Soybean salt-tolerant related gene GmNAT12, and coding protein and application thereof
By cloning and overexpressing the soybean salt tolerance-related gene GmNAT12 and its encoded protein, the problem of insufficient soybean salt tolerance was solved, and the growth and yield of soybeans under salt stress were enhanced.
Patent Information
- Application Number
- CN202410711229.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-04
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2044-06-04
AI Technical Summary
Existing technologies are insufficient to effectively improve the salt tolerance of soybeans. Traditional breeding strategies are time-consuming and have limited effects. There is a lack of in-depth understanding of the molecular mechanisms of soybean salt tolerance, which affects soybean yield and quality.
The soybean salt tolerance-related gene GmNAT12 and its encoded protein were cloned and overexpressed. Recombinant expression vectors and recombinant bacteria were constructed and introduced into soybean plants. Enhanced promoters and translation control signals were used to ensure gene expression. Salt-tolerant soybean varieties were bred by combining stress screening technology.
It significantly improves the salt tolerance of soybeans, enhances their growth ability under salt stress, and promotes plant development and yield increase.
Smart Images
Figure HDA0004874088470000011 
Figure HDA0004874088470000012
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering, specifically relating to a soybean salt tolerance-related gene GmNAT12, its encoded protein, and its applications. Background Technology
[0002] Soybeans, as a major source of oil and protein, are not only an important economic oilseed crop but also one of the most widely cultivated and utilized economic and food crops globally. The yield and quality of soybeans are crucial to global food security and economic development. However, with the increasing severity of global climate change, soil salinization is becoming a prominent problem, posing a significant challenge to agricultural production. Therefore, utilizing modern biological technologies to enhance the salt tolerance of soybeans and screening for salt-tolerant soybean plants is of profound significance for mitigating the negative impacts of salinized soils on soybean production.
[0003] Salt stress primarily originates from high concentrations of sodium and chloride ions in the soil. It manifests in various forms, including osmotic stress, ion stress, and secondary stress. When the salt content in the external environment is too high, plants are severely affected. Osmotic stress makes it difficult for plant root cells to absorb water, causing a sharp increase in intracellular osmotic pressure, cell shrinkage, and an inability to meet the plant's normal water needs. Simultaneously, a high-salt environment inhibits the function of ion channels and transport proteins on plant cell membranes, further limiting the entry of water and nutrients, thus inhibiting the growth and development of soybean plants and ultimately leading to a significant decrease in soybean yield.
[0004] The response of soybean to salt stress is a complex molecular regulatory network, resulting from multiple physiological and biochemical reactions. Traditional breeding strategies are time-consuming and have limited effectiveness in improving salt-tolerant soybeans. To accelerate the cultivation of new salt-tolerant soybean varieties, we have turned to molecular breeding techniques. However, our understanding of the molecular mechanisms of soybean salt tolerance is still insufficient, and related research progress is slow. Therefore, we urgently need to explore new genes with the potential to enhance soybean salt tolerance. Such research is of vital value for strengthening soybean salt tolerance and ultimately breeding new varieties that can grow in saline-alkali environments. Summary of the Invention
[0005] To address the aforementioned technical problems in the prior art, the present invention aims to disclose a soybean salt tolerance-related gene GmNAT12, its encoded protein, and its applications.
[0006] The first objective of this invention is to provide a salt tolerance-related gene GmNAT12, said gene GmNAT12 being a DNA molecule as described in 1) or 2) or 3) below:
[0007] 1) A DNA molecule with a gene sequence as shown in SEQ ID NO.1;
[0008] 2) DNA molecules with CDS sequences as shown in SEQ ID NO.2;
[0009] 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein.
[0010] A second objective of this invention is to provide a protein encoded by the aforementioned gene GmNAT12.
[0011] Specifically, the protein provided by this invention is selected from any one shown in (a) or (b):
[0012] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.3;
[0013] (b) Proteins derived from the target sequence selected from SEQ ID NO.2.
[0014] SEQ ID NO.3 in the sequence listing consists of 767 amino acids.
