CN112646818B

CN112646818B - Soybean gene GmTCM1 as well as obtaining method and application thereof

Info

Publication number: CN112646818B
Application number: CN202011641417.8A
Authority: CN
Inventors: 赵雪; 韩英鹏; 常宏; 井妍; 赵克臻; 战宇航; 李文滨
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-06-28
Anticipated expiration: 2040-12-31
Also published as: CN112646818A

Abstract

A soybean gene GmTCM1 and an obtaining method and application thereof belong to the technical field of soybean genetic breeding. In order to improve the content of soybean isoflavone and obtain a transgenic soybean plant with high isoflavone, the invention clones a soybean gene GmTCM1 from a soybean 27 of a soybean variety with high isoflavone content, and biological information analysis and preliminary analysis of gene functions of the gene show that the GmTCM1 gene can improve the content of soybean isoflavone and participate in resistance reactions to abiotic stress such as drought and salt and biotic stress such as sclerotinia sclerotiorum, phytophthora and botrytis cinerea. The invention has important theoretical significance and practical value for accelerating the breeding process of the new high isoflavone disease-resistant and stress-resistant soybean variety and improving the breeding efficiency.

Description

Soybean gene GmTCM1 as well as obtaining method and application thereof

Technical Field

The invention belongs to the technical field of soybean genetic breeding, and particularly relates to a soybean gene GmTCM1, and an obtaining method and application thereof.

Background

Soybean (Glycine max) is an important grain and economic crop in China and even the world. Meanwhile, soybeans are the main source of natural isoflavones. The isoflavone is used as an important secondary metabolite of the soybean, has an important effect on the growth and development of the soybean, and is also used as a health food and a medicinal application, thereby being beneficial to the health of human beings. Therefore, the isoflavone content of the soybeans becomes an important breeding target trait of the soybeans.

The seedlings of rape are researched by Meimeirong (2008), the low-concentration soybean isoflavone is found to reduce the damage of the seedlings under drought stress, and the Chenghao (2008) research finds that the higher the isoflavone content is, the lower the incidence rate of mosaic virus of the material is. Van Etten HD (1961) found soybean plants infected with the plant pathogenic fungus Sclerotinia sclerotiorum isoflavone levels significantly increased. Hershman (1945) found that isoflavone concentration of resistant and susceptible soybean varieties 2-3 days after soybean cyst nematode infection is higher than that of healthy plants. However, in the reported studies relating to soybean isoflavones, the studies confirmed the resistance function of isoflavones, but no stable over-expression positive plants were definitely obtained.

Because the traditional breeding method has the limitations of long period, small variation range and the like, a new soybean variety with high isoflavone content is difficult to obtain. Therefore, the gene for controlling the isoflavone content of the soybean is screened and identified by utilizing a molecular means and is applied to breeding, and the molecular method has important significance for accelerating the breeding of varieties with high isoflavone content and stress resistance.

Disclosure of Invention

1. In order to increase the isoflavone content of soybean and obtain transgenic soybean plant with high isoflavone. The invention provides a soybean gene GmTCM1, wherein the nucleic acid sequence of the soybean gene GmTCM1 is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2.

Further limiting, the soybean gene GmTCM1 is cloned from soybean 27 in a soybean variety with high isoflavone content.

The invention also provides an obtaining method of the soybean gene GmTCM1, which comprises the following steps:

step one, extracting total RNA from soybean leaves of a variety of Zhongdou 27 and performing reverse transcription to synthesize a cDNA first chain;

step two, designing a pair of cloning primers by taking a full-length gene sequence with the accession number of Glyma.20G114200 as a reference sequence of a gene GmTCM1, modifying an upstream primer by using restriction enzyme BstEII to obtain an upstream primer GmTCM1-S, and modifying a downstream primer by using restriction enzyme BglII to obtain a downstream primer GmTCM 1-A;

step three, taking the cDNA obtained in the step one as a template, performing PCR amplification by using the upstream and downstream primers obtained in the step two, and purifying a PCR product by using glue recovery;

and step four, connecting the purified PCR product with a pGEM-T vector, converting the connecting product into an escherichia coli competent cell, picking a single spot, and carrying out PCR and sequencing verification to finally obtain the GmTCM1 gene.

Further limited, the sequence of the upstream primer GmTCM1-S is shown as SEQ ID NO.3, and the sequence of the downstream primer GmTCM1-A is shown as SEQ ID NO. 4.

Further limited, the reference sequence of the gene GmTCM1 is shown as SEQ ID No. 5.

The invention also provides application of the soybean gene GmTCM1 in biotic stress resistance and abiotic stress resistance.

Further defined, biotic stress refers to soybean fungal diseases.

Further defined, abiotic stresses refer to drought and salt stresses.

Further defined, the fungal disease refers to one or more than two of soybean diseases caused by sclerotinia sclerotiorum, phytophthora and grey speck.

Further limited, the application is that transgenic soybean plants are obtained by over-expressing GmTCM1 gene, and then the obtained transgenic soybean plants are subjected to subsequent subculture propagation and identification to obtain stable high isoflavone disease-resistant and stress-resistant genetic materials.

Advantageous effects

In the invention, the GmTCM1 gene is screened out by fine positioning in the early stage, and the function of the GmTCM1 gene is preliminarily identified by means of bioinformatics analysis, correlation analysis of candidate gene pathways, treatment of biotic stress and abiotic stress, dynamic expression pattern analysis in the grain development process, subcellular positioning, genetic transformation and the like.

According to the research, a target gene GmTCM1 is transferred into soybean hairy roots and whole soybean plants through an agrobacterium rhizogenes and agrobacterium tumefaciens mediated soybean genetic transformation method, and identification results show that as a C4H gene family member, a gene GmTCM1 can respond to infection of pathogenic bacteria microorganisms, the content of isoflavone in transgenic positive plants is higher than that of wild plants, the over-expression of the transgenic positive plants in soybeans is presumed to possibly cause the increase of C4H enzyme activity, so that the synthesis of isoflavone is promoted, and the function of isoflavone phytoprotectant is exerted, so that the resistance of the soybeans to fungal diseases is improved; furthermore, studies in arabidopsis have shown that GmTCM1 can improve salt and drought tolerance of plants to some extent.

