A soybean salt-tolerance-related gene GmCNGC29, its encoded protein, and application

Abstract

The present invention aims to disclose a soybean salt-tolerance-related gene GmCNGC29, its encoded protein, and its application. The gene GmCNGC29 is a DNA molecule as described in 1) or 2) or 3) below: 1) a DNA molecule whose genomic sequence is shown in SEQ ID NO.1; 2) a DNA molecule whose CDS sequence is shown in SEQ ID NO.2; 3) a DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes the DNA molecule. The present invention provides a genetic engineering application of the gene GmCNGC29 in regulating soybean salt tolerance, specifically overexpressing the aforementioned gene GmCNGC29 to improve soybean salt tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a soybean salt-tolerance-related gene GmCNGC29, its encoded protein and application. Background Art

[0002] Soybeans are a major source of oil and protein, an important economic oilseed crop, and one of the most widely cultivated and utilized cash and food crops worldwide. Their yield and quality are directly linked to global food security and economic development. With the intensification of global climate change, soil salinization is becoming increasingly serious, posing a significant threat to agricultural production. Therefore, utilizing modern biological methods to improve soybean salt tolerance and select plants with excellent salt tolerance traits is crucial for addressing the impact of salinized soils on soybean production.

[0003] Salt stress is usually caused by high concentrations of sodium and chloride ions in the soil. Salt stress includes osmotic stress, ionic stress, and secondary stress. When the salt content in the external environment is high enough to significantly change the water potential, it will affect the plant. This osmotic stress makes it difficult for plant root cells to absorb water, resulting in excessive intracellular osmotic pressure, cell shrinkage, and water absorption that cannot meet the plant's needs. At the same time, in a high-salt environment, ion channels and transport proteins on the plant cell membrane will also be inhibited, thereby limiting the entry of water and nutrients, which will inhibit the growth and development of soybean plants and lead to reduced soybean yields.

[0004] Soybean's response to salt stress is finely regulated by an intricate molecular network. This salt tolerance is the result of the interweaving and combined effects of multiple physiological and biochemical reactions. Improving soybean salt tolerance using traditional breeding methods is often time-consuming and has limited effects, and faces great challenges. Molecular breeding technology can accelerate the breeding of new salt-tolerant soybean varieties, but because the current research on the molecular mechanism of soybean salt tolerance is not in-depth enough, related work is progressing relatively slowly. Therefore, we urgently need to discover more new genes with salt tolerance and further analyze the molecular mechanisms by which these genes regulate salt tolerance. These studies are of great significance for improving soybean salt tolerance and breeding new varieties that are more adaptable to saline-alkali environments. Summary of the Invention

[0005] In order to solve the above technical problems in the prior art, the purpose of the present invention is to disclose a soybean salt tolerance related gene GmCNGC29 and its encoded protein and application.

[0006] The first object of the present invention is to provide a salt tolerance-related gene GmCNGC29, wherein the gene GmCNGC29 is a DNA molecule as described in 1) or 2) or 3) below:

[0007] 1) A DNA molecule having a gene sequence as shown in SEQ ID NO. 1;

[0008] 2) a DNA molecule having a CDS sequence as shown in SEQ ID NO. 2;

[0009] 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein.

[0010] The second object of the present invention is to provide the protein encoded by the aforementioned gene GmCNGC29.

[0011] Specifically, the protein provided by the present invention is selected from any one of (a) or (b):

[0012] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3;

[0013] (b) Protein derived from the target sequence selected from SEQ ID NO.2.

[0014] SEQ ID NO. 3 in the sequence listing consists of 778 amino acids.

[0015] The third object of the present invention is to provide a recombinant expression vector, expression cassette or recombinant bacteria comprising the aforementioned gene GmCNGC29.

[0016] Furthermore, the recombinant expression vector or expression cassette is obtained by inserting the gene GmCNGC29 into the recombination site of the vector pBA002 cut with XbaI; the recombinant expression vector or expression cassette is transferred into the engineered bacteria to obtain the recombinant bacteria.

