Application of soybean GmMADS3 gene in improving salt tolerance of plants

Abstract

The invention discloses application of a soybean GmMADS3 gene in improvement of plant salt tolerance, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the soybean GmMADS3 gene is shown as SEQ ID NO: 1, and the coded amino acid sequence of the soybean GmMADS3 gene is shown as SEQ ID NO: 2. The GmMADS3 gene is used for constructing a plant expression vector to respectively transform arabidopsis thaliana and soybeans to obtain transgenic arabidopsis thaliana and transgenic soybeans, and the salt stress resistance of the arabidopsis thaliana and the soybeans can be remarkably improved. Therefore, the GmMADS3 gene has important application in improving the salt tolerance of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of soybean GmMADS3 gene in improving plant salt tolerance. Background Art

[0002] In plants, salt tolerance refers to the ability to maintain normal growth, development, and lifecycle processes in high-salt environments through physiological and metabolic regulation. Due to changes in the natural environment and the impact of human activities, soil salinization has become a significant factor affecting soybean yield. Different levels of salt stress can affect seed germination, plant height, number of main stem nodes, number of branches, number of pods per plant, 100-grain weight, and other key productivity indicators.

[0003] Currently, the rapid development of genetic engineering technology has opened up new directions for biological genetic improvement. By genetically transforming genes that play a key role in salt stress response, new salt-tolerant germplasms can be effectively obtained. Summary of the Invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide an application of the soybean GmMADS3 gene in improving plant salt tolerance.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The soybean GmMADS3 gene is used to improve plant salt tolerance. The amino acid sequence encoded by the gene is shown in SEQ ID NO: 2.

[0007] Based on the above scheme, the nucleic acid sequence of the GmMADS3 gene is shown in SEQ ID NO: 1.

[0008] On the basis of the above scheme, the tolerance of plants to salt stress is improved by increasing the expression level of the GmMADS3 gene in plants.

[0009] Based on the above solution, the plant is at least one of soybean and Arabidopsis thaliana.

[0010] A method for improving plant salt tolerance, comprising increasing the expression level of a GmMADS3 gene in a plant to improve the plant's tolerance to salt stress; the amino acid sequence encoded by the GmMADS3 gene is shown in SEQ ID NO: 2.

[0011] Based on the above scheme, the nucleic acid sequence of the gene is shown in SEQ ID NO: 1.

[0012] On the basis of the above scheme, a plant expression vector of the GmMADS3 gene is constructed and transformed into plants to express the gene in the plants, thereby increasing the expression level of the GmMADS3 gene in the plants.

[0013] Based on the above solution, the plant is at least one of soybean and Arabidopsis thaliana.

[0014] A preparation for improving plant salt tolerance, wherein the active ingredient of the preparation is a recombinant expression vector, expression cassette, recombinant bacteria, recombinant virus or transgenic cell line containing a GmMADS3 gene sequence; the nucleic acid sequence of the GmMADS3 gene is shown in SEQ ID NO: 1.

[0015] Advantages of the technical solution of the present invention:

[0016] The MADS gene family comprises numerous genes, most of which are associated with plant growth and development, as well as the formation and development of floral organs. Reports on MADS family genes associated with plant stress resistance are very limited. The present invention cloned a GmMADS3 gene from soybean, which is associated with plant salt tolerance. Plant expression vectors constructed from the gene of the present invention were used to transform Arabidopsis thaliana and soybean, respectively, to obtain transgenic Arabidopsis thaliana and soybean, which significantly improved the salt stress tolerance of Arabidopsis thaliana and soybean. Therefore, the GmMADS3 gene has important applications in improving plant salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 24-hour diurnal expression analysis of the GmMADS3 gene (A is the 24-hour diurnal expression analysis under long-day conditions; B is the 24-hour diurnal expression analysis under short-day conditions);

[0018] Figure 2 Analysis of GmMADS3 gene transcriptional activation activity;

[0019] Figure 3 Subcellular localization of GmMADS3 gene;

[0020] Figure 4 qRT-PCR level detection of Arabidopsis transgenic positive plants;