The experimental result of the invention proves that the GmTCM1 gene can participate in the abiotic stress resistance reaction of drought resistance and salt resistance and the biotic stress resistance reaction of sclerotinia sclerotiorum, phytophthora and botrytis cinerea, and the obtained transgenic material can become stable homoisoflavone and disease-resistant and stress-resistant genetic material through the subsequent subculture propagation and identification. The invention has important theoretical significance and practical value for accelerating the breeding process of the new high isoflavone disease-resistant and stress-resistant soybean variety and improving the breeding efficiency.

Drawings

FIG. 1 shows the restriction enzyme digestion identification result of pCAMBIA3301-GmTCM1 recombinant plasmid;

FIG. 2 alignment of amino acid sequence of GmTCM1 gene and soybean homologous gene thereof;

FIG. 3.TCM protein phylogenetic tree;

FIG. 4 shows the expression of GmTCM1 gene in the period of R5-R8 in soybean seeds, wherein A is the expression of GmTCM1 gene in the period of R5-R8 in the seeds of Zhongdou 27 and Jiunnong 20; b is the expression condition of the GmTCM1 gene in R5-R8 period in seeds of the strain 1 and the strain 2;

FIG. 5 shows the expression level of GmTCM1 in soybean leaves under different stress treatment conditions, wherein A is the expression level of GmTCM1 gene under NaCl treatment; b is the expression of GmTCM1 gene under PEG treatment; c is the expression of GmTCM1 gene under jasmonic acid treatment; d is the expression of GmTCM1 gene under the treatment of salicylic acid;

FIG. 6 shows PCR detection of specific primers of soybean plants transformed with GmTCM1 gene;

FIG. 7 Bar primer PCR detection of soybean plants transformed with GmTCM1 gene;

FIG. 8. qRT-PCR detection of over-expressed plants at T1 generation;

FIG. 9 Western blotting detection of T1 transgenic soybean plants;

FIG. 10 shows the change of leaves of a T2-generation GmTCM1 gene plant inoculated with sclerotinia rot of soybean, wherein a is the change of leaves of a T2-generation GmTCM1 gene plant inoculated with sclerotinia rot of soybean;

FIG. 11 shows the change of leaves of T2 transgenic GmTCM1 plants inoculated with soybean grifola frondosa;

FIG. 12. Phytophthora sojae resistance root rot identification of transgenic hairy roots;

FIG. 13 shows the change of leaves of T2 transgenic GmTCM1 gene plants inoculated with Phytophthora sojae;

FIG. 14 specific primer PCR detection of Arabidopsis thaliana overexpressing GmTCM1 gene;

FIG. 15 Bar primer PCR detection of Arabidopsis thaliana overexpressing the GmTCM1 gene;

FIG. 16 phenotypic analysis of Arabidopsis plants under salt stress;

FIG. 17 phenotypic analysis of Arabidopsis plants under drought stress;

FIG. 18 root length of Arabidopsis plants under salt stress;

FIG. 19 root length of Arabidopsis plants under drought stress.

Detailed Description

The present invention will be described in further detail with reference to the following examples and accompanying drawings, which are included to facilitate a better understanding of the invention and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The pharmaceutical reagents used in the following examples were purchased from conventional biochemical reagent stores, unless otherwise specified. In addition, sclerotinia: the public can collect the plants in the diseased plant stems of any soybean field;

phytophthora sojae, botrytis cinerea, fusarium oxysporum: publicly available from northeast university of agriculture; soybean 27 in the high isoflavone content soybean variety of the soybean variety, and soybean 20 in the low isoflavone content soybean variety jinong: the public is available from northeast university of agriculture.

pCAMBIA3301 expression vector: purchased from Youbao biology, product number: VT1386, web site:http:// www.youbio.cn/。

the public is available from northeast university of agriculture. Agrobacterium rhizogenes, agrobacterium tumefaciens, escherichia coli competence: the public is available from northeast university of agriculture.

Example 1, obtaining of GmTCM1 Gene and construction of plant expression vector

Step one, taking soybean leaves of a variety of Zhongdou 27 as a material to extract total RNA and performing reverse transcription to synthesize a cDNA first chain.

The reverse transcription step is as follows: after mixing 2. mu.L of RNA, 1. mu.L of oligo (dT) (50. mu.M) and 1. mu.L of Super Pure dNTP (10mM), DPEC water was added to 10. mu.L, the tube was incubated at 65 ℃ for 5min, followed by cooling on ice; mu.L of 5 XPrimeScript II buffer, 0.5. mu.L of RNase-Free DNase and 1. mu.L of PrimeScript II RTase were mixed, DPEC water was added to 20. mu.L, the mixture was incubated at 42 ℃ for 1h, at 95 ℃ for another 5min, and then cooled on ice until ready for use.

And step two, searching the full-length sequence of the gene with the accession number Glyma.20G114200(GmTCM1) in a Phytozome v11.0 database, wherein the full-length sequence is shown as SEQ ID No. 4. A pair of cloning primers GmTCM1-S/A is designed according to the sequence of Glyma.20G114200(GmTCM1) gene, and the 5' ends of an upstream primer and a downstream primer are respectively modified by using BstEII and BglII site sequences: GmTCM1-S sequence: 5'-GGAAGATCTTCCATTAAATTAAATCCTAGCTACG-3': GmTCM1-A sequence: 5'-GGGTTACCTAAGTGCTTGTTTTGTTATGACCTA-3' are provided.

Taking the first strand of cDNA obtained in the step one as a template, performing PCR amplification by using the upstream primer GmTCM1-S and the downstream primer GmTCM1-A obtained in the step two, and purifying a PCR product by using glue recovery, wherein a PCR system is as follows:

PCR reaction procedure: 94 ℃ for 2 min; 35 cycles: 94 ℃ for 10s, 63 ℃ for 30s and 72 ℃ for 1 min; the PCR product was run on a 1% agarose gel electrophoresis run at 72 ℃ for 10min, stored at 4 ℃ and purified.

Step four, connecting the purified PCR product with a pGEM-T vector by using a pGEM-T cloning kit of Promega company, wherein the system is as follows: recovering the product by glue by 5 mu L; pGEM-T Easy (50 ng/. mu.L) 1. mu.L; 10 XT₄ DNA Ligation Buffer 1μL；T₄ DNA Ligase(3U/μL)1μL；ddH₂O2. mu.L, 16 ℃ overnight ligation.

