CN117778413A - A GLMFR1 gene for improving soybean's ability to withstand biotic and abiotic stresses and its application - Google Patents

Abstract

A GLMFR1 gene that improves the biotic and abiotic stress capabilities of soybeans and its application, which relates to the field of soybeans. The nucleotide sequence of the GLMFR1 gene of the present invention is shown in SEQ ID NO: 1. The application of the GLMFR1 gene of the present invention is to improve the ability of soybeans to respond to salt stress, alkali stress, and Phytophthora soja root rot. The GLMFR1 gene of the present invention has certain resistance to salt stress, alkali stress, and Phytophthora sojae root rot stress. The GLMFR1 gene identified in the present invention has reference value for future research on soybean salt tolerance, alkali tolerance, and Phytophthora root rot resistance. The transgenic material of the present invention provides a basic basis for future research on variety cultivation and enrichment of germplasm resources. , this gene has great market potential and application prospects.

Description

A GLMFR1 gene for improving soybean's ability to withstand biotic and abiotic stresses and its application

Technical field

The invention belongs to the field of soybeans, and specifically relates to a GLMFR1 gene that improves the biotic and abiotic stress capabilities of soybeans and its application.

Background technique

Adversity stress will inhibit the growth and development of soybeans. Salt stress and alkali stress among abiotic stresses will lead to a decrease in soybean quality and yield when severe. Phytophthora root rot among biotic stresses is a worldwide disease that can It can reduce soybean production by 10-40%, and even produce no crops in severe cases. In recent years, molecular biology has developed rapidly, and many stress resistance genes have been discovered, including genes for salt-alkali tolerance and disease resistance. Using molecular biology methods to explore the function of stress resistance genes, improve the stress resistance of soybeans, and avoid the shortcomings of traditional breeding such as low efficiency, long cycle, and poor variety stability, play an important role in soybean stress resistance breeding. Application The prospects are very bright.

Summary of the invention

The purpose of the present invention is to solve the problem of poor soybean stress ability under salt stress, alkali stress and soybean Phytophthora root rot, and to provide a GLMFR1 gene that improves the biotic and abiotic stress ability of soybean and its application.

The present invention provides a GLMFR1 gene that improves the biotic and abiotic stress capabilities of soybeans. The nucleotide sequence of the GLMFR1 gene is shown in SEQ ID NO: 1.

The invention discloses an application of a GLMFR1 gene for improving the biological and abiotic stress resistance of soybeans. The GLMFR1 gene is applied to improve the ability of soybeans to resist salt stress, alkali stress and soybean phytophthora root rot pathogen stress.

Further, the concentration of the salt solution in the salt stress is 100 to 300 mmol·L ^-1 .

Furthermore, the concentration of the alkaline solution in the salt stress is 50 to 200 mmol·L ^-1 .

The present invention provides a kit containing the GLMFR1 gene for improving soybean biotic and abiotic stress capabilities.

The present invention adopts CRISPR/Cas9 technology, Agrobacterium tumefaciens-mediated method, uses Dongnong 50 as the receptor, and transgenic positive plants; through molecular biological testing of transgenic plants and their progeny, 6 GLMFR1 gene-edited plant lines are screened out ; The agronomic traits of the transgenic plants were analyzed, and they all grew normally. The GLMFR1 gene knockout plants were phenotypicly identified under salt and alkali stress and infected by Phytophthora sojae root rot fungus, and the results showed that the plants were under salt stress, alkali stress, and Phytophthora sojae root rot. After the three phenotypes of root rot fungus infection were identified, the expression levels of related genes were analyzed. The functional verification of the GLMFR1 gene has been initially completed, proving that the GLMFR1 gene has certain resistance to salt stress, alkali stress, and Phytophthora soybean root rot stress. The GLMFR1 gene identified in the present invention has reference value for future research on soybean salt tolerance, alkali tolerance, and Phytophthora root rot resistance. The transgenic material of the present invention provides a basic basis for future research on variety cultivation and enrichment of germplasm resources. , this gene has great market potential and application prospects.