[0015] A third objective of this invention is to provide a recombinant expression vector, expression cassette, or recombinant bacterium containing the aforementioned gene GmNAT12.
[0016] Furthermore, the recombinant expression vector or expression cassette is obtained by inserting the gene GmNAT12 into the recombination site of the vector pBA002 using XbaI single enzyme digestion; the recombinant expression vector or expression cassette is then transformed into engineered bacteria to obtain the recombinant bacteria shown.
[0017] Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.
[0018] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.
[0019] The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment. These vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the Nos gene for lipase) and plant genes (such as the soybean storage protein gene).
[0020] When constructing recombinant plant expression vectors using the aforementioned genes, any type of enhancing promoter or constitutive promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter, can be added before the transcription initiation nucleotide. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.
[0021] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.
[0022] The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene GmNAT12 into the recombination site of the vector pBA002, which is digested with the restriction endonuclease XbaI. pBA002 containing GmNAT is named pBA002-GmNAT12.
[0023] Expression cassettes, transgenic cell lines, and recombinant bacteria containing any of the above-mentioned genes GmNAT12 are all within the scope of protection of this invention.
[0024] A fourth objective of this invention is to provide primers for amplifying the aforementioned gene GmNAT12. Primer pairs for amplifying the full length or any fragment of the gene GmNAT12 are also within the scope of protection of this invention. In a specific example, the primers are shown in SEQ ID NO.4 and SEQ ID NO.5.
[0025] A fifth objective of this invention is to provide the application of the aforementioned gene GmNAT12, the aforementioned protein, the aforementioned recombinant expression vector, expression cassette or recombinant bacteria, or the aforementioned primers, expression vector or recombinant bacteria in improving the salt tolerance of soybeans.
[0026] Furthermore, overexpression of the aforementioned gene GmNAT12 improves the salt tolerance of soybeans.
[0027] Preferably, the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria are introduced into soybean to overexpress the aforementioned gene GmNAT12.
[0028] The sixth objective of this invention is to provide a method for improving the salt tolerance of soybeans by overexpressing the aforementioned gene GmNAT12 in soybean plants, thereby improving the salt tolerance of soybeans.
[0029] The aforementioned gene GmNAT12 in soybean plants can be overexpressed by introducing it into soybeans using the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria.
[0030] Expression vectors carrying the aforementioned genes can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.
[0031] A seventh objective of this invention is to provide a method for breeding salt-tolerant soybean varieties, wherein the method involves overexpressing the aforementioned gene GmNAT12 in soybean plants to obtain salt-tolerant soybean varieties. Preferably, the overexpression of the aforementioned gene GmNAT12 in soybean plants can be achieved by introducing the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria into soybeans to overexpress the aforementioned gene GmNAT12 in soybean plants.
[0032] Beneficial effects:
[0033] This invention marks the first discovery and cloning of a novel plant salt tolerance-related protein gene, GmNAT12. This salt tolerance-related protein influences the salt tolerance of plants. Introducing the gene encoding this protein into plants can improve their salt tolerance, thereby enabling the cultivation of salt-tolerant transgenic plants. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description
[0034] Figure 1 The expression level of the GmNAT12 gene in soybean rooted plants of the GmNAT12-Empty vector and GmNAT12-OE.
[0035] Figure 2 Phenotypic (Fig. a) and root length (Fig. b) of soybean plants with rooting in GmNAT12-Empty vector and GmNAT12-OE combination under salt stress. Detailed Implementation
[0036] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores, and the soybean variety Williams82 used for transgenic purposes is a publicly available variety.
[0037] Example 1: Cloning of the soybean gene GmNAT12
[0038] Design the following primers:
[0039] Primer1: 5'-ATGGAAACCGGGTCGAGTTCGGA-3';
[0040] Primer2: 5'-TCAGACTCCCAAACATTTCGCCCAA-3'.
[0041] Using primers 1 and 2, and root cDNA from Williams82 seedlings as a template, PCR amplification was performed to obtain the target gene GmNAT12.