[0017] The recombinant expression vector containing any of the above genes also falls within the scope of protection of the present invention.

[0018] Existing plant expression vectors can be used to construct a recombinant expression vector containing the gene.

[0019] Plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment. Plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the 3' transcribed untranslated region of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the rouge synthase Nos gene) and plant genes (such as the soybean storage protein gene) all have similar functions.

[0020] When using the gene construction recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (Ubiquitin), which can be used alone or in combination with other plant promoters; In addition, when using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers, and 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 source of the translation control signal and the start codon is extensive, and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

[0021] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For the safety of transgenic plants, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0022] The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene GmCNGC29 into the recombination site of the vector pBA002 cut with restriction endonuclease XbaI. The pBA002 containing GmCNGC29 is named pBA002-GmCNGC29.

[0023] The expression cassette, transgenic cell line and recombinant bacteria containing any of the above-mentioned genes GmCNGC29 all fall within the scope of protection of the present invention.

[0024] The fourth object of the present invention is to provide primers for amplifying the aforementioned gene GmCNGC29. Primer pairs for amplifying the full length or any fragment of the gene GmCNGC29 also fall within the scope of protection of the present invention. In a specific example, the primers are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0025] The fifth object of the present invention is to provide the use of the aforementioned gene GmCNGC29, 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, the aforementioned gene GmCNGC29 was overexpressed to improve the salt tolerance of soybean.

[0027] Preferably, the aforementioned recombinant expression vector, expression cassette or recombinant bacteria is introduced into soybean to overexpress the aforementioned gene GmCNGC29.

[0028] The sixth object of the present invention is to provide a method for improving the salt tolerance of soybeans, overexpressing the aforementioned gene GmCNGC29 in soybean plants, thereby improving the salt tolerance of soybeans.

[0029] To overexpress the aforementioned gene GmCNGC29 in soybean plants, the aforementioned recombinant expression vector, expression cassette or recombinant bacteria can be introduced into soybeans to overexpress the aforementioned gene GmCNGC29 in soybean plants.

[0030] The expression vector carrying the gene can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissue can be cultivated into plants.

[0031] A seventh object of the present invention is to provide a method for cultivating a salt-tolerant soybean variety, wherein the method comprises overexpressing the gene GmCNGC29 in soybean plants to obtain a salt-tolerant soybean variety. Preferably, the overexpression of the gene GmCNGC29 in soybean plants can be achieved by introducing the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria into soybeans to overexpress the gene GmCNGC29 in the soybean plants.

[0032] Beneficial effects:

[0033] This invention, for the first time, discovered and cloned a new plant salt-tolerance-related protein gene, GmCNGC29. This plant salt-tolerance-related protein affects plant salt tolerance. Introducing the gene encoding this protein into plants can improve their salt tolerance, thereby enabling the cultivation of salt-tolerant transgenic plants. This protein and its encoding gene can be used in plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The expression level of GmCNGC29 gene in the rooting plants of soybean combination of GmCNGC29-Empty vector and GmCNGC29-OE.

[0035] Figure 2 Comparison of the phenotypes, root length and plant height of rooting plants of soybean combinations expressing GmCNGC29-Empty vector and GmCNGC29-OE under salt stress. DETAILED DESCRIPTION

[0036] The following examples facilitate a better understanding of the present invention but are not intended to limit the present invention. The experimental methods used in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The soybean variety Williams 82 used for transgenic production is a publicly available variety.

[0037] Example 1. Cloning of soybean gene GmCNGC29

[0038] The following primers were designed:

[0039] Primer1: 5'-ATGGCTAATTTTGAGAAAGATGAGG-3';

[0040] Primer2: 5'-TTACAGTGAATTGTAAATGTTTGATG-3'.