The nucleotide sequence of the GLMFR1 gene according to the present invention is as follows:

GGTAATTATCATTAACGAGCAAGCAGTTACTTTTCTTTTCTGTTTTCAATCATGGATTCAAGAGTAGTT CAAAGTGTAGTGCATGAGAACGATGAACATTACCCCCACATTGTTGCCTTAGAGCAAGGTATATATGAATCACATTTGGAAGCAACAAGTTGTATTATATTTGAAGGCATCTTCATTAATGTTCCTATGATGTTTTTTCTTTTCTTTGAATAATTTGCTTTGATTCTTTTTCTACTTCAAATTTTTTGGATATAGTGACACACGGTGCAGAAGAAGACCACTTTGATCATG AGAAGAAGTCTGTTCTGAATAAGGTCAAGGCAAAGGCTAAGAAAATTAAGGACACGATTAAGAAGCATGGCCATCAA GTGCTTGATCGTGGTCATGAGTATAACAATGAAGATCAGCATACCCTTGATGATCATGACTTAGACGAGGATGATGA AATGGCTAAGACCCACAAGTTCATAAAACACCAAGTATGTTCTTTTCTAAATCATCTGGAATTAGAGGATGAGTATTAGTTCGCCATAAAGTGAAATAAGATCCGGGGTTCAAACACCTTTTCAGATCATTATGATTAAATTTGATTTTATTCCGTTAATTTTTTTCCTATTGATTATGATCTTCATAAATTTTAAATTTTTAAAAAATAATTCCTATTATCATTCAACAATAACGTGACATATGGTGACTATTATGAGATAACATATGATGTAACATGTTTGAATGCATGTGACGTTATCA ATAAAATCTATGAGGAGTAAAATCAATTGGAGGGAGTAAAATCAAAATTAATCAGGGATAAAAAACATCACTAAAAAATATTACCTTAGAATTTGACATTTACTCGTCATAGATACTTTTCTAATTTTTCTTCAAAACAGAAGGAACTCAGAACCATTTCTTTTTTGAAACTTAAAACCGTTTAATAACGGGAATTTACTAATAATAAAAATAAATCTGATTTAAGTTGGACTATGTACTTGAATGTTGTGCAGTTCACGAAAGTGAAGATGTTAAAACTGCAACACCTACATCTGAGCAAGTCGAAAATTTG GGGAAGTCAGGAATTGATTCTGGAGGCACAAGAGGTACAACAGTTATGGGAGAGGAACCCCGTCATGATGCACTACT TGGAGGTGTTTCTTCAACTACTGAAATTGATCAAAACATAGCCACTGACTCGGCCAAAACATTTTCTGTGGAAGAAA AGGCAGGGCCACCAAAGGACAATTTGGAGAAGTCAATAGGCTTGGACTTGGAGGAAGAGCCTCACGCTCCAGGAAGT AGACCTGAGGCATATCCCCCTACCAATTATCAAACCAAAATCACTGATCCAAGTGTGATAGGTAAGTAAGACAATTGAACAAAGTTGTATAACTCCATTTTGTGCTATGGACTTTTTGCATTATGCATTAATATTAGTGTACCTTTTAAAAGATTTGATGGTTAAATTGATTAAGTTGGATTTGACTCCCCATTAATTTCAGATTCCTTATTTTACTATATACATGCATGTGTTCAAATTCCATATTTTACTGTTGTAATTCTGCGATCAATTTTTTGGAGTTGAATTCTTTTACTGTCCATTCAACATACCATCAATTTTGTTTTCAAATTATAC CTTTAAATGTGTTCACGCTTAATGATATTTATGTACTATGGTCTATGTGATTAAGATTTTCATCTGTGGTGGCTTACAGGAAAGGATGAAATAGAAGAAATCACACCAGTTGAGGAATCTTTTGC AAAAATGAATATGCATGATGAGCCAAAACCTACTCCAGAACCAAATATCCAAGCAACTGTTGTTGATTCTGAATACC CTCCTGTTGGAAACCACGATCAGTTTGTGCCACACCTCTCTGCTGCAACACAAACTCAGTATCCTTCTGCTGAAAGT CATGATCAGTTCAATCAGGAAACAACATCCACAAATATCAACAGAAACCTGGTAAATCCCACAGAAACTGGACAAAC TTTCAACACCATCACAACCACAATTGAAGAAAAACCACTCTATGAAGCAAAGACTGATGAAGTTATCTCTCCTAAAG ATGTCATAGCTTCTGAGGCTGGTTCAGGAGAGAAAGATGCCATAAAGGACAAGGTGGTAACAAATAAAAGAGCAACAA AAAATTGGGGATGCTTCTAACATGTCTGGCTCAACTGCACAACATGGAAAGAACATTGCTCACTCTCTGACTGAGAA ATTAGCTCCAGTTTATGATAAGGTTGCAGTGGTAGGAAGTGCAGTGAAGTCCAAAGTGACTGGAACTAGCACTGGTG GTGTTGGAACTGAGACAAAGAATGAGGTTTCTGTGAAGGACTATTTGGCTGAGAAGCTAAAGCCTGGTGAAGAAGAC AAGGCACTTTCTGAGTTAATTTCAGAAGCTTTACATAAGAAAAAGGAAGAGCCAGTGAAAAATGAGGATGGAAACTT GGATGACGGCAATGATAAAATGTGTGAAGAGATCAGTGTGAAGAGTCCAGGGAAAGGTGTGGTTGGCAAGCTTAAGG GTGTTGTTGGCTCTTGGTTTGGCAAAGCCGAGGAAAAAGGTGAATGAGGTTTCTTTTGCTAAGTACATTTTCAAAATGGTGTGTTATGTATATGTATAATTCTGATTTTGAGTTTGCCGTTGTTATGGTTGCAGGAGGTGAAGATTTATCCAA GAATACAAATTCTGGTGCAGAAGTGGAACAGGTTCACCAGGTTGTAGGTGAAATCAAGAGTGGTCCAATTGAAGAAC AAGGGACTGGCTGAATTTCTTCATGGACATTGGATAATACATGTGTGCTGTGCTTTCCTTGTGATTAGATATTTGTA TATGTTGGATTCATGTGTAAGTTATTATATGTTTGTGTAGATGTGGAATTTACATTATGATATCGTTGTACTGGAGC CGAGAATGAGACTTGGTTATTACAATGTTAAATGTAAAAAAAAGTGGCAATATATAATTGTTTTCTGTGATTGTATT TTACTTAATTCAATGTCCCATGGTAATGTGTATTTTTCTTTTTTCC

