A soybean salt tolerance-related gene GmCIPK12, its encoded protein, and its applications.
By cloning and overexpressing the soybean salt tolerance-related gene GmCIPK12 and its encoded protein, the problem of soybean sensitivity to salt stress was solved, and the salt tolerance and growth performance of soybean were improved, especially the plant height and root length were enhanced.
Patent Information
- Application Number
- CN202410711235.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-04
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2044-06-04
AI Technical Summary
Soybeans are sensitive to salt stress, and traditional breeding methods are time-consuming and difficult to improve. Existing technologies have not been able to thoroughly study the molecular mechanisms of soybean salt tolerance, resulting in reduced soybean yield and quality.
The soybean salt tolerance-related gene GmCIPK12 and its encoded protein were cloned and overexpressed. The recombinant expression vector and recombinant bacteria were used to introduce the protein into soybean plants. The enhanced promoter and translation control signals ensured correct translation, thereby promoting soybean salt tolerance and growth.
Improve the salt tolerance and growth performance of soybeans, especially plant height and root length, and enhance the survival ability of soybeans under salt stress.
Smart Images
Figure HDA0004874089230000011 
Figure HDA0004874089230000012
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering, specifically relating to a soybean salt tolerance-related gene GmCIPK12, its encoded protein, and its applications. Background Technology
[0002] Soybeans are an important oilseed crop and play a vital role in soybean product production. However, soil salinization leads to severe yield reduction and quality degradation in soybeans. Therefore, screening and breeding salt-tolerant varieties and utilizing saline-alkali land are effective measures to increase soybean yield. Thus, using modern biological methods to screen for salt-tolerant genes in soybeans and obtain salt-tolerant germplasm is of great significance.
[0003] Excessive sodium and chloride ions in the soil are the main factors causing salt stress. High salt concentrations in saline soils can cause damage to crops such as soybeans in four ways: osmotic stress, ion toxicity, nutrient imbalance, and imbalance of reactive oxygen species regulation. Soybeans are among the crops most sensitive to salt stress, and its effects on soybean growth and development occur throughout their entire growth cycle. During germination and emergence, salt stress severely affects seed germination and significantly reduces emergence rate. In the seedling stage, salt stress leads to stunted growth, reduced branching, and even yellowing and wilting of leaves. During flowering and grain filling, salt stress significantly reduces pollen viability, resulting in lower pollination rates, underdeveloped grains, and fruit abortion, ultimately leading to reduced yield. Furthermore, under high salt stress, soybean quality deteriorates, with significantly reduced processing quality, cooking and eating quality, and altered oil, protein, and mineral content.
[0004] The response of soybean to salt stress is regulated by a complex molecular network. Salt tolerance is a comprehensive manifestation of various physiological and biochemical responses, and improving soybean salt tolerance using traditional breeding methods is time-consuming and difficult. While molecular design breeding technology can accelerate the development of new salt-tolerant soybean varieties, research on the molecular mechanisms of soybean salt tolerance is still insufficient, and related work is progressing slowly. Therefore, it is particularly important to discover novel genes with superior salt tolerance and elucidate their regulatory pathways and molecular mechanisms. 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 GmCIPK12, its encoded protein, and its applications.
[0006] The first objective of this invention is to provide a salt tolerance-related gene, GmCIPK12, wherein the gene GmCIPK12 is a DNA molecule as described in 1), 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 the protein encoded by the aforementioned gene GmCIPK12.
[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 512 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 GmCIPK12.
[0016] Furthermore, the recombinant expression vector or expression cassette is obtained by inserting the gene GmCIPK12 into the recombination site of the vector pBA002 by Xba I 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 GmCIPK12 into the recombination site of the vector pBA002 by digestion with the restriction endonuclease Xba I. pBA002 containing GmCIPK12 is named pBA002-GmCIPK12.
[0023] Expression cassettes, transgenic cell lines, and recombinant bacteria containing any of the above-mentioned genes GmCIPK12 are all within the scope of protection of this invention.
[0024] The fourth objective of this invention is to provide primers for amplifying the aforementioned gene GmCIPK12. Primer pairs for amplifying the full length or any fragment of the gene GmCIPK12 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 an expression vector or recombinant bacteria that overexpress the aforementioned gene GmCIPK12.