The ligation product transformed DH 5. alpha. competent cells by the following procedure: after thawing DH 5. alpha. competent cells on ice, the desired DNA was pipetted into the bottom of the centrifuge tube and mixed with 100. mu.L competent cells well, and ice-cooled for 30 min. After incubation at 42 ℃ for 45s, the cells were immediately transferred to ice, and 700. mu.L of LB liquid medium was added. Incubate at 37 ℃ for 1h with shaking at 220 rpm. Centrifuge at 5,000rpm for 1min and discard the supernatant. The pellet was resuspended in 100. mu.L of LB liquid medium and then spread on LB solid medium containing kanamycin resistance (Kana (50mg/ml)) with a sterile spreading bar, and incubated overnight at 37 ℃. And selecting a single clone for PCR and sequencing verification. The result is shown in FIG. 1, and the GmTCM1 gene with the target fragment size of 1706bp is finally obtained, and the sequence is shown in SEQ ID NO. 1.

BstEII and BglII are used for respectively carrying out double digestion on pCAMBIA3301 plasmid and pGEM-T-GmTCM1 vector, the digestion reaction system refers to a double digestion recommendation system and conditions provided by New EnliandBi labs (https:// www.neb.com /), and T4 DNA ligase is used for carrying out ligation reaction on the digestion fragment of the pCAMBIA3301 plasmid and the GmTCM1 gene sequence fragment, so that the pCAMBIA3301-GmTCM1 vector is obtained.

Analysis of GmTCM1 Gene

Bioinformatic analysis of GmTCM1 Gene

Homology alignment analysis was performed on the amino acid sequences encoding the homologous genes of GmTCM1, and the results showed that the homology of GmTCM1 (Glyma.20G114200) to Glyma.02G236500, Glyma.10G275600 and Glyma.14G205200 proteins was 58%, 81% and 59%, respectively. Among them, GmTCM1 showed the highest protein homology to glyma.10g275600. Homology alignment analysis is carried out on the sequences of the candidate gene GmTCM1, and a homology evolutionary tree is constructed through MEGA5.2 software. The results indicate that GmTCM1 has high similarity to homologous genes from kidney beans and alfalfa, presumably functionally. GmTCM1 has high homology with soybean Glyma.10G275600, belongs to different branches with Glyma.02G236500 and Glyma.14G205200 genes, and is far away from relativity. The GmTCM1 is presumed to have great functional differentiation with Glyma.02G236500 and Glyma.14G205200 genes in the evolution process; analyzing the amino acid sequences of different species, GmTCM1 has high similarity with homologous genes from kidney beans and alfalfa, and presumably has similarity in function. Inputting a 1000bp upstream sequence of ATG of GmTCM1 gene in plantare software to predict promoter elements, finding that a plurality of stress-related cis-acting elements exist upstream of the gene, TCA-element plays an important role in responding to biotic and abiotic stress, MYB, I-box and ARE ARE stress and stress-related elements, and MYB transcription factor regulates and controls accumulation of isoflavone, so that the gene is presumed to be possibly involved in synthesis of isoflavone, sensing biotic and abiotic stress and participating in stress resistance or disease resistance processes. The results of some of the experiments are shown in fig. 2, fig. 3 and table 1.

TABLE 1 GmTCM1 promoter element analysis

Analysis of expression Pattern of GmTCM1 Gene

The following primer pairs were used for 1), 2), 3) below: fluorescent quantitative primer pair of GmTCM1 gene: qGmTCM1-F: 5'TTTGTACCCTTGCTCCG 3': qGmTCM 11-R5 'TTCACTGATTTCTCCCT 3'; soybean reference gene fluorescent quantitative primer pair: GmActin 4-F: 5'-GTGTCAGCCATACTGTCCCCATTT-3', GmActin 4-R: 5' - ' GTTTCAAGCTCTTGCTCGTAATCA-3 '. By using 2^-ΔΔCTThe method calculates the relative expression level of the gene.

1) Dynamic expression of GmTCM1 gene in soybean grain development process

The research adopts a qRT-PCR method, takes bean 27 and strain 1 in a high isoflavone variety, and jinong 20 and strain 2 in a low isoflavone variety as objects, and researches the expression dynamics of GmTCM1 genes in the soybean grain development process. The results show that the high-level expression of the GmTCM1 gene in the later reproductive growth stage of soybeans can be involved in the accumulation process of isoflavone in grains. From the stage R6 to the stage R8, the expression level of GmTCM1 in the high isoflavone material is always higher than that in the low isoflavone material, which indicates that the GmTCM1 gene participates in the accumulation process of isoflavone.

2) Expression pattern analysis of GmTCM1 gene under abiotic stress and signal factor treatment

The promoter region of the GmTCM1 gene has a cis-acting element for sensing stress, so that the expression mode of the GmTCM1 gene in soybean leaves under different abiotic stress is analyzed in the research. Results show that salt stress, drought stress, jasmonic acid and salicylic acid treatment can induce the up-regulated expression of the GmTCM1 gene in the soybeans.

3) Expression pattern analysis of GmTCM1 gene under stress of three pathogenic microorganisms

Analyzing the expression patterns of the GmTCM1 gene in two varieties of soybean leaves under the stress of 3 pathogenic microorganisms. The result shows that the leaf can induce the expression of the GmTCM1 gene in the soybean under the treatment of sclerotinia sclerotiorum, phytophthora and gray spot, the expression level of the GmTCM1 gene in the soybean leaf after the sclerotinia sclerotiorum treatment is rapidly increased, and the soybean leaf reaches an extreme value when treated for 12 hours; under the stress of phytophthora, the expression level of the GmTCM1 gene in a root system shows a tendency of increasing firstly and then decreasing, reaches an extreme value when treated for 12h and then decreases; under the stress of the alternaria alternata, the expression pattern of the GmTCM1 gene is similar to that of the two diseases. The expression of the gene is shown to respond to various pathogenic fungi of the soybean and can be induced by pathogens in different tissues of the soybean. Partial results are shown in fig. 4 and 5.

Example 2 application of GmTCM1 in resistance to biotic stress

1. Agrobacterium rhizogenes mediated transformation of hairy roots

The plasmid of the expression vector pCAMBIA3301-GmTCM1 is transferred into the agrobacterium rhizogenes competent cell, and the donnong 50 of the susceptible soybean variety is used as a receptor for transformation, and the specific method comprises the following steps:

1) recipient soybean culture

Mixing soil and vermiculite according to a ratio of 1:1, filling into a small pot, and thoroughly watering with water before planting. Uniformly planting 50 seeds of the soybean variety Dongnong with smooth and no-insect bite in a small pot, and culturing in a culture room with proper temperature conditions for 4d in a dark environment.