The dotted underlined sequence is the 5'UTR (5' untranslated region), the single underlined sequence is the CDS (coding sequence), the wavy underlined sequence is the 3'UTR (3' untranslated region), and the ununderlined sequence is the intron.

Description of the drawings

Figure 1 shows the PCR results of T ₃ generation specific primers; Note: M: DL2000 DNA molecular weight standard; 1-6: transgenic plant QF01-06; 7: negative control, water;

Fig. 2 is a diagram showing the DNA sequence comparison results;

Figure 3 is a photo of the phenotypic results of the recipient control and transgenic plants after salt stress treatment;

FIG4 is a photograph of the phenotypic results of the receptor control and transgenic plants after alkaline stress treatment;

Figure 5 is a photo of the resistance to infection after hypocotyl wound inoculation; Figure A: before inoculation with Phytophthora sojae; Figure B: 7 days after inoculation with Phytophthora sojae;

Figure 6 is a graph showing the survival rate of recipient control and transgenic plants after salt stress and alkali stress treatment; Note: Significant difference analysis: One-way analysis of variance (* represents P<0.05, ** represents P<0.01); Standard error: Std. error bars;

Figure 7 is a graph showing the expression levels of related stress resistance genes under salt stress conditions; Note: Significant difference analysis: one-way analysis of variance (* represents P<0.05, ** represents P<0.01); standard error: standard error bar;

Figure 8 shows the expression levels of related stress resistance genes under alkali stress conditions; Note: Significant difference analysis: one-way analysis of variance (* represents P<0.05, ** represents P<0.01); standard error: standard error bar;

Figure 9 is a graph showing the expression levels of related stress resistance genes under Phytophthora sojae infection conditions; Note: Significant difference analysis: one-way analysis of variance (* represents P<0.05, ** represents P<0.01); standard error: standard error bar

Figure 10 is an analysis chart of GLMFR1 gene transcription levels under different stress treatment conditions; Note: Significant difference analysis: one-way analysis of variance; standard error: standard error bar.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the spirit of the disclosed contents of the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can understand it from the contents of the present invention. The techniques taught are subject to changes and modifications without departing from the spirit and scope of the invention.