[0026] A sixth object of the present invention is to provide the application of the aforementioned gene GmCIPK12, 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 and / or promoting soybean growth.
[0027] Furthermore, overexpression of the aforementioned gene GmCIPK12 improves the salt tolerance of soybeans.
[0028] Preferably, the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria are introduced into soybean to overexpress the aforementioned gene GmCIPK12.
[0029] The seventh objective of this invention is to provide a method for improving the salt tolerance of soybeans or promoting soybean growth, wherein the method is to overexpress the aforementioned gene GmCIPK12 in soybean plants, thereby improving the salt tolerance of soybeans or promoting soybean growth.
[0030] The aforementioned gene GmCIPK12 in soybean plants can be overexpressed by introducing the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria into soybean plants.
[0031] By overexpressing the gene encoding the protein using any vector that is used in plants, transgenic cell lines and transgenic plants can be obtained. The expression vector carrying the gene 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, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.
[0032] In some embodiments, promoting soybean growth according to the present invention specifically means increasing the plant height and root length of soybeans.
[0033] The eighth objective of this invention is to provide a method for breeding salt-tolerant soybean varieties, wherein the method involves overexpressing the aforementioned gene GmCIPK12 in soybean plants to obtain salt-tolerant soybean varieties. Preferably, the overexpression of the aforementioned gene GmCIPK12 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 GmCIPK12 in soybean plants.
[0034] Beneficial effects:
[0035] This invention marks the first discovery and cloning of a novel plant salt tolerance-related protein gene, GmCIPK12. This salt tolerance-related protein influences plant salt tolerance. Overexpression of the gene encoding this protein can enhance plant salt tolerance, thereby enabling the breeding of salt-tolerant transgenic plants. It can also promote plant growth, particularly increasing plant height. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description
[0036] Figure 1 The expression level of the GmCIPK12 gene in the rooted soybean plants of the GmCIPK12-OE combination.
[0037] Figure 2 The phenotype of the soybean hybrid GmCIPK12-OE under salt stress (Fig. a) and the height of the aboveground parts of the plants (Fig. b). Detailed Implementation
[0038] 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.
[0039] Example 1: Cloning of the soybean gene GmCIPK12
[0040] Design the following primers:
[0041] Primer1: 5'-ATGGCCGACGTCGTTTCCAAGTC-3';
[0042] Primer2: 5'-TCAATCATCAGAAGGTATACAGA-3'.
[0043] Using Primer1 and Primer2 as primers and Williams82 seedling root cDNA as a template, PCR amplification was performed to obtain the target gene GmCIPK12.
[0044] PCR amplification was performed in a Bio-rad T100 PCR instrument. The reaction system (50 μL) consisted of: 25 μL 2×Phanta Max Buffer, 1 μL dNTP Mix (10 mM), 1.5 μL Primer 1 (10 μM), 1.5 μL Primer 2 (10 μM), 2 μL template cDNA (50 ng / μL), 1 μL Phanta Max Super-Fidelity DNA Polymerase, and 18 μL 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℃.
[0045] 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.
[0046] Sequencing results showed that the GmCIPK12 gene fragment obtained by PCR reaction had the nucleotide sequence shown in SEQ ID NO.2, encoding a protein composed of 512 amino acid residues (SEQ ID NO.3), and its whole genome sequence is shown in SEQ ID NO.1.
[0047] Example 2: Obtaining and identifying rooting combinations of soybean GmCIPK12 gene overexpression
[0048] I. Construction of GmCIPK12 gene overexpression vector
[0049] Using the root genomic cDNA of Williams82 seedlings as a template, PCR amplification was performed using Primer1 and Primer2 to obtain the full-length CDS sequence fragment of the GmCIPK12 gene (SEQ ID NO.2).
[0050] Primer1: 5'-ATGGCCGACGTCGTTTCCAAGTC-3' (SEQ ID NO.4);
[0051] Primer2: 5'-TCAATCATCAGAAGGTATACAGA-3' (SEQ ID NO. 5).
[0052] The amplified product was ligated into the pBA002 vector by Xba I 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-GmCIPK12.
[0053] II. Obtaining Recombinant Agrobacterium
[0054] pBA002-GmCIPK12 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-GmCIPK12.