[0021] Figure 5 Potted salt tolerance phenotype and chlorophyll fluorescence index determination of Arabidopsis thaliana overexpressing the GmMADS3 gene;

[0022] Figure 6 Determination of fluorescence parameters of salt tolerance in Arabidopsis thaliana overexpressing the GmMADS3 gene (A is the photoprotection index - Y(NPQ); B is the maximum optical efficiency - Fv / Fm; C is the PSⅡ quantum efficiency; D is the photodamage index - Y(NO));

[0023] Figure 7 NBT and DAB staining of salt tolerance in Arabidopsis thaliana overexpressing the GmMADS3 gene;

[0024] Figure 8 Salt tolerance pot phenotype of soybean overexpressing GmMADS3 gene;

[0025] Figure 9 Determination of chlorophyll fluorescence index in soybeans overexpressing the GmMADS3 gene;

[0026] Figure 10 Determination of fluorescence parameters of salt tolerance in soybeans overexpressing the GmMADS3 gene (A is the photoprotection index - Y(NPQ); B is the maximum optical efficiency - Fv / Fm; C is the PSⅡ quantum efficiency; D is the photodamage index - Y(NO));

[0027] Figure 11 NBT and DAB staining of salt tolerance in soybean overexpressing the GmMADS3 gene. DETAILED DESCRIPTION

[0028] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0029] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0030] In the following examples,

[0031] Soybean Williams82 was provided by the Crop Applied Functional Genomics Laboratory, College of Life Sciences, Qingdao Agricultural University;

[0032] The pGAL4 plasmid and pGBKT7 vector were provided by the Crop Applied Functional Genomics Laboratory, College of Life Sciences, Qingdao Agricultural University;

[0033] Yeast Y2HGold was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0034] The pPTN1171 vector was provided by the Crop Applied Functional Genomics Laboratory, College of Life Sciences, Qingdao Agricultural University;

[0035] Agrobacterium GV3101 and Agrobacterium LBA4404 were purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0036] DH5α was purchased from Tolo Biotechnology Co., Ltd.

[0037] Columbia-0 Arabidopsis thaliana was provided by the Crop Functional Genomics Laboratory, College of Life Sciences, Qingdao Agricultural University.

[0038] Example 1

[0039] Cloning of the GmMADS3 gene

[0040] First, RNA was extracted from soybean Williams82 leaves and reverse-transcribed into cDNA. The resulting cDNA was then amplified using primers GmMADS3-F / R designed by NCBI. The full-length cDNA sequence of the GmMADS3 gene was obtained, as shown in SEQ ID NO:1. The gene coding sequence contains 621 nucleotides and encodes 207 amino acids. The encoded amino acid sequence is shown in SEQ ID NO:2.

[0041] SEQ ID NO: 1 (5'→3'):

[0042] ATGACTAGAAAGAGGATACAGATCAAGAAAATCGACAACATCAGTTCGAGGCAAGTGACTTTCTCCAAGAGAAGGAAAGGGCTTTTCAAGAAGGCTCAGGAGTTGTCAACCCTTTGTGATGCAGACATAGCTCTCATAGTCTTTTCTGCAACTAGCAAGCTCTTTGAGTATGCCAGTTCAAGCATGCATCAAGTAATTGAAAGGCACGATCGCTATTCAGCAATCCATAGATTGGATCGCCCCTCTATTGAGCTGCAGATTGAGAGTGACTCCAACAACATTCTGCGCAAGAAAGTAGAAGATAAGACTCGTGAACTGAGGCAGATGAATGGGGAAGATCTGCAAGGATTGACATTACAAGAACTGCAGAAACTAGAGGAACATCTTAAAAGAAGTTTGACCAATGTTTCAAAAGTAAAGGATGCAAAATTTATGCAAGAGATCAGCACCTTTAAGAGAAAGGGAGTGGAACTAATGGAGGAAAACCAAAGACTGAAACAGGTGCCGAGCCTTATCCATGCACATAGCTATAGGCAATCATCGGAATCCATTCTTAGCAATTCATCTAACCTTCCTGAAGATGGTGGTAGCAACACATCTCTCAAGTTGGGGTTACCTTAA