The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but are not used to limit the present invention.

Example 1

1. Obtaining soybean T _3rd generation plants

1.1 GLMFR1 gene target design

The Huanong CRISPR-P website was used for design (http://crispr.hzau.edu.cn/CRISPR2/), and targets with high target scores, low off-target rates, and appropriate locations were selected, as shown in Table 1.

Table 1GLMFR1 gene targets

1.2 Construction of plant CRISPR/Cas9 expression vector

1.2.1 Construction of recombinant expression vector pYLCRISPR/Cas9

The CRISPR/Cas9 gene knockout construct was developed using the [pCBSG015(Basta)] vector (Weimi Biotechnology Co., Ltd.).

1.2.2 Detection of soybean _T3 plants transformed with pYLCRISPR/Cas9

The constructed recombinant expression vector pYLCRISPR/Cas9 was transferred into Dongnong 50 (DN50), and after greenhouse generation, _T3 transgenic seeds were obtained. PCR amplification was performed using the specific primers in Table 3 (both target sites were included between the primers), and the agarose gel electrophoresis results are shown in Figure 1. The PCR products were sent for sequencing, and the sequencing results were analyzed. The results are shown in Figure 2. All editing occurred, that is, the soybean GLMFR1 gene was successfully knocked out. Table 2 shows the editing type detected in the transgenic _T3 plants, and the deletion between the target sites is 4bp-205bp.

Table 2 Transgenic T ₃ plant detection and editing types

Table 3 Specific primer sequences

2. Identification of abiotic stress in soybean transformed with pYLCRISPR/Cas9 in the T3 _generation

2.1 Treatment of salt stress and alkali stress in soybean seedling stage

The soybean transgenic line QF01-06 of _{the 3rd} generation of soybean T that was transformed with pYLCRISPR/Cas9 in 1.2.2 and the control plant (Dongnong 50 plant that was not transformed with pYLCRISPR/Cas9) were subjected to salt stress and alkali stress at the seedling stage (the control group was defined as DN50). , Salt stress conditions: 18 days after sowing, stress treatment was started. The treatment solution was used once every 3 days for a total of three treatments. The treatment solution was 200mmol·L ^-1 NaCl solution. Alkali stress conditions: 18 days after sowing, stress treatment was started. The treatment solution was used once every 3 days for a total of three treatments. The treatment solution was 100mmol·L ^-1 Na ₂ CO ₃ solution.

The results of salt stress in soybean seedling stage are shown in Figure 3. After 3 days of salt stress treatment, the leaves of the control plants and T _3rd generation pYLCRISPR/Cas9 transformed soybean plants were slightly yellowed; after 6 days of salt stress treatment, the control plants could be clearly observed. The difference between the phenotypes of soybean plants transformed with T _3rd generation pYLCRISPR/Cas9 is that 70% of the leaves of "DN50" turned yellow and wilted, while the leaves of QF01-06, the soybean plant transformed with _T3rd generation pYLCRISPR/Cas9, became more yellowing, and a small number of leaves Withering; after 9 days of treatment, the survival rates were counted. The results are shown in Figure 5(A). The survival rates of DN50 and QF01-06 were 10%, 30%, 33.33%, 33.33%, 26.67%, 33.33%, and 43.33 respectively. %, pYLCRISPR/Cas9 transformed soybean plants QF01-03, QF05, and QF06 were extremely significantly higher than the control plant "DN50", and QF04 was significantly higher than the control plant "DN50".

The results of alkali stress in soybean seedling stage are shown in Figure 4. After 3 days of alkali stress treatment, the control "DN50" and T _3rd generation pYLCRISPR/Cas9 transformed soybean plants did not show stress phenotypes. After 6 days of alkali stress treatment, all the leaves of the control "DN50" plants were wilted, and some of the leaves of the transgenic plants were wilted. After 9 days of treatment, the survival rates were calculated. The results are shown in Figure 6(B). The survival rates of DN50 and QF01-06 were respectively 3.33%, 53.33%, 43.33%, 53.33%, 63.33%, 53.33%, 76.67%, pYLCRISPR/Cas9 transformed soybean plant QF01-06 was extremely significantly higher than the control plant "DN50".