2) Preparation of Agrobacterium rhizogenes infection solution

The day before the agrobacterium rhizogenes is cultured, the transformation bacteria carrying the pCAMBIA3301-GmTCM1 plasmid are activated by a vibration method, the activated bacteria liquid is centrifuged, and 5mL of sterilized water is added into a centrifuge tube after centrifugation to uniformly mix the precipitate.

3) Infection by infection

And (3) infecting the plant with proper seedling age, and injecting thallus at a hypocotyl position close to a cotyledonary node. Carefully puncturing the plant with a needle, and smearing the bacterial liquid on the wound of the plant; wiping non-infected bacteria liquid at the wound with sterilized filter paper, covering the wound with a thin layer of vermiculite, and placing in an incubator at 25 ℃ for 16h of illumination and 8h of dark treatment.

4) Inducing the generation of adventitious roots

Washing off vermiculite attached to the roots with clear water, cutting off the main root part of the soybeans cultured for about two weeks, and putting the cut soybean roots into a wide-mouth bottle to be added with sterilized water for adventitious root induction.

2. Agrobacterium tumefaciens mediated transformation method for soybean cotyledon node

The recombinant plasmid pCAMBIA3301-GmTCM1 is transferred into agrobacterium EHA105, and the donnong 50 of the susceptible soybean variety is used as a receptor for soybean cotyledonary node transformation, and the specific method is as follows:

1) seed sterilization: the full, sterile spotted soybean variety Dongnong 50 was placed in a petri dish, and 96mL of NaCl and 6mL of concentrated HCl were added and sterilized in a well ventilated fume hood.

2) Seed germination: sterilized soybean seeds were seeded in MS0 germination medium in a sterile operating station.

3) Preparation of transformed receptors: when the seeds just have the signs of germination, the seed coat is removed by a scalpel, the cotyledon is cut into two halves, the axillary buds are scraped off, and 4-5 wounds with the diameter of about 2-4mm are lightly scratched at the cotyledonary node.

4) Infection and co-culture: the over-expressed cells were activated in liquid medium to OD600 of 0.6-0.8 at 5000rpm, centrifuged for 10min, and then the pellet was resuspended in YEP in liquid CCM medium of the same volume. Immersing the cotyledonary node explant in the infection solution, and placing the cotyledonary node explant in a shaking table at 200rpm for 30min for infection. Washing the infected explant with sterilized distilled water, and culturing in LCCM co-culture medium for about 2-3 days in dark, and proceeding to the next step according to the condition of the explant turning green.

5) Inducing and screening cluster buds: and (3) placing the cotyledonary node explants subjected to dark culture into a sterile culture flask, cleaning the cotyledonary node explants for 3 times by using sterile distilled water, sucking liquid on the surfaces of cotyledonary nodes by using sterile paper, and inserting the cotyledonary node explants into a recovery culture medium at an angle of 45 degrees to the surfaces of the culture medium to perform bud induction. After 7-10 days of induction, the clumpy shoots were transferred to screening medium containing 5mg/L PPT and screened for 7-10 days.

6) Elongation and rooting of cluster buds: the selected cluster buds are taken, the large buds are removed, the blackheads of the cluster buds are scraped off by a scalpel, and then the cluster buds are inserted into an elongation culture medium. After the soybean seedlings are stretched for about 20 to 30 days, cutting the soybean seedlings growing out 2 groups of three leaves from the cluster buds, transferring the soybean seedlings to a rooting culture medium, and after about 15 days, transferring the soybean seedlings to soil.

3, creation and disease-resistant identification of GmTCM1 gene overexpression hairy roots and plants

The GmTCM1 gene overexpression vector is transferred into a Dongnong 50 root line of an infected variety by utilizing an agrobacterium rhizogenes-mediated soybean cotyledon node transformation method. 100 soybean cotyledonary nodes are co-transformed to obtain 80 healthy rooting seedlings. Extracting the DNA of the roots of the GmTCM1 gene transferred rooting seedlings by using an improved SDS method as a template, identifying the transgenic hairy roots by using a PCR amplification technology, wherein the size of a target fragment is 695bp, and obtaining 20 GmTCM1 gene transferred rooting seedlings. The GmTCM1 gene overexpression vector is transferred into a soybean Dongnong 50 variety through an agrobacterium tumefaciens mediated soybean genetic transformation method, and finally a resistant plant 3 is obtained through PPT screening. And (3) harvesting T0-generation plant seeds identified as PPT resistance, sowing, taking plant leaves to extract DNA when the first group of three compound leaves of the T1-generation plant are completely unfolded, and performing PCR detection, qRT-PCR detection and Western blotting detection by using Bar gene primers (primer 3) and GmTCM1 (primer 4) gene specific primers to obtain 3 positive T1-generation transgenic plants. Partial results are shown in fig. 6, 7, 8 and 9.

The soybean identified as a transgenic plant in the T1 generation is collected and sown, and a fungal pathogen inoculation experiment is carried out when the T2 generation plants completely expand the live leaves, and the results show that: inoculating sclerotinia sclerotiorum: the disease spots of the control plant leaves are expanded and enlarged, almost half of the control plant leaves are distributed, the disease spots are irregular, the center is dark brown, the periphery is light brown, yellow halos are arranged on the outer portion and are rotten, light yellow halos are arranged on the outer portion of the soybean leaves of the GmTCM1 transgenic plant, the disease spots are changed into dark brown, the outer light yellow halos are changed into dark yellow, and the disease spots are not greatly expanded and changed. The GmTCM1 gene is transferred to improve the resistance of Dongnong 50 of the susceptible variety to sclerotinia sclerotiorum. In-vitro inoculation experiment of the plaque fungus, dark brown frog eye-shaped speckles are generated at the inoculated part of the control plant leaf, the center of the lesion spot is gray, then the lesion spot is gradually enlarged, and hypha is generated at the center of the lesion spot; the leaf inoculation part of an over-expression plant transformed with the GmTCM1 gene generates dark green frog-eye disease spots, the peripheries of the disease spots are brown, the disease spots are not obviously expanded, and the GmTCM1 gene is preliminarily proved to generate certain disease resistance to soybean gray leaf spot. The transgenic hairy roots and the wild soybean roots are inoculated with phytophthora sojae and then take the wilting state, and the wilting state of the wild soybean roots is serious and black compared with the transgenic hairy roots; the wilting and blackening degree of the roots after the transgenic hairy roots are inoculated with fusarium oxysporum is lighter compared with the roots of wild soybeans. The disease resistance effect of the hairy root of the GmTCM1 gene is more obvious than that of the hairy root of an empty vector, which shows that the GmTCM1 gene can improve the resistance of soybean root systems to soybean phytophthora root rot and soybean fusarium oxysporum root rot. The phytophthora is not obviously attacked in leaves, but the transgenic leaves and wild type are attacked after inoculation, the wild type leaves turn yellow, brown water stain-like stripes are generated at the inoculated part of the leaves, the center of the disease spot is yellow brown, the yellow of the later-stage leaves is deepened, and the disease spot is enlarged; the inoculated part of the leaf of the GmTCM1 gene-transferred plant generates dark green water stain-like scab which is locally browned, the leaf is not yellowed, and the scab spreading speed is slow. The GmTCM1 gene is transferred to improve the disease resistance of the Dongnong 50 of the susceptible variety. Partial results are shown in fig. 10, 11, 12 and 13.