[0043] SEQ ID NO:2:

[0044] MTRKRIQIKKIDNISSRQVTFSKRRKGLFKKAQELSTLCDADIALIVFSATSKLFEYASSSMHQVIERHDRYSAIHRLDRPSIELQIESDSNNILRKKVEDKTRELRQMNGEDLQGLTLQELQKLEEHLKRSLTNVSKVKDAKFMQEISTFKRKGVELMEENQRLKQVPSLIHAHSYRQSSESILSNSSNLPEDGGSNTSLKLGLP

[0045] The GmMADS3-F / R sequences are as follows:

[0046] GmMADS3-F: 5'-ATGACTAGAAAGAGGATACA-3' (SEQ ID NO: 3);

[0047] GmMADS3-R: 5'-TTAAGGTAACCCCAACTTGA-3' (SEQ ID NO: 4);

[0048] The PCR reaction system was as follows: upstream primer (F) 1 μL, downstream primer (R) 1 μL, template 1 μL, DNA polymerase 15 μL, and ddH2O 12 μL.

[0049] The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 58°C for 15 sec, extension at 72°C for 10 sec / kb, 32 cycles; and final extension at 72°C for 3 min.

[0050] Example 2

[0051] 24-hour circadian expression analysis of the GmMADS3 gene

[0052] Soybean Williams82 was treated with a 15-day LD / SD photoperiod. Samples were taken at 0, 4, 8, 12, 16, 20, and 24 hours. Real-time quantitative PCR (qRT-PCR) was used to observe the expression of the GmMADS3 gene, and the rhythmic changes of the GmMADS3 gene over 24 hours were observed. qRT-PCR primers were designed online using NCBI primer blast, and the amplified product range was 100 to 150 bp. Gmcons4 was used as an internal reference gene, and the expression of the GmMADS3 gene was observed according to 2 -ΔΔCT The algorithm calculates the relative expression level of the gene. The expression of the GmMADS3 gene reaches its peak before entering darkness in different light cycles ( Figure 1 ).

[0053] The primer sequences for qRT-PCR of the GmMADS3 gene and the Gmcons4 internal reference gene are as follows:

[0054] qRT-GmMADS3-F: 5'-AGGCACGATCGCTATTCAGC-3' (SEQ ID NO: 5);

[0055] qRT-GmMADS3-R: 5'-CTCGGCACCTGTTTCAGTCT-3' (SEQ ID NO: 6);

[0056] GmCons4-F: 5'-CGGTGGTTTCTATCTTGGCATC-3' (SEQ ID NO: 7);

[0057] GmCons4-R: 5'-GTCTTTCGCTTCAATAACCCTA-3' (SEQ ID NO: 8);

[0058] The reaction system for qRT-PCR was as follows: upstream primer (F) 0.3 μL, downstream primer (R) 0.3 μL, template (cDNA) 0.3 μL, 2× SYBR Green Pro Taq HS Premix 5 μL, and RNase-Free ddH2O 4.1 μL.

[0059] The reaction program of qRT-PCR was as follows: 95°C for 30 sec; 95°C for 5 sec, 60°C for 30 sec, 40 cycles; 95°C for 15 sec, 60°C for 60 sec, and 95°C for 1 sec.

[0060] Example 3

[0061] Analysis of GmMADS3 gene transcriptional activation activity and subcellular localization

[0062] First, the full-length cDNA fragment of the GmMADS3 gene obtained in Example 1 was ligated to the pGBKT7 vector to construct the GmMADS3-BD plasmid, which is driven by the ADH1 promoter reporter gene. The pGAL4 plasmid, GmMADS3-BD plasmid, and pGBKT7 plasmid were transformed into the Y2HGold yeast strain, plated on SD / -Trp medium, and grown in a 28°C oven for 2-3 days until single colonies of uniform size were grown. Yeast with pGAL4, GmMADS3-BD, and pGBKT7 were streaked on SD / -His / Trp / x-ɑ-gal medium and cultured at 28°C for 5 hours to observe the growth of the colonies. Figure 2 It can be seen that the GmMADS3 gene has no transcriptional activation activity.