[0055] III. Obtaining Transgenic Plants
[0056] The K5-pBA002-GmCIPK12 strain was transformed into the soybean variety Williams82. The specific method was as follows:
[0057] (1) Select large, plump, and disease-free mature seeds and wash them with deionized water. Then, take a clean filter paper, spray it with water, and place it in a clean petri dish. Spread the selected soybean seeds evenly on the filter paper and germinate them in the dark at 26℃.
[0058] (2) After the soybean seeds germinate, select seeds with uniform growth and sow them in a turnover box containing vermiculite. Cover the surface of the seeds 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 there are lateral roots growing), hairy root infection can be carried out.
[0059] (3) When the seeds germinate for 2-3 days, culture the K5-pBA002-GmCIPK12 strain overnight at 28℃ and 200rpm for about 12 hours; then inoculate the bacterial solution 1:100 into a new LB medium and culture at 28℃ and 200rpm with shaking until OD. 600 =0.6-0.8, collect bacterial cells;
[0060] (4) Resuspend the centrifuged cells in an equal volume of prepared and sterilized co-culture medium (CCM).
[0061] (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. Then, transplant the infected explants into moist vermiculite and incubate them in a 26°C culture room.
[0062] (6) After one week of culture, 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 culture, soybean roots grow at the callus tissue, and soybean plants with transgenic roots are obtained.
[0063] IV. Identification of Transgenic Plants
[0064] 1. PCR molecular identification
[0065] 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:
[0066] Primer1: 5'-ATGGCCGACGTCGTTTCCAAGTC-3';
[0067] Primer2: 5'-TCAATCATCAGAAGGTATACAGA-3'.
[0068] The primers Primer1 and Primer2 are located in the CDS sequence of the GmCIPK12 gene shown in SEQ ID NO.2.
[0069] The PCR products were detected by 1% agarose gel electrophoresis. The target band could be detected in positive plants, but not in negative plants.
[0070] 2. Detection of GmCIPK12 gene expression level
[0071] Root samples were collected from soybean rooting hybrid plants (GmCIPK12). 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. 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 GmCIPK12 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 GmCIPK12 gene (Primer 5 and Primer 6) are as follows:
[0072] Primer3: 5'-GTTGAAAAGCCAGGGGACAC-3';
[0073] Primer4: 5'-TCTTACCCCTTGAGCGTGG-3'.
[0074] Primer5:5'-TTTTGTGGGACACCTGCGTA-3';
[0075] Primer6: 5'-CACAAACAACAACCACCCCAC-3'.
[0076] 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 (GmCIPK12-OE1, GmCIPK12-OE2, GmCIPK12-OE3).
[0077] Example 3: Salt tolerance identification of rooting combination plants overexpressing GmCIPK12 gene
[0078] The successfully identified GmCIPK12-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 100mM NaCl for further culture. After 7-10 days of continued growth, the phenotype of soybean rooting combination plants was observed by taking pictures, and relevant physiological indicators were measured.
[0079] Figure 2The experimental results showed that, compared with the soybean rooting combination transformed with empty strains, the GmCIPK12-OE soybean rooting plants exhibited milder leaf chlorosis and wilting symptoms under salt stress, and the plant height was significantly higher than that of the control group.
[0080] Therefore, based on these results, it is clear that the GmCIPK12 gene plays an important positive regulatory role in soybean salt tolerance. Overexpression of this protein-encoding gene can improve the salt tolerance of soybean plants and promote plant growth.
Claims
1. The application of the gene GmCIPK12 shown in SEQ ID NO.1 or SEQ ID NO.2, the protein shown in SEQ ID NO.3, the recombinant expression vector containing the gene GmCIPK12 shown in SEQ ID NO.1 or SEQ ID NO.2, or the recombinant bacteria in improving soybean salt tolerance and / or promoting soybean growth, characterized in that, Overexpress the gene GmCIPK12 shown in SEQ ID NO.1 or SEQ ID NO.2 in soybean.
2. A method for improving the salt tolerance of soybeans or promoting soybean growth, characterized in that, The method involves overexpressing the gene GmCIPK12 shown in SEQ ID NO.1 or SEQ ID NO.2 in soybean plants, thereby improving the salt tolerance of soybeans or promoting soybean growth.
3. A method for breeding salt-tolerant soybean varieties, characterized in that, The method involves overexpressing the gene GmCIPK12 shown in SEQ ID NO.1 or SEQ ID NO.2 in soybean plants to obtain salt-tolerant soybean varieties.