[0042] PCR amplification was performed in a Bio-rad T100 PCR instrument. The reaction system (50 μL) consisted of: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix (10 mM), 1.5 μL of primer 1 (10 μM), 1.5 μL of primer 2 (10 μM), 2 μL of template cDNA (50 ng / μL), 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 18 μL of ddH2O. The program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, and 72℃ extension for 2 min, for 35 cycles; 72℃ extension for 5 min; and storage at 15℃.
[0043] The PCR products were recovered and purified, then ligated into pEASY-Blunt (Beijing TransGen Biotech Co., Ltd.), transformed into E. coli DH5α competent cells (Beijing Tiangen CB101), and positive clones were selected and sequenced.
[0044] Sequencing results showed that the GmNAT12 gene fragment obtained by PCR reaction had the nucleotide sequence shown in SEQ ID NO.2, encoding a protein composed of 767 amino acid residues (SEQ ID NO.3), and its whole genome sequence is shown in SEQ ID NO.1.
[0045] Example 2: Obtaining and identifying rooting combinations of soybean GmNAT12 gene overexpression
[0046] I. Construction of GmNAT12 gene overexpression vector
[0047] Using root genomic cDNA from Williams82 seedlings as a template, PCR amplification was performed using primer1 and primer2 to obtain the full-length CDS sequence fragment of the GmNPF7.5 gene (SEQ ID NO.2).
[0048] Primer1: 5'-ATGGAAACCGGGTCGAGTTCGGA-3' (SEQ ID NO.4);
[0049] Primer2: 5'-TCAGACTCCCAAACATTTCGCCCAA-3' (SEQ ID NO. 5).
[0050] The amplified product was ligated into the pBA002 vector by XbaI single enzyme digestion, transformed into E. coli DH5α, and the positive plasmid was extracted and sequenced. The sequencing results showed that a recombinant expression vector containing the sequence shown in SEQ ID NO.2 was obtained and named pBA002-GmNAT12.
[0051] II. Obtaining Recombinant Agrobacterium
[0052] pBA002-GmNAT12 was transformed into Agrobacterium K599 using the heat shock method to obtain recombinant strains. Plasmids were extracted and identified by PCR and enzyme digestion. The correctly identified recombinant strains were named K5-pBA002-GmNAT12.
[0053] III. Obtaining Transgenic Plants
[0054] The K5-pBA002-GmNAT12 strain was transformed into the soybean variety Williams82. The specific method was as follows:
[0055] (1) Select large, plump, and disease-free mature seeds and sow them in thoroughly moist vermiculite. Cover the seed surface with another layer of vermiculite and place them in a 26℃ incubation room for 3-4 days to germinate. When the soybean cotyledons are about to open but have not yet opened (the color is light green and lateral roots are growing), hairy root infection can be carried out.
[0056] (2) When the seeds are 2-3 days after germination, inoculate the K5-pBA002-GmCNGC300 strain into a solution containing the antibiotics str and spec. + Streak the bacteria on LB agar plates, incubate at 28°C for 2 days, then pick a single colony (the day before infection) and inoculate it into 5 mL of solution containing antibiotics streptomycin and spectinomycin. + In liquid LB, incubate overnight at 28°C and 200 rpm for about 12 hours;
[0057] (3) Inoculate the bacterial culture at a ratio of 1:100 into a new LB medium, and culture at 28°C and 200 rpm with shaking until OD600 = 0.6-0.8, then collect the bacterial cells;
[0058] (4) Resuspend the centrifuged cells in an equal volume of prepared and sterilized co-culture medium (CCM).
[0059] (5) Select uncontaminated, intact, and undamaged germinated seeds, cut them off at the base with a scalpel, leaving about 2 cm of the hypocotyl, and immerse the soybean hypocotyl in the above-mentioned resuspended bacterial solution for 1 hour at 150 rpm and 28°C. Then transplant the infected explants into moist vermiculite, seal them with a film, and incubate them in a 26°C culture room.
[0060] (6) After one week of cultivation, when white callus tissue grows at the wound of the soybean hypocotyl, it is cultured in 1 / 2 Hoagland nutrient solution. After another week of cultivation, soybean roots grow at the callus tissue, and a transgenic soybean plant with roots is obtained.