Example 4 application of GmTCM1 in abiotic stress resistance

1) GmTCM1 gene transformed arabidopsis thaliana and screening after transformation

Transforming the GmTCM1 gene into wild arabidopsis thaliana, harvesting seeds, sowing the transgenic arabidopsis thaliana seeds on an MS culture medium containing 5mg/L of glufosinate-ammonium for screening to obtain 5 normally-growing arabidopsis thaliana plants with PPT resistance, transplanting the plants into nutrient soil, and culturing in a 22 ℃ constant-temperature incubator.

2) PCR detection of transgenic Arabidopsis

Selecting arabidopsis thaliana rosette leaves of overexpression plants transformed with GmTCM1 genes to extract DNA, performing PCR identification by using a primer 3 and a primer 4, detecting to obtain target fragments with the sizes of 695bp and 400bp, identifying 2 positive arabidopsis thaliana plants from 5 PPT positive plants, harvesting single positive plants, and performing PCR detection on each generation until obtaining homozygous T3 generation transgenic plants. Partial results are shown in fig. 14 and 15.

3) Phenotypic analysis of transgenic Arabidopsis under abiotic stress

Homozygous T3-generation transgenic Arabidopsis lines and wild-type seeds were selected and sown in MS medium containing 0mmol/L, 125mmol/L NaCl and 125 mmol/LPEG. Compared with transgenic Arabidopsis thaliana plants, the inhibition of seed germination of wild type plants in 125mmol/LNaCl and 125mmol/LPEG culture media is more serious, and the germination rate of transgenic plants is obviously higher than that of wild type plants. In addition, in the root length experiment, it was found that the root length of transgenic Arabidopsis under stress was relatively less affected by NaCl and PEG. Preliminarily shows that the gene participates in the resistance reaction of arabidopsis thaliana to salt and drought stress. Partial results are shown in fig. 16, 17, 18 and 19.

SEQ ID NO.1

Name: nucleic acid sequence of GmTCM1

ATGGGTCTTCAAATCAAGGAACCGCTCCTTTTCACTCTTGTAACAATATCACTTATT TCAATTACAAAACTCTTGCATTCTTATTTTTCTATACCTTTCTCTCCATCCAATCTTT CCATTGCTATTGCCACCCTCATTTTTGTTCTAATCTCATACAAATTTTCCTCATCCTC TATAAAACACTCTTCCACTACTCTGCCCCCAGGTCCTCTATCTGTTCCAATATTTGG TAACTGGCTACAAGTTGACAATGACCTTAACCACCGTCTTCTAGCATCAATGTCTCA AACCTATGGTCCCGTGTTCCTACTCAAACTAGGTTCCAAAAACTTGGTCGTGGTCTC TGACCCCGAGCTTGCCACCCAAGTGCTCCACGCACAAGGCGTAGAATTTGGCTCTC GCCCACGGAACGTTGTGTTTGATATCTTCACGGGGAATGGCCAAGACATGGTTTTC ACCGTCTACGGCGACCACTGGCGCAAAATGCGAAGAATAATGACACTGCCATTCTT CACCAACAAGGTTGTCCACAATTACAACAACATGTGGGAGGAGGAGATGGACTTG GTGGTGCGTGACCTCAACGTGAATGAGAGGGTGAGGAGCGAAGGGATAGTTATCA GAAGGCGGCTTCAGCTGATGCTGTACAATATCATGTATAGGATGATGTTTGATGCC AAGTTTGAGTCTCAAGAAGACCCTTTGTTCATTCAGGCCACCAGGTTTAACTCCGA GAGAAGCCGTTTGGCGCAGAGTTTTGAATACAATTACGGGGATTTTATACCCTTGC TCCGGCCATTCTTGAGAGGGTACCTCAACAAGTGCAAGGACTTGCAGTCTAGGAGG TTGGCATTTTTCAACACCCACTACGTTGAGAAAAGAAGACAAATAATGGCTGCCAA TGGGGAGAAGCACAAGATCAGCTGTGCAATGGATCACATCATAGATGCTCAGATG AAGGGAGAAATCAGCGAAGAGAATGTGATCTACATAGTAGAAAACATCAACGTTG CAGCAATTGAGACAACACTATGGTCCATAGAGTGGGCAGTAGCAGAGTTGGTGAA CCATCCAACCGTCCAAAGCAAGATTCGTGATGAGATATCAAAAGTGCTAAAAGGG GAGCCAGTTACAGAATCCAACCTACACGAGCTACCATACTTACAAGCCACGGTGAA AGAGACACTGAGACTTCACACC

CCAATTCCTCTTCTGGTGCCCCACATGAACCTGGAAGAAGCAAAGCTAGGAGGGCA CACTGTTCCAAAAGAGTCAAAGGTGGTGGTGAATGCTTGGTGGCTTGCCAACAACC CTTCATGGTGGAAGAACCCAGAGGAGTTCAGGCCAGAAAGGTTCTTGGAAGAGGA ATGTGCAACAGATGCAGTTGCAGGAGGAAAAGTTGACTTTAGGTTCGTGCCATTTG GTGTGGGAAGGAGGAGTTGCCCTGGGATCATACTTGCATTGCCAATACTGGGGCTT GTGATTGCAAAGTTGGTGAAAAGTTTTCAGATGAGTGCTCCAGCGGGGACAAAGAT TGATGTGAGTGAAAAAGGAGGGCAATTCAGCTTGCACATTGCCAACCACTCCACTG TGTTGTTCCATCCAATTAAGACACTATGA

