutting a sclerotinia sclerotiorum (hypha is in a growth period) which is bred in advance into 10 × 5mm fungus blocks, aligning the front of the hypha to the inner side of the hypocotyl, winding the hypocotyl with a sealing film to fix the fungus blocks, respectively placing the inoculated Maple Arrow 'and the Hefeng 25' for 0h, 4h, 8h, 12h and 24h for timed sampling of the hypocotyl, and repeating the steps for three times at each time point.

(3) Sowing susceptible variety 'Hefeng 25' in vermiculite, growing to V2 stage, and performing Jasmonic Acid (JA) 50mmol/L, Salicylic Acid (SA) 0.5mmol/L, ethylene (CT) 0.5mmol/L, and hydrogen peroxide (H) 0.5mmol/L₂O₂) Treatment ofSoybean leaves treated for 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h were taken, respectively, and repeated three times at each time point.

And 3, synthesizing cDNA.

Total RNA from the sample from step 2 was extracted (Trizol Total RNA extraction reagent, TIANGEN BIOTECH, DP 405-02) and reverse transcribed to synthesize the first strand of cDNA (ReverTra Ace qpcr RT Master Mix with gDNA remover, TOYOBO, FSQ-301).

4. And (4) performing fluorescent quantitative PCR analysis.

1) The soybean housekeeping gene GmActin 4(Genbank No: AF049106), quantitative primers (primer pair 2) of the candidate gene GmPR5 were designed according to the qRT-PCR method, loading was performed according to the reaction system of the fluorescence quantitative SYBR Green kit of TIANGEN corporation, using a rowse LightCycler 480 quantitative instrument, PCR reaction conditions: 5min at 95 ℃; 10s at 95 ℃,20 s at 60 ℃, 32s at 72 ℃ and 40 cycles; 95 ℃ for 1min, 65 ℃ for 30s and 97 ℃ for 30 s.

2) Tissue specific expression analysis

The GmPR5 gene is expressed in all tissues and organs of soybean, has the highest expression level in hypocotyl, is followed by root and stem, and has the lowest expression level in cotyledon and petiole, thus proving that the gene is possibly involved in the anti-soybean sclerotiniose reaction (figure 2).

3) Sclerotinia sclerotiorum stress-induced expression analysis

Carrying out sclerotinia sclerotiorum stress treatment on hypocotyls of a soybean disease-resistant variety 'Maple Arrow' and a susceptible variety 'Hefeng 25', analyzing the expression conditions of GmPR5 in the sclerotinia sclerotiorum stress treatment and the disease-resistant variety 'Maple Arrow' and the susceptible variety 'Hefeng 25' which are not subjected to stress treatment by a qRT-PCR method, wherein the result shows that the expression quantity of a GmPR5 gene in the disease-resistant variety and the susceptible variety which are subjected to sclerotinia sclerotiorum treatment has an obvious rising trend compared with an untreated contrast, and shows that the candidate gene GmPR5 responds to sclerotinia sclerotiorum stress, wherein the expression quantities of the GmPR5 genes of the treated disease-resistant variety and the susceptible variety reach the maximum value within 2 h; the amplification of the GmPR5 gene of the disease-resistant variety is obviously higher than that of the disease-resistant variety 'Hefeng 25' at each time point after inoculation, which indicates that the gene interacts with sclerotinia sclerotiorum more strongly in the disease-resistant variety 'Maple Arrow' and is probably the reason for causing the resistance difference of the disease-resistant variety (figure 3 and figure 4).

4) Analysis of hormone stress-induced expression

SA, JA and ET are important signal molecules in the signal transduction pathway of plant defense responses, while H₂O₂And also as a signaling molecule involved in regulating plant response to biotic and abiotic stress. SA, JA, ET and H are carried out on the susceptible variety 'Hefeng 25' of the soybean V2 stage₂O₂Hormone treatment, namely extracting and treating soybean leaf RNA for 0h, 2h, 4h, 8h, 12h, 24h, 36h and 48h respectively, analyzing the relative expression quantity of the GmPR5 gene by a qRT-PCR method (figure 5 and figure 6), and indicating that the expression quantity of the GmPR5 gene is obviously changed under SA treatment, the expression quantity is increased firstly and then decreased, and reaches the maximum value after 4h treatment; the GmPR5 gene is in JA, ET and H₂O₂The expression amount is not obviously changed under the treatment; the SA can quickly activate the expression of GmPR5 gene in the Hefeng 25, JA, ET and H₂O₂Has no influence on the expression of the GmPR5 gene.