[0041] Using primer1 and primer2 as primers and Williams82 seedling root cDNA as template, PCR amplification was performed to obtain the target gene GmCNGC29.

[0042] PCR amplification was performed in a Bio-rad T100 PCR instrument. The reaction system (50 μL) was as follows: 2× Phanta Max Buffer 25 μL, dNTP Mix (10 mM) 1 μL, primer 1 (10 μM) 1.5 μL, primer 2 (10 μM) 1.5 μL, template cDNA (50 ng / uL) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O 18 μL. The program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 2 min; extension at 72°C for 5 min; and storage at 15°C.

[0043] The PCR product was recovered and purified, then ligated to pEASY-Blunt (Beijing Quanshijin Biotechnology Co., Ltd.), transformed into Escherichia coli DH5α competent cells (Beijing Tiangen Co., Ltd. CB101), and positive clones were selected and sequenced.

[0044] The sequencing results showed that the GmCNGC29 gene fragment obtained by PCR reaction had the nucleotide sequence shown in SEQ ID NO.2, encoding a protein composed of 778 amino acid residues (SEQ ID NO.3), and its full genome sequence was shown in SEQ ID NO.1.

[0045] Example 2: Obtaining and Identifying Root-Growing Combination Plants Overexpressing the GmCNGC29 Gene in Soybeans

[0046] 1. Construction of GmCNGC29 gene overexpression vector

[0047] Using the root genomic cDNA of Williams82 seedlings as a template, primer1 and primer2 were used for PCR amplification to obtain the full-length CDS sequence fragment of the GmCNGC29 gene (SEQ ID NO. 2).

[0048] Primer1: 5'-ATGGCTAATTTTGAGAAAGATGAGG-3' (SEQ ID NO.4);

[0049] Primer2: 5'-TTACAGTGAATTGTAAATGTTTGATG-3' (SEQ ID NO. 5).

[0050] The amplified product was digested with XbaI and ligated into the pBA002 vector, 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-GmCNGC29.

[0051] 2. Obtaining recombinant Agrobacterium

[0052] pBA002-GmCNGC29 was transformed into Agrobacterium K599 strain by heat shock method to obtain recombinant strain. The plasmid was extracted and identified by PCR and enzyme digestion. The correctly identified recombinant strains were named K5-pBA002-GmCNGC29.

[0053] 3. Obtaining transgenic plants

[0054] The K5-pBA002-GmCNGC29 strain was transformed into soybean variety Williams82 by the following method:

[0055] (1) Select large, plump, and spot-free mature seeds and sow them in moistened vermiculite. Cover the surface of the seeds with a layer of vermiculite and place them in a 26°C culture room for germination for 3-4 days. 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 germinated for 2-3 days, the K5-pBA002-GmCNGC29 strain was inoculated with antibiotics str and spec. + Streak on LB plate, culture at 28℃ for 2 days, pick a single colony (one day before infection) and inoculate it into 5mL containing antibiotics str and spec+ Incubate in liquid LB at 28°C and 200 rpm overnight for about 12 h.

[0057] (3) Inoculate the bacterial solution into new LB medium at a ratio of 1:100 and culture at 28°C and 200 rpm until the OD 600 =0.6-0.8, collect bacteria;

[0058] (4) Resuspend the centrifuged cells in an equal volume of sterilized co-culture medium (CCM).

[0059] (5) Select germinated seeds that are uncontaminated and have intact cotyledons. Cut them off along the base with a scalpel, leaving about 2 cm of the hypocotyl. Soak the soybean hypocotyl in the resuspended bacterial solution and infect it for 1 hour at 150 rpm and 28°C. Then transplant the infected explants into moist vermiculite, seal them with film, and incubate them in a culture room at 26°C.

[0060] (6) After one week of culture, white callus tissue grows from the wound of the soybean hypocotyl, and the callus tissue is cultured in 1 / 2 Hoagland nutrient solution. After another week of culture, soybean roots grow from the callus tissue, and a transgenic soybean plant with transgenic roots is obtained.