[0063] The coding sequence of GmMADS3 was amplified from soybean Williams82, and the PCR fragment was cloned into the modified pPTN1171 vector to produce the GmMADS3-GFP fusion protein. Next, the constructed GmMADS3-GFP plasmid was transformed into Agrobacterium GV3101 for transient expression in 4-week-old tobacco leaves. After injection, the lower epidermis of the tobacco was cultured under low light for 2 days, and slides were prepared. The GFP signal of GmMADS3-GFP was detected using a laser confocal microscope (Olympus, Tokyo, Japan) and compared with the control GFP. Figure 3 It can be seen that the laser confocal imaging results showed that the GmMADS3-GFP fusion protein was mainly located in the nucleus of tobacco cells.

[0064] Example 4

[0065] Construction of GmMADS3 overexpression vector and recombinant strain

[0066] First, the full-length GmMADS3 cDNA fragment obtained in Example 1 and the overexpression vector pPTN1171 were analyzed for enzyme cleavage sites. Xho I and Xba I restriction endonucleases were then used to double-digest the full-length GmMADS3 cDNA fragment and the overexpression vector pPTN1171 to ensure ligation between the fragment and the vector. The enzyme digestion system was placed in a PCR instrument, set at 37°C, and the digestion time was set to 5 hours. After the digestion was completed, the fragment and vector were gel-recovered using the Eric Bio Steady Pure DNA Gel Recovery Kit. The recovered fragment and vector were ligated using the T4 DNA ligation method. After the system was constructed, the reaction was allowed to proceed at 16°C overnight to obtain the overexpression vector (GmMADS3-pPTN1171).

[0067] The constructed overexpression vector (GmMADS3-pPTN1171) was transformed into DH5α by heat shock. After the bacteria were cultured overnight at 37°C, single clones were picked and sent to Qingke Company for sequencing to obtain the correct bacterial solution containing the overexpression vector (GmMADS3-pPTN1171-DH5α), i.e., the recombinant bacteria.

[0068] Example 5

[0069] The steps for constructing GmMADS3 transgenic Arabidopsis are as follows:

[0070] 1) Extract the E. coli plasmid containing the correct overexpression vector in Example 4 and transform it into Agrobacterium GV3101 by freeze-thaw method. Use the verified correct Agrobacterium to infect Columbia-type Arabidopsis thaliana (Columbia-0) to obtain transgenic Arabidopsis plants, and harvest T0 generation seeds after maturity. The T0 generation Arabidopsis seeds are screened by adding PPT screening marker to MS culture medium. The screened seeds are placed in a 4°C refrigerator for vernalization for 3 days and then placed in a light incubator for light culture. After about two weeks of culture, the Arabidopsis seedlings are transferred to nutrient soil for planting and cultivation. Be careful not to break the roots of the seedlings during transplanting. The seedlings that have just been transplanted need to be covered with plastic wrap and the plastic wrap should be removed after the seedlings grow to a certain size.

[0071] 2) Because the Arabidopsis plants screened above may contain false positives, it is necessary to perform DNA testing on the screened plants to verify that the plants obtained are successfully transformed. The specific steps are: First, DNA testing is performed on the obtained seedlings. DNA from the leaves of the Arabidopsis seedlings is extracted as a template for identifying positive seedlings. Primers for identification are designed at both ends of the CDS of the GmMADS3 gene. The primer sequences for identifying positive seedlings are as follows:

[0072] The forward primer was Gm3-2024-F: 5′-ATGACTAGAAAGAGGATACA-3′ (SEQ ID NO: 3);

[0073] The reverse primer was Gm3-2024-R: 5'-TTAAGGTAACCCCAACTTGA-3' (SEQ ID NO: 4).