The expression quantity of the GmPR5 gene is obviously changed under the SA treatment, the expression quantity is increased and then decreased, and the maximum value is reached after the treatment for 4 hours; the GmPR5 gene is in JA, ET and H₂O₂The expression amount is not obviously changed under the treatment; the result is consistent with the result of the susceptible variety 'Hefeng 25' under hormone treatment, and the GmPR5 gene is preliminarily deduced to possibly play a role in an SA-mediated disease-resistant signal transduction pathway to participate in the disease-resistant process of plants.

Example 3 subcellular localization analysis of GmPR5 Gene

pCambia1302 as a backbone vector and In-Fusion seamless ligation kit (Clontech)

HD Cloning Kit), according to the provided operation instruction, completes the construction of pCambia1302-GmPR5-GFP fusion expression vector, specifically: the full-length CDS primer of the GmPR5 gene was designed and 15bp flanking the Nco I cleavage site from pCambia1302 was added as a modification at the 5' end of the primer (primer pair 5) in the reaction program: 3min at 98 ℃; 38 cycles: 10s at 98 ℃, 10s at 55 ℃ and 5s at 72 ℃; 10min at 72 ℃. To proceed withPCR reaction, recovering gel, single digestion of pCambia1302 plasmid with Nco I enzyme, recovering gel, and mixing recovered gel with carrier fragment

The HD Cloning Kit operation shows that the ligation reaction is completed, escherichia coli is transformed, single-spot PCR verification is finally picked, the obtained positive single spots are extracted, positive plasmids are extracted, sequencing analysis is carried out, tobacco epidermal cells are expressed homeopathically through an injection method, fluorescence detection is carried out, and the result shows that the GmPR5 gene is mainly positioned on cell membranes, cytoplasm and cell skeletons and is accompanied with partial nuclear localization.

Example 4 construction of transgenic soybean plants with GmPR5 gene.

1) Constructing a recombinant vector: the pCambia3300 vector plasmid and the pGM-T-GmPR5 vector plasmid prepared in example 1 are subjected to double enzyme digestion by restriction enzymes BamH I and Spe I respectively, after enzyme digestion, agarose gel electrophoresis detection is carried out, purified pCambia3300 vector skeleton and GmPR5 gene enzyme digestion fragments are recovered, and the purified products are connected to obtain a recombinant vector pCambia3300-GmPR5 containing GmPR5 gene. After the recombinant vector is transformed into escherichia coli, a single spot is picked, PCR amplification identification is carried out, and the result shows that a band of the GmPR5 gene exists at the position of 996bp, which shows that the GmPR5 gene is connected with the expression vector and is successfully transformed into the escherichia coli (figure 7).

2) Recombinant vector transformation of agrobacterium tumefaciens

The pCambia3300-GmPR5 vector is transferred into Agrobacterium tumefaciens EHA105 by a freeze-thaw method, and the transferred bacterial liquid is identified by PCR to obtain a target band with the length of 996bp (figure 8), which proves that pCambia3300-GmPR5 has been successfully transferred into Agrobacterium tumefaciens EHA 105.

3) Transformation of soybean by agrobacterium tumefaciens mediated method:

a. a GmPR5 gene over-expression material is created by a soybean stem tip transformation method mediated by agrobacterium tumefaciens, and an over-expression transformation receptor is a susceptible variety 'Hefeng 25' and a partially resistant variety 'Maple Arrow'.

b. Preparing bacterial liquid: prepared bacterial liquid is taken to be respectively streaked on YEP solid plates (50mg/mL Str, 50mg/mL Kan and 25mg/mL Rif) for culture at 28 ℃, single colonies are selected to be inoculated in YEP liquid culture medium (50mg/mL Str, 50mg/mL Kan and 25mg/mL Rif), shaking culture is carried out at the temperature of 28 ℃ and 200rpm for 1-2 days, and 1-2mL of bacterial liquid is taken to be inoculated in 50mL of fresh YEP liquid culture medium for shaking culture until OD600 is 0.6-0.8.

