Soybean salt-tolerant related gene GmABCG14, and coding protein and application thereof

Abstract

The application discloses a soybean salt-tolerant related gene GmABCG14, a coding protein and application thereof. The gene GmABCG14 is an NDA molecule as described in 1), 2) or 3) below: 1) a DNA molecule with a genomic sequence as shown in SEQ ID NO. 2; 2) a DNA molecule with a CDS sequence as shown in SEQ ID NO. 2; 3) a DNA molecule hybridized with the DNA sequence defined in 1) or 2) under stringent conditions and encoding the DNA molecule. The application provides a genetic engineering application of the gene GmABCG14 in regulating soybean salt tolerance, specifically overexpressing the aforementioned gene GmABCG14 to improve the salt tolerance of soybean.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a soybean salt tolerance-related gene GmABCG14, its encoded protein, and its applications. Background Technology

[0002] Soybean (Glycine max) is the world's fourth largest crop and the most widely planted oilseed crop. Mature soybean seeds are rich in plant protein and oil, and have extremely high utilization value. Currently, due to global climate change, improper irrigation, and inappropriate fertilizer application, more than 20% of arable land is affected by salt stress, and soil salinization is becoming increasingly prominent, seriously threatening food security. Therefore, exploring the impact of salt stress on soybeans is of significant theoretical and practical importance for researching and developing salt-tolerant soybean varieties, improving soybean yield and quality, and increasing land utilization.

[0003] Salt stress affects plants primarily in two ways: firstly, it reduces the plant's water absorption capacity, thus inhibiting growth and causing osmotic stress; secondly, ionic toxicity occurs when salt concentration reaches a critical point, preventing the plant from maintaining ion balance and causing secondary damage. Salt stress affects plant germination, growth, photosynthetic pigments, photosynthesis, ion balance, and nutrient balance, which severely impacts normal plant growth and development and crop yield.

[0004] Screening and breeding superior salt-tolerant soybean varieties is of great significance for improving the utilization rate of saline-alkali land. Discovering salt-tolerant genes can reveal their molecular functions in the salt-alkali tolerance process, which helps to elucidate the salt-alkali tolerance mechanism of soybeans, and also helps to provide high-quality gene sources for the breeding of salt-tolerant soybean varieties, thus accelerating the process of soybean resistance breeding. 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 GmABCG14, its encoded protein, and its applications.

[0006] The first objective of this invention is to provide a salt tolerance-related gene GmABCG14, wherein the gene GmABCG14 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 GmABCG14.

[0011] Specifically, the protein sequence provided by this invention is shown in SEQ ID NO.3 and consists of 651 amino acids.

[0012] A third objective of this invention is to provide a recombinant expression vector, expression cassette, or recombinant bacterium containing the aforementioned gene GmABCG14.

[0013] Furthermore, the recombinant expression vector or expression cassette is obtained by inserting the gene GmABCG14 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.

[0014] Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.

[0015] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0016] 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).

[0017] 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.

[0018] 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.

[0019] The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene GmABCG14 into the recombination site of the vector pBA002 by digestion with the restriction endonuclease XbaI. pBA002 containing GmABCG14 is named pBA002-GmABCG14.

[0020] Expression cassettes, transgenic cell lines, and recombinant bacteria containing any of the above-mentioned genes GmABCG14 are all within the scope of protection of this invention.

[0021] The fourth objective of this invention is to provide primers for amplifying the aforementioned gene GmABCG14. Primer pairs for amplifying the full length or any fragment of the gene GmABCG14 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.

[0022] A fifth objective of this invention is to provide the application of the aforementioned gene GmABCG14, 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.

[0023] Furthermore, overexpression of the aforementioned gene GmABCG14 improves the salt tolerance of soybeans.

[0024] Preferably, the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria are introduced into soybean to overexpress the aforementioned gene GmABCG14.

[0025] The sixth objective of this invention is to provide a method for improving the salt tolerance of soybeans, wherein the method involves overexpressing the aforementioned gene GmABCG14 in soybean plants, thereby improving the salt tolerance of soybeans.

[0026] The aforementioned gene GmABCG14 in soybean plants can be overexpressed by introducing the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria into soybean plants.

[0027] 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 GmABCG14 in soybean plants to obtain salt-tolerant soybean varieties. Preferably, the overexpression of the aforementioned gene GmABCG14 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 GmABCG14 in soybean plants.

[0028] Beneficial effects:

[0029] This invention marks the first discovery and cloning of a novel plant salt tolerance-related protein gene, GmABCG14. 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. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0030] Figure 1 The expression level of the GmABCG14 gene in the rooted soybean plants of the GmABCG14-OE combination.

[0031] Figure 2 The phenotype of soybean plants with rooting in the GmABCG14-OE combination under salt stress (Figure A) and the chlorophyll content of the plants (Figure B). Detailed Implementation

[0032] 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.