[0074] The enzyme used in the experiment was 2× Accurate Taq Master Mix from Accurate. After PCR, analysis was performed on a 1% agarose gel electrophoresis. The sizes of PCR fragments amplified from plant DNA and the constructed overexpression vector (GmMADS3-pPTN1171) plasmid were compared. If the size of the fragment amplified from the plant DNA matched the size of the band amplified from the plasmid control, the plant was preliminarily identified as transgenic. Positive seedling testing revealed that three T1-generation positive Arabidopsis lines were generated from the transformed Arabidopsis. Two of these lines were selected and analyzed for expression of the target gene GmMADS3 in these two transgenic Arabidopsis lines using qRT-PCR. After 2-3 weeks of growth, RNA was sampled from leaves of three positive plants from each line, ensuring that no significant differences in growth were observed between the three plants. Samples were collected from Columbia-0 Arabidopsis as a control. The samples were immediately stored in liquid nitrogen and then RNA was extracted. After the samples were extracted, the RNA A 260 and A 280 The concentration and purity of the RNA were recorded, and 1 μg of RNA was used for reverse transcription. The qRT-PCR specific primer sequences for detecting the expression of the GmMADS3 gene and the primer sequences for the internal reference Actin gene are as follows:

[0075] qRT-GmMADS3-F: 5'-AGGCACGATCGCTATTCAGC-3' (SEQ ID NO: 9);

[0076] qRT-GmMADS3-R: 5'-CTCGGCACCTGTTTCAGTCT-3' (SEQ ID NO: 10);

[0077] Atactin-F: 5'-GCTCCTCTTAACCCAAAGGC-3' (SEQ ID NO: 11);

[0078] Atactin-R: 5'-CACACCATCACCAGAATCCAGG-3' (SEQ ID NO: 12).

[0079] Primer design requires spanning exons, and the amplified product size is about 150-180bp, not exceeding 300bp. The qRT-PCR system includes enzymes, cDNA samples, upper and lower primers, and RNase-free water. Add all components of the system to a 96-well plate, centrifuge to remove bubbles, and place the 96-well sample plate in an ABI PCR instrument for amplification. The resulting qRT-PCR melting curve and C T The values were analyzed and the relative expression levels were calculated. The results showed that the expression level of the GmMADS3 gene in the overexpression strain GmMADS3-2 was higher than that in GmMADS3-1, and no expression of the GmMADS3 gene was detected in the Columbia-0 Arabidopsis thaliana ( Figure 4 ).

[0080] Example 6

[0081] Analysis of salt tolerance in GmMADS3 transgenic Arabidopsis thaliana

[0082] (1) Phenotypic observation of GmMADS3 transgenic Arabidopsis

[0083] The obtained Arabidopsis thaliana overexpressing the GmMADS3 gene and the wild-type Arabidopsis thaliana seeds of Columbia were inoculated into 1 / 2MS culture medium for germination, and the seedlings were transferred to pots after 10 days to observe the growth of the seedlings. When the Arabidopsis thaliana grew to four weeks old, they were treated with 150mM NaCl stress for one week and the phenotypic data were statistically analyzed. It can be clearly seen from the phenotypic observation that the Arabidopsis thaliana in the control group grew well, with tender green leaves, and there was no significant difference in the various phenotypic indicators. On the contrary, the Arabidopsis thaliana in the treatment group showed varying degrees of damage such as yellowing and curling of the leaves. Fluorescence measurement showed that the chlorophyll content of the Columbia-type Arabidopsis thaliana in the treatment group was seriously less than that of the Arabidopsis thaliana overexpressing the GmMADS3 gene ( Figure 5 ).