[0061] 4. Identification of transgenic plants

[0062] 1. PCR molecular identification

[0063] DNA was extracted from the roots of the soybean hairy root combination plants and used as a template for PCR amplification. The PCR primers were as follows:

[0064] Primer1: 5'-ATGGCTAATTTTGAGAAAGATGAGG-3';

[0065] Primer2: 5'-TTACAGTGAATTGTAAATGTTTGATG-3'.

[0066] The PCR products were detected by 1% agarose gel electrophoresis. The target bands were detected in positive plants but not in negative plants.

[0067] 2. Detection of GmCNGC29 gene expression

[0068] Take the root sample of GmCNGC29 soybean hairy root combination plant, freeze the sample in liquid nitrogen and extract RNA by TRIzol method. Take appropriate amount of RNA and use hairy root transcription kit to obtain cDNA as template for fluorescence quantitative RT-PCR detection. Take appropriate amount of template cDNA and use soybean GmELF as internal reference gene. TM qPCR Green Master Mix (No RoxPlux) kit was used to detect the expression of the GmCNGC29 gene in a Bio-Rad CFX96 fluorescent quantitative PCR instrument. The sequences of the primers used for quantitative detection of the GmELF gene (primer 3 and primer 4) and the GmCNGC29 gene (primer 5 and primer 6) are as follows:

[0069] Primer3: 5'-GTTGAAAAGCCAGGGGACAC-3';

[0070] Primer4: 5'-TCTTACCCCTTGAGCGTGG-3'.

[0071] Primer5: 5'-ATGGCTAATTTTGAGAAAGA-3';

[0072] Primer6: 5'-CTTTCGTATGGCTCCGTGAG-3'.

[0073] The relative expression level was 2 -△△CT The quantitative calculation was performed by the method, and the results were as follows Figure 1 As shown ( Figure 1 GmCNGC29-OE in .

[0074] Example 3: Identification of salt tolerance of GmCNGC29 gene overexpressing rooting combination plants

[0075] The successfully identified GmCNGC29-OE rooting combination plants were further cultured in 1 / 2 Hoagland nutrient solution until the 2nd-3rd compound leaves unfolded. The soybean rooting combination plants transformed with the K599 empty strain were used as controls. Then, soybean rooting combination plants with relatively consistent growth were selected and moved to 1 / 2 Hoagland nutrient solution and 1 / 2 Hoagland nutrient solution containing 120 mM NaCl for further culture. After 7-10 days of growth, the phenotypes of the soybean rooting combination plants were 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 the empty strain, the GmCNGC29-OE soybean rooting combination plants showed milder leaf chlorosis and wilting symptoms under salt stress, and the plant height and root length were significantly higher than the control group.

[0077] Therefore, based on these results, it can be clearly seen that the GmCNGC29 gene has an important positive regulatory effect on soybean salt tolerance, and overexpression of this protein encoding gene can improve soybean salt tolerance.

Claims

1. Use of the gene GmCNGC29 shown in SEQ ID NO.1 or SEQ ID NO.2, the protein shown in SEQ ID NO.3, and a recombinant expression vector comprising the gene GmCNGC29 shown in SEQ ID NO.1 or SEQ ID NO.2 in improving soybean salt tolerance, specifically overexpressing the gene GmCNGC29 shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A method for regulating soybean salt tolerance, characterized in that: Overexpression of the gene GmCNGC29 shown in SEQ ID NO.1 or SEQ ID NO.2 in soybean plants improves the salt tolerance of soybeans.

3. A method for cultivating salt-tolerant soybean varieties, characterized in that: The method is to overexpress the gene GmCNGC29 shown in SEQ ID NO.1 or SEQ ID NO.2 in soybean plants to obtain a salt-tolerant soybean variety.

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