c. Seed sterilization: the method comprises the steps of selecting full and sterile spot seeds in a culture dish, placing the seeds in a dryer of a ventilation kitchen by adopting a chlorine sterilization method, pouring 96ml of sodium hypochlorite into a triangular flask of the dryer, quickly adding 6ml of concentrated hydrochloric acid, and quickly covering and sealing the flask. Sterilizing for 16h, placing in a clean bench, blowing off residual chlorine gas for about 30min, and sealing for use.

d. Seed germination: inoculating the sterilized seed to MS via navel₀In the culture medium, 10 grains were inoculated in each flask, and the germination was carried out for 5-6 days at 23 ℃ under 16h light/8 h dark conditions.

e. Preparing a stem tip explant: when the seed germinates to the point that the cotyledon breaks through the seed coat, the seed coat is removed by using a pair of tweezers and a scalpel, half of the two halves of the seed coat is cut off, axillary buds between the cotyledon and a growing point are slightly scraped by using the scalpel, 3-5 wounds are slightly scratched at the cotyledon node, and the rest part is used as an explant for infection.

f. Infection and co-culture: and putting the prepared explant into the invasion dye liquor for vacuumizing for 10min under the condition of 0.6 pa. Washing the explant after vacuum infection with sterilized distilled water for three times, sucking the infection liquid on the surface of the explant with sterile paper, inserting into MS₀Co-culturing for 3 days in the culture medium, observing the revival condition, and determining the transplanting time according to the green turning condition of the explant.

Example 5. identification of plants overexpressing the GmPR5 gene phenotypic analysis.

Identification of plants with overexpression of the GmPR5 gene.

1)T₁PCR detection of over-expressed plants

In the embodiment 4 of the invention, through a soybean stem tip transformation method mediated by agrobacterium tumefaciens, the GmPR5 gene overexpression vectors are respectively transferred into a disease-resistant variety 'mapleArrow' and a susceptible variety 'Hefeng 25', 300 and 200 plants are respectively transformed, DNA is extracted from the obtained plants,PCR identification is carried out by using Bar (primer pair 6) primers for 3 times, plants of target fragments with the size of 996bp are detected to be identified as positive, and 5 positive plant disease-resistant varieties of 'Maple Arrow' and 25 susceptible varieties of 'Hefeng 25' are finally obtained. T to be harvested₀Identifying as transgenic plant, sowing, waiting for T₁When the first three-leaf complex of the generation plant is completely unfolded, the leaves of the plant are taken to extract DNA, and the DNA is detected by PCR with Bar primer (primer pair 7), and the soybean plant transformed with GmPR5 gene is used for further screening (figure 9, figure 10).

2)T₁qRT-PCR detection of over-expressed plants

Quantitative primers are designed according to the gene sequence of GmPR5, and a qRT-PCR method is utilized to respectively treat T of a disease-resistant variety ' mapleArrow ' and a susceptible variety ' Hefeng 25₁And generation (FIG. 11 and FIG. 12), the relative expression amount of the GmPR5 gene in the soybean leaves transformed with the GmPR5 gene is detected to be higher than that of a control variety, which indicates that the GmPR5 gene generates certain disease resistance to soybean sclerotinia sclerotiorum. Taking the relative expression quantity which is 2.5 times more than that of the contrast as a reference, and counting out the disease-resistant variety 'mapleArrow' T ₁3 soybean plants transformed with the GmPR5 gene are obtained; susceptible variety 'Hefeng 25' T ₁3 soybean plants transformed with the GmPR5 gene are obtained.

3)T₂Western Blot analysis of transgenic plants, fresh leaves of partial T2-substituted plants are taken to extract total protein of soybean tissues, proteins with different sizes are separated by running electrophoresis and transferred to a PVDF membrane, and the proteins are subjected to hybridization and color development by a primary antibody and a secondary antibody, and finally Bar protein is not detected (figure 13).

Phenotypic identification of plants overexpressing the GmPR5 gene.

1)T₁And (3) carrying out inoculation phenotype analysis on the generation transgenic plant sclerotinia sclerotiorum.