SEQ ID NO.2

Name: amino acid sequence of GmTCM1

MGLQIKEPLLFTLVTISLISITKLLHSYFSIPFSPSNLSIAIATLIFVLISYKFSSSSIKHSSTTL PPGPLSVPIFGNWLQVDNDLNHRLLASMSQTYGPVFLLKLGSKNLVVVSDPELATQVL HAQGVEFGSRPRNVVFDIFTGNGQDMVFTVYGDHWRKMRRIMTLPFFTNKVVHNYN NMWEEEMDLVVRDLNVNERVRSEGIVIRRRLQLMLYNIMYRMMFDAKFESQEDPLFI QATRFNSERSRLAQSFEYNYGDFIPLLRPFLRGYLNKCKDLQSRRLAFFNTHYVEKRRQ IMAANGEKHKISCAMDHIIDAQMKGEISEENVIYIVENINVAAIETTLWSIEWAVAELVN HPTVQSKIRDEISKVLKGEPVTESNLHELPYLQATVKETLRLHTPIPLLVPHMNLEEAKL GGHTVPKESKVVVNAWWLANNPSWWKNPEEFRPERFLEEECATDAVAGGKVDFRFV PFGVGRRSCPGIILALPILGLVIAKLVKSFQMSAPAGTKIDVSEKGGQFSLHIANHSTVLF HPIKTL

SEQ ID NO.3

Name: sequence of upstream primer GmTCM1-S

GGAAGATCTTCCATTAAATTAAATCCTAGCTACG

SEQ ID NO.4

Name: sequence of downstream primer GmTCM1-A

GGGTTACCTAAGTGCTTGTTTTGTTATGACCTA

SEQ ID NO.5

Name: reference sequence of gene GmTCM1

ATGGGTCTTCAAATCAAGGAACCGCTCCTTTTCACTCTTGTAACAATATCACTTATTT CAATTACAAAACTCTTGCATTCTTATTTTTCTATACCTTTCT

CTCCATCCAATCTTTCCATTGCTATTGCCACCCTCATTTTTGTTCTAATCTCATACAAAT TTTCCTCATCCTCTATAAAACACTCTTCCACTACTCTGCC

CCCAGGTCCTCTATCTGTTCCAATATTTGGTAACTGGCTACAAGTTGGCAATGACCTT AACCACCGTCTTCTAGCATCAATGTCTCAAACCTATGGTCCC

GTGTTCCTACTCAAACTAGGTTCCAAAAACTTGGTCGTGGTCTCTGACCCCGAGCTT GCCACCCAAGTGCTCCACGCACAAGGCGTAGAATTTGGCTCTCGCCCACGGAACGT TGTGTTTGATATCTTCACGGGGAATGGCCAAGACATGGTTTTCACCGTCTACGGCGA CCACTGGCGCAAAATGCGAAGAATAATGACACTGCCATTCTTCACCAACAAGGTTGT CCACAATTACAGCAACATGTGGGAGGAGGAGATGGACTTGGTGGTGCGTGACCTCA ACGTGAATGAGAGGGTGAGGAGCGAAGGGATAGTTATCAGAAGGCGGCTTCAGCTG ATGCTGTACAATATCATGTATAGGATGATGTTTGATGCCAAGTTTGAGTCTCAAGAAG ACCCTTTGTTCATTCAGGCCACCAGGTTTAACTCCGAGAGAAGCCGTTTGGCGCAG AGTTTTGAATACAATTACGGGGATTTTATACCCTTGCTCCGGCCATTCTTGAGAGGGT ACCTCAACAAGTGCAAGGACTTGCAGTCTAGGAGGTTGGCATTTTTCAACACCCAC TACGTTGAGAAAAGAAGACAAATAATGGCTGCCAATGGGGAGAAGCACAAGATCA GCTGTGCAATGGATCACATCATAGATGCTCAGATGAAGGGAGAAATCAGCGAAGAG AATGTGATCTACATAGTAGAAAACATCAACGTTGCAGCAATTGAGACAACACTATGG TCCATAGAGTGGGCAGTAGCAGAGTTGGTGAACCATCCAACCGTCCAAAGCAAGAT TCGTGATGAGATATCAAAAGTGCTAAAAGGGGAGCCAGTTACAGAATCCAACCTAC ACGAGCTACCATACTTACAAGCCACGGTGAAAGAGACACTGAGACTTCACACCCCA ATTCCTCTTCTGGTGCCCCACATGAACCTGGAAGAAGCAAAGCTAGGAGGGCACAC TGTTCCAAAAGAGTCAAAGGTGGTGGTGAATGCTTGGTGGCTTGCCAACAACCCTT CATGGTGGAAGAACCCAGAGGAGTTCAGGCCAGAAAGGTTCTTGGAAGAGGAATG TGCAACAGATGCAGTTGCAGGAGGAAAAGTTGACTTTAGGTTCGTGCCATTTGGTG TGGGAAGGAGGAGTTGCCCTGGGATCATACTTGCATTGCCAATACTGGGGCTTGTGA TTGCAAAGTTGGTGAAAAGTTTTCAGATGAGTGCTCCAGCGGGGACAAAGATTGAT GTGAGTGAAAAAGGAGGGCAATTCAGCTTGCACATTGCCAACCACTCCACTGTGTT GTTCCATCCAATTAAGACACTATGA

SEQ ID NO.6

Name: fluorescent quantitative PCR (polymerase chain reaction) upstream primer qGmTCM1-F of GmTCM1 gene

5'-TTTGTACCCTTGCTCCG-3'

SEQ ID NO.7

Name: fluorescent quantitative PCR (polymerase chain reaction) downstream primer qGmTCM1-R for GmTCM1 gene

5'-TTCACTGATTTCTCCCT-3'

SEQ ID NO.8

Name: soybean reference gene fluorescent quantitative PCR (polymerase chain reaction) upstream primer GmActin4-F

5’-GTGTCAGCCATACTGTCCCCATTT-3'

SEQ ID NO.9

Name: soybean reference gene fluorescent quantitative PCR downstream primer GmActin4-R