[0033] Example 1: Cloning of the soybean gene GmABCG14

[0034] Design the following primers:

[0035] Primer1: 5'-ATGCCAGAAAACTGCATAGC-3' (SEQ ID NO.4);

[0036] Primer2: 5'-CTACCTCACTCTCAGTAGTGC-3' (SEQ ID NO. 5).

[0037] Using primers 1 and 2, and root cDNA from Williams82 seedlings as a template, PCR amplification was performed to obtain the target gene GmABCG14.

[0038] 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℃.

[0039] 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.

[0040] Sequencing results showed that the GmABCG14 gene fragment obtained by PCR reaction had the nucleotide sequence shown in SEQ ID NO.2, encoding a protein composed of 651 amino acid residues (SEQ ID NO.3), and its whole genome sequence is shown in SEQ ID NO.1.

[0041] Example 2: Obtaining and identifying rooting combinations of soybean GmABCG14 gene overexpression

[0042] I. Construction of GmABCG14 gene overexpression vector

[0043] 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 GmABCG14 gene (SEQ ID NO.2).

[0044] Primer1: 5'-ATGCCAGAAAACTGCATAGC-3' (SEQ ID NO.4);

[0045] Primer2: 5'-CTACCTCACTCTCAGTAGTGC-3' (SEQ ID NO. 5).

[0046] 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-GmABCG14.

[0047] II. Obtaining Recombinant Agrobacterium

[0048] pBA002-GmABCG14 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-GmABCG14.

[0049] III. Obtaining Transgenic Plants

[0050] The K5-pBA002-GmABCG14 strain was transformed into the soybean variety Williams82. The specific method was as follows:

[0051] (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℃.

[0052] (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.

[0053] (3) When the seeds germinate for 2-3 days, culture the K5-pBA002-GmABCG14 strain overnight at 28℃ and 200r for about 12 hours; then inoculate the bacterial solution 1:100 into a new LB medium and culture at 28℃ and 200r with shaking until OD. 600 =0.6-0.8, collect bacterial cells;

[0054] (4) Resuspend the centrifuged cells in an equal volume of prepared and sterilized co-culture medium (CCM).

[0055] (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.

[0056] (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.

[0057] IV. Identification of Transgenic Plants

[0058] 1. PCR molecular identification

[0059] 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:

[0060] Primer3: 5'-AAGTGGATTGATGTGATATCTCCA-3';

[0061] Primer4: 5'-CTACCTCACTCTCAGTAGTGC-3'.

[0062] The primers described above, primer 3 and primer 4, are located in the CDS sequence of the GmABCG14 gene shown in SEQ ID NO.2.

[0063] The PCR products were detected by 1% agarose gel electrophoresis. The target band could be detected in positive plants, but not in negative plants.

[0064] 2. Detection of GmABCG14 gene expression level

[0065] Root samples were collected from soybean plants in the GmABCG14 rooting combination. 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 GmABCG14 gene in a Bio-Rad CFX96 real-time PCR instrument. The sequences of the primers used for quantitative detection of the GmELF gene (primer 5 and primer 6) and the GmABCG14 gene (primer 7 and primer 8) are as follows:

[0066] Primer5:5'-GTTGAAAAGCCAGGGGACA-3';

[0067] Primer6: 5'-TCTTACCCCTTGAGCGTGG-3'.

[0068] Primer7:5'-GGCTCAAGCCTCATCCAGTT-3';

[0069] Primer8: 5'-ATGAAGGGAGGGATCTGTTGT-3'.

[0070] Relative expression level was 2 -△△CTThe method is used for quantitative calculation, and the results are as follows: Figure 1 As shown ( Figure 1 (ABCG14-OE1, ABCG14-OE2, ABCG14-OE3).

[0071] Example 3: Salt tolerance identification of rooting combination plants overexpressing GmABCG14 gene

[0072] The successfully identified GmABCG14-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 transfected with pBA002 empty vector 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 photographing, and relevant physiological indicators were measured.

[0073] Figure 2 The experimental results showed that, compared with the soybean rooting combination plants transformed with empty vectors, the GmABCG14-OE soybean rooting plants exhibited milder leaf chlorosis and wilting symptoms under salt stress, and the chlorophyll content of the plants was significantly higher than that of the control group.

[0074] Therefore, based on these results, it is clear that the GmABCG14 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 gene GmABCG14 in improving salt tolerance in soybeans, characterized in that, The sequence of the gene GmABCG14 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 gene GmABCG14 are introduced into soybeans to overexpress the gene GmABCG14, thereby improving the salt tolerance of soybeans.

3. A method for improving the salt tolerance of soybeans, characterized in that, The method involves overexpressing the gene GmABCG14 shown in SEQ ID NO. 1 or SEQ ID NO. 2 in soybean plants to improve the salt tolerance of soybeans.

4. A method for breeding salt-tolerant soybean varieties, characterized in that, The method involves overexpressing the gene GmABCG14 shown in SEQ ID NO. 1 or SEQ ID NO. 2 in soybean plants to obtain salt-tolerant soybean varieties.

Images