[0061] IV. Identification of Transgenic Plants
[0062] 1. PCR molecular identification
[0063] DNA was extracted from the roots of soybean rooting combination plants and used as a template for PCR amplification. The PCR primers are as follows:
[0064] Primer1: 5'-ATGGAAACCGGGTCGAGTTCGGA-3';
[0065] Primer1: 5'-TCAGACTCCCAAACATTTCGCCCAA-3'.
[0066] The PCR products were detected by 1% agarose gel electrophoresis. The target band could be detected in positive plants, but not in negative plants.
[0067] 2. Detection of GmNAT12 gene expression level
[0068] Root samples were collected from GmNAT12 soybean rooting hybrid plants. After homogenization in liquid nitrogen, RNA was extracted using the TRIzol method. An appropriate amount of RNA was used to obtain cDNA using a transcription kit, which was then used as a template for quantitative RT-PCR detection. An appropriate amount of template cDNA was taken, and soybean GmELF was used as an internal control gene for analysis using Hieff. TM qPCR The Green Master Mix (No RoxPlux) kit was used to detect the expression level of the GmNAT12 gene in a Bio-Rad CFX96 real-time PCR instrument. The sequences of the primers used for quantitative detection of the GmELF gene (primer 3 and primer 4) and the primers used for quantitative detection of the GmNAT12 gene (primer 5 and primer 6) are as follows:
[0069] Primer3: 5'-GTTGAAAAGCCAGGGGACAC-3';
[0070] Primer4: 5'-TCTTACCCCTTGAGCGTGG-3'.
[0071] Primer5: 5'-ATGGAAACCGGGTCGAGTTC-3';
[0072] Primer6: 5'-CAGTTCTCTTTGCCCATGAC-3'.
[0073] Relative expression level was 2 -△△CT The method is used for quantitative calculation, and the results are as follows: Figure 1 As shown ( Figure 1 (GmNAT12gf-OE in the middle).
[0074] Example 3: Salt tolerance identification of rooting combination plants overexpressing GmNAT12 gene
[0075] The successfully identified GmNAT12-OE rooting combination plants were further cultured in 1 / 2 Hoagland nutrient solution until the second and third compound leaves unfolded. Soybean rooting combination plants transformed with empty strain K599 were used as controls. Then, soybean rooting combination plants with relatively uniform growth were selected and transferred to 1 / 2 Hoagland nutrient solution and 1 / 2 Hoagland nutrient solution containing 120mM NaCl for further culture. After 7-10 days of continued growth, the phenotype of soybean rooting combination plants was observed by photographing, and relevant physiological indicators were measured.
[0076] Figure 2 The experimental results showed that, compared with the soybean rooting combination plants transformed with empty strains, the GmNAT12-OE soybean rooting combination plants exhibited milder leaf chlorosis and wilting symptoms under salt stress, and the plant height and root length were significantly higher than those of the control group.
[0077] Therefore, based on these results, it is clear that the GmNAT12 gene plays an important positive regulatory role in soybean salt tolerance, and overexpression of this protein-encoding gene can improve soybean salt tolerance.
Claims
1. The application of overexpression of the gene GmNAT12 in improving the salt tolerance of soybean, characterized in that, The sequence of the gene GmNAT12 is shown in SEQ ID NO. 1 or SEQ ID NO.
2.
2. The application according to claim 1, characterized in that, Recombinant expression vectors or recombinant bacteria containing the GmNAT12 gene are introduced into soybeans to overexpress the GmNAT12 gene, thereby improving the salt tolerance of soybeans.
3. A method for improving the salt tolerance of soybeans, characterized in that, Overexpression of the gene GmNAT12, shown in SEQ ID NO. 1 or SEQ ID NO. 2, in soybean plants improves the salt tolerance of soybeans.
4. A method for breeding salt-tolerant soybean varieties, characterized in that, The method involves overexpressing the gene GmNAT12 shown in SEQ ID NO. 1 or SEQ ID NO. 2 in soybean plants to obtain salt-tolerant soybean varieties.