[0084] (2) Determination of fluorescence parameters of salt tolerance in GmMADS3 transgenic Arabidopsis

[0085] Arabidopsis plants were first treated in darkness for more than 20 minutes, and chlorophyll fluorescence parameters were measured using the IMAG-K7 chlorophyll fluorescence imaging system from WALZ / WALZ, Germany. The maximum photochemical efficiency (Fv / Fm) of photosynthetic system II (PSII) reflects the plant's maximum potential photosynthetic activity. The quantum yield Y (NPQ) of photosynthetic system II's regulated energy dissipation is an important indicator of light protection. A larger value indicates a stronger self-protection ability. PSII quantum efficiency It reflects the efficiency of converting light energy absorbed by photosystem II (PSⅡ) into chemical energy. The fluorescence parameter results after dark treatment showed that the maximum photochemical efficiency of photosynthetic system II (PSII) (Fv / Fm) of the two overexpression lines was Figure 6 B), non-photochemical quenching coefficient NPQ ( Figure 6 A), PSⅡ quantum efficiency ( Figure 6 The quantum yield of non-regulated energy dissipation of photosynthetic system II, Y(NO), is an important indicator of light damage. The Y(NO) of the two overexpression lines was significantly higher than that of Williams82 line ( Figure 6 Middle D).

[0086] (3) NBT and DAB staining analysis of GmMADS3 transgenic Arabidopsis

[0087] To further investigate the role of GmMADS3 in regulating salt stress, H2O2 content was measured to reflect the plant's ability to respond to salt tolerance. Arabidopsis leaves were stained with nitro blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) to detect H2O2 content in two GmMADS3 transgenic lines, Col, under normal or treated conditions. Under normal conditions, no substantial differences were observed in NBT and DAB staining; however, under 150mM NaCl treatment, the color depth of the control Col was significantly higher than that of the two GmMADS3 transgenic lines. In contrast, the leaf color depth of the two GmMADS3 transgenic lines was significantly lower than that of the control Col ( Figure 7 ).

[0088] Example 7

[0089] The steps for constructing GmMADS3 transgenic soybean are as follows:

[0090] 1) The E. coli plasmid containing the correct overexpression vector described in Example 4 was extracted and transformed into Agrobacterium tumefaciens LBA4404 via the freeze-thaw method. The verified correct Agrobacterium was then used to transform soybean cultivar Williams 82. Regeneration shoots gradually grew from the cotyledonary nodes of the infected soybeans, and tissue culture was performed until regeneration of T0-generation transgenic soybean plants was obtained. Phenotypic observation of the stably inherited transgenic plants was performed, and gene function was analyzed.

[0091] 2) Identification of DNA-positive transgenic soybean plants

[0092] First, DNA testing was performed on the resulting T0 generation soybeans. Leaf DNA was extracted from soybean seedlings as a template for positive seedling identification. Primers were designed at either end of the CDS of the GmMADS3 gene. The forward and reverse primers for positive seedling identification are shown in SEQ ID NO:3 and SEQ ID NO:4. The enzyme used in the experiment was 2× Accurate Taq Master Mix from Acryl. After PCR, the results were analyzed by 1% agarose gel electrophoresis. The sizes of PCR fragments generated using plant DNA and the constructed overexpression vector (GmMADS3-pPTN1171) as templates were compared. If the size of the fragment amplified from the plant DNA matched that of the plasmid control, the plant was preliminarily identified as a transgenic plant. A total of eight T0 generation positive transgenic soybean plants were obtained from the GmMADS3 transformation experiment. These positive transgenic soybean plants were cultured in a laboratory greenhouse until mature seeds were harvested.

[0093] The seeds from the eight T0 transgenic soybean plants were subcultured, and each resulting soybean plant was identified as positive. Phenotypes of the T1 transgenic soybeans were also observed. The positive seedling identification results showed that three positive lines in the T1 generation were genetically stable.

[0094] Example 8

[0095] Analysis of salt tolerance of GmMADS3 transgenic soybean

[0096] (1) Phenotypic observation of GmMADS3 transgenic soybean

[0097] The obtained overexpressed GmMADS3 transgenic soybeans and wild-type soybean Williams82 were planted under SD light, and after the first three compound leaves grew out, they were treated with 500mM NaCl stress for one week and the phenotypic data were counted. After the first three compound leaves grew out, the normal treatment group was watered with the same amount of water for one week and the phenotypic data were counted. It can be clearly seen from the phenotypic statistical analysis that the soybeans in the normal group grew neatly and in good condition, and the various phenotypic indicators were basically consistent. However, all soybean plants in the salt treatment group suffered from salt damage to varying degrees, with yellowing of leaves, curling, and other damage to varying degrees ( Figure 8 ).