5’-'GTTTCAAGCTCTTGCTCGTAATCA-3' 。

SEQUENCE LISTING

<110> northeast university of agriculture

<120> soybean gene GmTCM1, and obtaining method and application thereof

<130>

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 1620

<212> DNA

<213> nucleic acid sequence of GmTCM1

<400> 1

atgggtcttc aaatcaagga accgctcctt ttcactcttg taacaatatc acttatttca 60

attacaaaac tcttgcattc ttatttttct atacctttct ctccatccaa tctttccatt 120

gctattgcca ccctcatttt tgttctaatc tcatacaaat tttcctcatc ctctataaaa 180

cactcttcca ctactctgcc cccaggtcct ctatctgttc caatatttgg taactggcta 240

caagttgaca atgaccttaa ccaccgtctt ctagcatcaa tgtctcaaac ctatggtccc 300

gtgttcctac tcaaactagg ttccaaaaac ttggtcgtgg tctctgaccc cgagcttgcc 360

acccaagtgc tccacgcaca aggcgtagaa tttggctctc gcccacggaa cgttgtgttt 420

gatatcttca cggggaatgg ccaagacatg gttttcaccg tctacggcga ccactggcgc 480

aaaatgcgaa gaataatgac actgccattc ttcaccaaca aggttgtcca caattacaac 540

aacatgtggg aggaggagat ggacttggtg gtgcgtgacc tcaacgtgaa tgagagggtg 600

aggagcgaag ggatagttat cagaaggcgg cttcagctga tgctgtacaa tatcatgtat 660

aggatgatgt ttgatgccaa gtttgagtct caagaagacc ctttgttcat tcaggccacc 720

aggtttaact ccgagagaag ccgtttggcg cagagttttg aatacaatta cggggatttt 780

atacccttgc tccggccatt cttgagaggg tacctcaaca agtgcaagga cttgcagtct 840

aggaggttgg catttttcaa cacccactac gttgagaaaa gaagacaaat aatggctgcc 900

aatggggaga agcacaagat cagctgtgca atggatcaca tcatagatgc tcagatgaag 960

ggagaaatca gcgaagagaa tgtgatctac atagtagaaa acatcaacgt tgcagcaatt 1020

gagacaacac tatggtccat agagtgggca gtagcagagt tggtgaacca tccaaccgtc 1080

caaagcaaga ttcgtgatga gatatcaaaa gtgctaaaag gggagccagt tacagaatcc 1140

aacctacacg agctaccata cttacaagcc acggtgaaag agacactgag acttcacacc 1200

ccaattcctc ttctggtgcc ccacatgaac ctggaagaag caaagctagg agggcacact 1260

gttccaaaag agtcaaaggt ggtggtgaat gcttggtggc ttgccaacaa cccttcatgg 1320

tggaagaacc cagaggagtt caggccagaa aggttcttgg aagaggaatg tgcaacagat 1380

gcagttgcag gaggaaaagt tgactttagg ttcgtgccat ttggtgtggg aaggaggagt 1440

tgccctggga tcatacttgc attgccaata ctggggcttg tgattgcaaa gttggtgaaa 1500

agttttcaga tgagtgctcc agcggggaca aagattgatg tgagtgaaaa aggagggcaa 1560

ttcagcttgc acattgccaa ccactccact gtgttgttcc atccaattaa gacactatga 1620

<210> 2

<211> 539

<212> PRT

<213> amino acid sequence of GmTCM1

<400> 2

Met Gly Leu Gln Ile Lys Glu Pro Leu Leu Phe Thr Leu Val Thr Ile

1 5 10 15

Ser Leu Ile Ser Ile Thr Lys Leu Leu His Ser Tyr Phe Ser Ile Pro

20 25 30

Phe Ser Pro Ser Asn Leu Ser Ile Ala Ile Ala Thr Leu Ile Phe Val

35 40 45

Leu Ile Ser Tyr Lys Phe Ser Ser Ser Ser Ile Lys His Ser Ser Thr

50 55 60

Thr Leu Pro Pro Gly Pro Leu Ser Val Pro Ile Phe Gly Asn Trp Leu

65 70 75 80

Gln Val Asp Asn Asp Leu Asn His Arg Leu Leu Ala Ser Met Ser Gln

85 90 95

Thr Tyr Gly Pro Val Phe Leu Leu Lys Leu Gly Ser Lys Asn Leu Val

100 105 110

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

115 120 125

Val Glu Phe Gly Ser Arg Pro Arg Asn Val Val Phe Asp Ile Phe Thr

130 135 140

Gly Asn Gly Gln Asp Met Val Phe Thr Val Tyr Gly Asp His Trp Arg

145 150 155 160

Lys Met Arg Arg Ile Met Thr Leu Pro Phe Phe Thr Asn Lys Val Val

165 170 175

His Asn Tyr Asn Asn Met Trp Glu Glu Glu Met Asp Leu Val Val Arg

180 185 190

Asp Leu Asn Val Asn Glu Arg Val Arg Ser Glu Gly Ile Val Ile Arg

195 200 205

Arg Arg Leu Gln Leu Met Leu Tyr Asn Ile Met Tyr Arg Met Met Phe

210 215 220

Asp Ala Lys Phe Glu Ser Gln Glu Asp Pro Leu Phe Ile Gln Ala Thr

225 230 235 240

Arg Phe Asn Ser Glu Arg Ser Arg Leu Ala Gln Ser Phe Glu Tyr Asn

245 250 255

Tyr Gly Asp Phe Ile Pro Leu Leu Arg Pro Phe Leu Arg Gly Tyr Leu

260 265 270

Asn Lys Cys Lys Asp Leu Gln Ser Arg Arg Leu Ala Phe Phe Asn Thr

275 280 285

His Tyr Val Glu Lys Arg Arg Gln Ile Met Ala Ala Asn Gly Glu Lys

290 295 300

His Lys Ile Ser Cys Ala Met Asp His Ile Ile Asp Ala Gln Met Lys

305 310 315 320

Gly Glu Ile Ser Glu Glu Asn Val Ile Tyr Ile Val Glu Asn Ile Asn

325 330 335

Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Ala Val Ala

340 345 350

Glu Leu Val Asn His Pro Thr Val Gln Ser Lys Ile Arg Asp Glu Ile

355 360 365

Ser Lys Val Leu Lys Gly Glu Pro Val Thr Glu Ser Asn Leu His Glu

370 375 380

Leu Pro Tyr Leu Gln Ala Thr Val Lys Glu Thr Leu Arg Leu His Thr

385 390 395 400

Pro Ile Pro Leu Leu Val Pro His Met Asn Leu Glu Glu Ala Lys Leu

405 410 415

Gly Gly His Thr Val Pro Lys Glu Ser Lys Val Val Val Asn Ala Trp

420 425 430

Trp Leu Ala Asn Asn Pro Ser Trp Trp Lys Asn Pro Glu Glu Phe Arg

435 440 445

Pro Glu Arg Phe Leu Glu Glu Glu Cys Ala Thr Asp Ala Val Ala Gly

450 455 460