Identification of Phytophthora root rot in soybean transformed with 2.2T _3rd generation pYLCRISPR/Cas9

Phytophthora sojae strains were used to identify the T _3rd generation pYLCRISPR/Cas9 transformed soybean plants by hypocotyl wound inoculation. The base of the stem of susceptible plants rots or appears water-soaked or turns reddish-brown, breaks above the inoculation site, and eventually dies. However, the resistant plants partially discolor at the hypocotyl inoculation wound and can grow normally (see Figure 5). QF01-06 was evaluated for disease resistance. The results showed that QF01, QF03, QF04, QF05, and QF06 were moderately resistant lines. QF02 also had surviving plants, but did not meet the evaluation standards and was designated as a susceptible variety. The recipient control DN50 was classified as susceptible. Variety. The specific test results are shown in Table 4.

Hypocotyl wound inoculation method, the specific method is as follows: when the opposite true leaves of the soybean seedlings are about to expand, use a sterile scalpel to gently draw a wound 1cm below the cotyledons with a depth of 1/3 of the stem thickness, and inoculate the cultured The Phytophthora sojae pieces were punched into 10 mm × 10 mm pieces with a hole punch and inoculated into the wound. They were cultured in an environment with 12 hours of light, a temperature of 28°C, and a relative air humidity of 60%. After 6 days, the lodging rate was investigated. The inoculation site of the susceptible plant changed color and had water stains. The part above the inoculation was broken, and the entire plant wilted and died. The inoculation site of the disease-resistant plant was partially discolored, and the plant did not die if touched lightly. It was normal. grow. Resistance standards: Lodging rate ≤30% is resistant (R), 30% <lodging rate <70% is intermediate (Interminate, I), lodging rate ≥70% is susceptible (Susceptible, S).

Table 4 Evaluation of resistance to infection after inoculation with hypocotyl wounds

2.3 Analysis of stress resistance gene expression in transformed soybeans after salt stress, alkali stress and inoculation with Phytophthora root rot

The expression regulation of genes is interactive in most cases. Since GLMFR1 gene knockout can improve the ability of plants to cope with stress, in order to explore whether the related stress resistance genes in transgenic plants have changed, fluorescence quantitative PCR technology was used to measure the related stress resistance under salt stress, alkali stress and inoculation with Phytophthora sojae. Gene expression levels were identified.

Under salt stress, the results are shown in Figure 7. Compared with the control, the expression levels of 5 editing types of the GmNHX1 gene increased, among which the QF02, QF03, and QF04 materials increased significantly, the QF01 and QF06 materials increased significantly, and the QF05 Materials were significantly reduced; the expression levels of 5 editing types of the GmST1 gene were extremely significantly increased, namely QF01, QF02, QF03, QF04, and QF05, while the QF06 material was significantly reduced; all 6 editing types of the GmAKT1 gene were increased, among which QF02 and QF03 , QF05 material increased significantly, QF01, QF04, and QF06 materials did not reach a significant level; the 6 editing types of the GmNCED1 gene all increased, and all reached a very significant level; the 6 editing types of the GmFDL19 gene all increased, including QF01 and QF02 , QF03 and QF04 materials reached extremely significant levels, QF05 materials reached significant levels, and QF06 materials did not reach significant levels; the GmWRKY13 gene had 4 editing types with increased expression levels, among which QF03 and QF04 materials reached extremely significant levels, and QF05 materials reached significant levels. , QF02 material did not reach a significant level, while QF01 and QF06 were similar to the control; all six editing types of the GmERF3 gene were increased, among which QF03 and QF04 materials reached extremely significant levels, QF01, QF02, and QF06 materials reached significant levels, and QF05 material did not reach a significant level.