When T is₁When the generation plant is in the R3 stage, carrying out an inoculation experiment on the leaves of the transgenic plant, carrying out inoculation experiment on the sclerotinia sclerotiorum cultured for 4 days, taking a1 cm-diameter agar block on a solid culture medium for growth of the sclerotinia sclerotiorum, and inoculating the agar block on the leaves. Transferred to GmPR5 Gene "The soybean leaf of the Maple Arrow' plant generates an unobvious water stain-like scab on the leaf surface at the initial stage, the scab at the later stage becomes dark brown, and finally, the leaf turns yellow and the scab expansion stops. "Hefeng 25" contrasts soybean leaves and produces bigger dark green water stain-like speckles in the initial stage, and the later stage scabs are expanded faster and are almost covered on the whole leaves. The soybean leaf of the 'Hefeng 25' plant transformed with the GmPR5 gene generates dark green water stain-like scabs on the early leaf surface, the scabs on the later stage become dark brown, yellow halos are formed on the outer part, finally, the leaf turns yellow, a small amount of white hypha is generated when the humidity is high, and then the scabs are expanded and stopped. Indicating that the GmPR5 gene is resistant to soybean sclerotinia sclerotiorum (FIGS. 14 and 15).

2)T₁And (3) carrying out phytophthora inoculation phenotype analysis on the transgenic plants.

T₁Carrying out phenotype analysis on the generation transgenic plant phytophthora inoculation, and taking T₁Carrying out an phytophthora in-vitro inoculation experiment on three compound leaves of a transgenic plant R3 period, culturing the phytophthora for 4 days, taking a1 cm-diameter agar block on a solid culture medium for growth of the phytophthora, and inoculating the agar block on leaves, wherein the result shows that the 'Hefeng 25' contrast plant leaves become yellow after 3 days of inoculation, the inoculated part of the leaves generate brown water stain-like speckles, the center of each scab is yellow brown, the dark green water stain-like speckles are generated, the periphery is light brown, yellow scabs are locally formed, the later-stage scab becomes yellow, and the scab is not obviously expanded; the leaf inoculation part of the plant of the 'Hefeng 25' transformed with the GmPR5 gene generates dark green water stain-like scab, the part of the scab is browned, the dark green water stain-like scab is not obvious, the color of the scab is lightened at the later stage, and the scab is expanded and stopped. This indicates that the GmPR5 gene is involved in the resistance response of soybean to phytophthora blight (FIGS. 16 and 17).

In conclusion, the obtained GmPR5 is over-expressed in soybean, and the gene is proved to be capable of remarkably improving the resistance of a receptor soybean germplasm to sclerotinia sclerotiorum, and has a function in an SA-mediated disease-resistant signal transduction pathway and a certain resistance to phytophthora sojae. Provides effective molecular markers and gene resources for the molecular design and breeding of the soybean sclerotinia rot resistance.