Gly Lys Val Asp Phe Arg Phe Val Pro Phe Gly Val Gly Arg Arg Ser

465 470 475 480

Cys Pro Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Leu Val Ile Ala

485 490 495

Lys Leu Val Lys Ser Phe Gln Met Ser Ala Pro Ala Gly Thr Lys Ile

500 505 510

Asp Val Ser Glu Lys Gly Gly Gln Phe Ser Leu His Ile Ala Asn His

515 520 525

Ser Thr Val Leu Phe His Pro Ile Lys Thr Leu

530 535

<210> 3

<211> 34

<212> DNA

<213> sequence of upstream primer GmTCM1-S

<400> 3

ggaagatctt ccattaaatt aaatcctagc tacg 34

<210> 4

<211> 33

<212> DNA

<213> sequence of downstream primer GmTCM1-A

<400> 4

gggttaccta agtgcttgtt ttgttatgac cta 33

<210> 5

<211> 1620

<212> DNA

<213> reference sequence of the gene GmTCM1

<400> 5

atgggtcttc aaatcaagga accgctcctt ttcactcttg taacaatatc acttatttca 60

attacaaaac tcttgcattc ttatttttct atacctttct ctccatccaa tctttccatt 120

gctattgcca ccctcatttt tgttctaatc tcatacaaat tttcctcatc ctctataaaa 180

cactcttcca ctactctgcc cccaggtcct ctatctgttc caatatttgg taactggcta 240

caagttggca atgaccttaa ccaccgtctt ctagcatcaa tgtctcaaac ctatggtccc 300

gtgttcctac tcaaactagg ttccaaaaac ttggtcgtgg tctctgaccc cgagcttgcc 360

acccaagtgc tccacgcaca aggcgtagaa tttggctctc gcccacggaa cgttgtgttt 420

gatatcttca cggggaatgg ccaagacatg gttttcaccg tctacggcga ccactggcgc 480

aaaatgcgaa gaataatgac actgccattc ttcaccaaca aggttgtcca caattacagc 540

aacatgtggg aggaggagat ggacttggtg gtgcgtgacc tcaacgtgaa tgagagggtg 600

aggagcgaag ggatagttat cagaaggcgg cttcagctga tgctgtacaa tatcatgtat 660

aggatgatgt ttgatgccaa gtttgagtct caagaagacc ctttgttcat tcaggccacc 720

aggtttaact ccgagagaag ccgtttggcg cagagttttg aatacaatta cggggatttt 780

atacccttgc tccggccatt cttgagaggg tacctcaaca agtgcaagga cttgcagtct 840

aggaggttgg catttttcaa cacccactac gttgagaaaa gaagacaaat aatggctgcc 900

aatggggaga agcacaagat cagctgtgca atggatcaca tcatagatgc tcagatgaag 960

ggagaaatca gcgaagagaa tgtgatctac atagtagaaa acatcaacgt tgcagcaatt 1020

gagacaacac tatggtccat agagtgggca gtagcagagt tggtgaacca tccaaccgtc 1080

caaagcaaga ttcgtgatga gatatcaaaa gtgctaaaag gggagccagt tacagaatcc 1140

aacctacacg agctaccata cttacaagcc acggtgaaag agacactgag acttcacacc 1200

ccaattcctc ttctggtgcc ccacatgaac ctggaagaag caaagctagg agggcacact 1260

gttccaaaag agtcaaaggt ggtggtgaat gcttggtggc ttgccaacaa cccttcatgg 1320

tggaagaacc cagaggagtt caggccagaa aggttcttgg aagaggaatg tgcaacagat 1380

gcagttgcag gaggaaaagt tgactttagg ttcgtgccat ttggtgtggg aaggaggagt 1440

tgccctggga tcatacttgc attgccaata ctggggcttg tgattgcaaa gttggtgaaa 1500

agttttcaga tgagtgctcc agcggggaca aagattgatg tgagtgaaaa aggagggcaa 1560

ttcagcttgc acattgccaa ccactccact gtgttgttcc atccaattaa gacactatga 1620

<210> 6

<211> 17

<212> DNA

<213> GmTCM1 gene fluorescent quantitative PCR upstream primer qGmTCM1-F

<400> 6

tttgtaccct tgctccg 17

<210> 7

<211> 17

<212> DNA

<213> fluorescent quantitative PCR downstream primer qGmTCM1-R of GmTCM1 gene

<400> 7

ttcactgatt tctccct 17

<210> 8

<211> 24

<212> DNA

<213> soybean reference gene fluorescent quantitative PCR upstream primer GmActin4-F

<400> 8

gtgtcagcca tactgtcccc attt 24

<210> 9

<211> 24

<212> DNA

<213> soybean reference gene fluorescent quantitative PCR downstream primer GmActin4-R

<400> 9

gtttcaagct cttgctcgta atca 24

Claims

1. A soybean gene GmTCM1 is characterized in that the nucleic acid sequence of the soybean gene GmTCM1 is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2.

2. The method for obtaining soybean gene GmTCM1 of claim 1, wherein the steps of the method for obtaining the soybean gene GmTCM1 are as follows:

3. The obtaining method of claim 2, wherein the sequence of the upstream primer GmTCM1-S is shown as SEQ ID NO.3, and the sequence of the downstream primer GmTCM1-A is shown as SEQ ID NO. 4.

4. The method for obtaining a gene GmTCM1, wherein the reference sequence of the gene GmTCM1 is shown as SEQ ID No. 5.

5. The application of the soybean gene GmTCM1 in biotic stress resistance and abiotic stress resistance as claimed in claim 1, wherein the biotic stress refers to soybean fungal diseases, specifically refers to one or more than two of soybean diseases caused by sclerotinia sclerotiorum, phytophthora and botrytis cinerea; the abiotic stress refers to drought and salt stress.

6. The application of claim 5, wherein the application is to obtain a transgenic soybean plant by over-expressing GmTCM1 gene, and then obtain the transgenic soybean plant through subsequent subculture propagation and identification to obtain the stable homoisoflavonoid disease-resistant and stress-resistant genetic material.