Under alkali stress, the results are shown in Figure 8. Compared with the control, the 6 editing types of the GmNHX1 gene were all increased, among which the QF02 and QF05 materials reached extremely significant levels, while the others did not reach significant levels; the GmST1 gene had 3 editing types expressed. The expression levels of QF01, QF03 and QF04 were significantly increased, respectively. The QF02 and QF05 materials were similar to the control. The expression of QF06 material was reduced, but did not reach a significant level. Five editing types of the GmAKT1 gene were increased, among which the QF05 material reached At the extremely significant level, the QF02 material reached the significant level, while the QF03, QF04, and QF06 materials did not reach the significant level, while the expression of the QF01 material decreased; all six editing types of the GmNCED1 gene increased, among which the QF01, QF02, QF03, and QF06 materials reached the extremely significant level. level, QF04 and QF05 materials did not reach a significant level; the expression levels of 4 editing types of the GmFDL19 gene were significantly increased, namely QF01, QF02, QF03, and QF04, and the expression levels of 2 editing types were reduced, among which QF05 was extremely significantly reduced, and QF06 The GmWRKY13 gene has not reached a significant level; the expression level of 2 editing types of the GmWRKY13 gene increased, among which QF03 increased significantly and QF06 increased significantly, and the expression level of 2 editing types decreased, among which QF04 decreased significantly and QF01 did not reach a significant level, while The QF02 and QF05 materials are not much different from the control; 5 editing types of the GmERF3 gene are increased, among which the QF02 and QF05 materials reach a very significant level, the QF06 material reaches a significant level, the QF03 and QF04 materials do not reach a significant level, and the expression level of the QF01 material A slight decrease.

Under the condition of inoculation with soybean Phytophthora, the results are shown in Figure 9. The expression levels of CYP82C4 and GmERF5 genes in QF01-06 materials were significantly increased, and the expression levels of salicylic acid signaling pathway related genes GmPR10 and GmNPR1 were also higher than those of the receptor control plants; WRKY transcription factors can participate in the disease resistance signal transduction pathways mediated by SA, JA and ET. Under the condition of soybean Phytophthora infection, the expression level of GmWRKY22 gene was upregulated to varying degrees in QF01-06 materials, among which it was extremely significant in QF02-05 materials; the expression level of GmWRKY40 gene was extremely significant in QF01-06 materials.

3. Expression analysis of GLMFR1 gene under adverse stress

In order to verify the response of GLMFR1 gene to biotic and abiotic stress, real-time fluorescence quantitative qRT-PCR method was used to analyze the expression changes of GLMFR1 gene mRNA level under salt stress and alkali stress infection treatment conditions. Opposite true leaves were selected to expand (V1 The expression analysis of GLMFR1 gene under stress was performed on the "Dongnong 50" material from the period). The results showed that under salt treatment (200mmol·L ^-1 NaCl solution), alkali treatment (100mmol·L ^-1 Na ₂ CO ₃ solution), salt treatment Alkali mixed treatment (80mmol·L ^-1 salt-alkali mixed solution) was used to extract soybean roots at 0h, 1h, 6h, 12h, 24h, 36h, and 48h respectively, and then quickly frozen in liquid nitrogen to extract RNA for use in real-time fluorescence quantitative PCR experiments. The data analysis results are shown in Figure 10.

1 hour after salt stress treatment, the expression of GLMFR1 gene in soybean roots increased slowly, decreased slowly from 1 to 9 hours after treatment, and dropped to the lowest value at 9 hours, then increased rapidly, and reached the highest value at 24 hours, which was significantly higher than that of the treatment before, then the expression level decreased, and then slowly increased from 36 to 48h. Overall, the expression level at 12-48h was relatively higher than that at 1-9h; 1-9h after alkali stress treatment, the expression level of GLMFR1 gene in roots It increased with the increase of treatment time, slowly decreased 12h after treatment, then increased rapidly, rose to the highest value at 24h, which was significantly higher than before treatment, and then decreased slowly; 1-24h after Phytophthora sojae inoculation, the expression of GLMFR1 gene The amount increased slightly, and then increased rapidly at 48 hours.

Claims (5)

1. A GLMFR1 gene that improves the biotic and abiotic stress capabilities of soybeans, characterized in that the nucleotide sequence of the GLMFR1 gene is shown in SEQ ID NO: 1. 2. A kind of GLMFR1 gene application for improving the biotic and abiotic stress ability of soybeans as claimed in claim 1, characterized in that the GLMFR1 gene is used to improve soybeans' response to salt stress, alkali stress, and Phytophthora sojae root rot stress. Application of abilities. 3. The use according to claim 2, characterized in that the concentration of the salt solution in the salt stress is 100 to 300 mmol·L ^-1 . 4. The application according to claim 2, characterized in that the concentration of the alkali solution in salt stress is 50 to 200 mmol·L ^-1 . 5. A kit comprising the GLMFR1 gene for improving soybean biotic and abiotic stress capabilities according to claim 1.