[0098] (2) Determination of chlorophyll fluorescence index of GmMADS3 transgenic soybean

[0099] Soybean plants were first placed in darkness for more than 20 minutes, and chlorophyll fluorescence parameters were measured using the IMAG-K7 chlorophyll fluorescence imaging system from WALZ / WALZ, Germany. Fluorescence measurements showed that the chlorophyll content of the Williams82 treated group was significantly lower than that of soybeans overexpressing the GmMADS3 gene ( Figure 9 The maximum photochemical efficiency of photosynthetic system II (PSII) (Fv / Fm) reflects the potential maximum photosynthetic activity of the plant. The quantum yield Y (NPQ) of photosynthetic system II's regulated energy dissipation is an important indicator of light protection. The larger the value, the stronger the self-protection ability. PSⅡ quantum efficiency It reflects the efficiency of converting light energy absorbed by photosystem II (PSII) into chemical energy. The fluorescence parameter results after dark treatment showed that the maximum photochemical efficiency (Fv / Fm) of photosynthetic system II (PSII) of the three overexpression lines ( Figure 10 B), non-photochemical quenching coefficient NPQ ( Figure 10 A) and PSⅡ quantum efficiency ( Figure 10 The photoprotection ability of the Williams82 strain decreased sharply after salt treatment, and its value was 0. The quantum yield of non-regulated energy dissipation of photosynthetic system II, Y(NO), is an important indicator of light damage. The Y(NO) value of the Williams82 strain was the highest, and the Y(NO) values of the three overexpression strains were all lower than that of the Williams82 strain ( Figure 10 Middle D).

[0100] (3) NBT and DAB staining analysis of GmMADS3 transgenic soybean

[0101] To further investigate the role of GmMADS3 in regulating salt stress, H2O2 content was measured to reflect the plant's ability to respond to salt tolerance. Soybean leaves were stained with nitro blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) to detect H2O2 content in three GmMADS3 transgenic lines, Williams82, under normal or treated conditions. Under normal conditions, no substantial differences were observed in NBT and DAB staining; however, under 500 mM NaCl treatment, the color depth of Williams82 soybean was significantly higher than that of the three GmMADS3 transgenic lines. In contrast, the leaf color depth of the three GmMADS3 transgenic lines was significantly lower than that of Williams82 soybean ( Figure 11 ).

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of soybean GmMADS3 gene in improving plant salt tolerance, characterized in that, The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The nucleic acid sequence of the gene is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that By increasing the expression level of the GmMADS3 gene in plants, the tolerance of plants to salt stress is improved.

4. The use according to any one of claims 1 to 3, characterized in that The plant is at least one of soybean and Arabidopsis thaliana.

5. A method for improving plant salt tolerance, characterized in that: By increasing the expression level of the GmMADS3 gene in the plant, the plant's tolerance to salt stress is improved; the amino acid sequence encoded by the GmMADS3 gene is shown in SEQ ID NO:

2.

6. The method for improving plant salt tolerance according to claim 5, wherein: The nucleic acid sequence of the gene is shown in SEQ ID NO:

1.

7. The method for improving plant salt tolerance according to claim 5, wherein: By constructing a plant expression vector of the GmMADS3 gene and transforming the plant, the GmMADS3 gene is expressed in the plant, thereby increasing the expression level of the GmMADS3 gene in the plant.

8. The method for improving plant salt tolerance according to any one of claims 5 to 7, characterized in that: The plant is at least one of soybean and Arabidopsis thaliana.

9. A preparation for improving plant salt tolerance, characterized in that The active ingredient of the preparation is a recombinant expression vector, expression cassette, recombinant bacteria, recombinant virus or transgenic cell line containing the GmMADS3 gene sequence; the nucleic acid sequence of the GmMADS3 gene is shown in SEQ ID NO: 1.

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