Nucleotide sequence listing

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<210> 1

<211> 996

<212> DNA

<213> GmPR5 CDS

<400> 1

atgtatgaaa gtattgaaaa ctatctagaa caaaacaact tgatgcctat tagatactca 60

tacaaggaaa ttaagaagat gggtgggggt ttcaaagaca agttgggcga aggaggatat 120

ggacatgtgt tcaagggaaa gttgcgtagt gggcctagtg tggcaattaa aatattgggt 180

aaattaaaag gtaatggaca agattttatc aatgaagttg caactattgg aagaatacat 240

catcaaaatg tagtacaatt aattggattt tgtgttgagg gatcaaaacg tgctttgctt 300

tgtgaattta tgcccagtgg atctctcgat aaatttattt tttctaaaga tggaagtaag 360

catttaagct atgacaaaat atataatata tcaattggag ttgctcgtgg gatttcttat 420

ctccaccatg ggtgtgagat gcagattttg cattttgata tcaagcccca caacatctta 480

ctagatgaaa attttatccc aaaaatctct gactttggat tggcaaagct atatccaata 540

gataatagca ttgtcacaat gactggagta agagggacaa ttgggtacat ggctccagaa 600

ttattttata aaaatattgg aggaatatcc tataaggctg atgtttatag ttttggaatg 660

cttttgatgg agatggcaag taaaaggaaa aacctaaatc cttatgcaga gcgttcaagc 720

caactatact atcctttttg gatttataat catcttgtag aagagaaaga tatagaaacg 780

aaagatgtca cagaggagga aaataaaata gcaaagaaga tgatcatagt tgcactatgg 840

tgcatacaat tgaaaccaaa tgatcgtcca tcgatgaaca aggtagtgga aatgcttgaa 900

ggagacattg agaacctaga aataccccca aagcctactc tatatccaca tgaaacgacg 960

ataagggatc aaagaacacc aaacaatatc gaatga 996

<210> 2

<211> 32

<212> DNA

<213> 1S

<400> 2

ggatccatgt atgaaagtat tgaaaactat ct 32

<210> 3

<211> 30

<212> DNA

<213> 1A

<400> 3

actagttcat tcgatattgt ttggtgttct 30

<210> 4

<211> 27

<212> DNA

<213> 2S

<400> 4

agaacaaaac aacttgatgc ctattag 27

<210> 5

<211> 22

<212> DNA

<213> 2A

<400> 5

cgcaactttc ccttgaacac at 22

<210> 6

<211> 21

<212> DNA

<213> 3S

<400> 6

tttggcgatg agactgagga a 21

<210> 7

<211> 22

<212> DNA

<213> 3A

<400> 7

tcccataacc aacaggaacc ag 22

<210> 8

<211> 24

<212> DNA

<213> 4S

<400> 8

gtgtcagcca tactgtcccc attt 24

<210> 9

<211> 24

<212> DNA

<213> 4A

<400> 9

gtttcaagct cttgctcgta atca 24

<210> 10

<211> 37

<212> DNA

<213> 5S

<400> 10

cgggggactc ttgacatgta tgaaagtatt gaaaact 37

<210> 11

<211> 37

<212> DNA

<213> 5A

<400> 11

gtcagatcta ccatgcattc gatattgttt ggtgttc 37

<210> 12

<211> 20

<212> DNA

<213> 6S

<400> 12

gcggtaccgg caggctgaag 20

<210> 13

<211> 20

<212> DNA

<213> 6A

<400> 13

ccgcaggaac cgcaggagtg 20

<210> 14

<211> 21

<212> DNA

<213> 7S

<400> 14

atgccatcat tgcgataaag g 21

<210> 15

<211> 22

<212> DNA

<213> 7A

<400> 15

tccttttact tgccatctcc at 22

<210> 16

<211> 24

<212> DNA

<213> 8S

<400> 16

gtttcaagct cttgctcgta atca 24

<210> 17

<211> 24

<212> DNA

<213> 8A

<400> 17

gtgtcagcca tactgtcccc attt 24

Claims

1. The soybean sclerotinia sclerotiorum resistant gene GmPR5 is characterized in that the nucleotide sequence of the soybean sclerotinia sclerotiorum resistant gene GmPR5 is shown as SEQ ID No. 1.

2. The primer for amplifying the soybean sclerotinia rot resistant gene GmPR5 as claimed in claim 1, wherein the sequence of the primer is shown as SEQ ID No.2-SEQ ID No. 3.

3. A recombinant vector comprising the soybean sclerotinia sclerotiorum resistant gene GmPR5 according to claim 1.

4. A recombinant bacterium containing the soybean sclerotinia sclerotiorum resistant gene GmPR5 according to claim 1.

5. A method for constructing a soybean sclerotiniose-resistant transgenic plant is characterized by comprising the following steps:

1) constructing a recombinant vector for over-expressing the soybean sclerotinia sclerotiorum resistant gene GmPR5 of claim 1;

6. The method according to claim 5, wherein the intermediate vector used in the recombinant vector construction of step 1) is pCambia 3300.

7. The method according to claim 5, wherein the Agrobacterium of step 2) is Agrobacterium tumefaciens EHA 105.

8. The method according to claim 5, wherein the plant of step 3) is soybean.

9. The method according to claim 8, wherein the soybean is selected from the group consisting of MapleArrow, Hefeng 25 and Williams 82.

10. The use of the soybean sclerotinia sclerotiorum resistant gene GmPR5 according to claim 1 in the breeding of sclerotinia sclerotiorum resistant crops.