Application of GmCIPK6 gene in regulating soybean salt tolerance

Abstract

The application relates to the technical field of plant genetic engineering, and specifically provides GmCIPK6 application of a gene in regulating salt tolerance of soybeans. GmCIPK6 The application clones a gene from soybeans, and obtains transgenic soybean plants with overexpression and CRISPR / Cas9 knockout of the gene through an agrobacterium-mediated genetic transformation technology. GmCIPK6 Results show that under salt stress, the leaf damage degree of the overexpression plants is significantly lower than that of wild types, and the damage degree of the knockout plants is significantly aggravated; meanwhile, the activities of antioxidant enzymes CAT, POD and APX in the overexpression plants are significantly increased, and the activities of the antioxidant enzymes in the knockout plants are significantly decreased. Transcriptome analysis shows that the differentially expressed genes are significantly enriched in a hydrogen peroxide catalytic pathway and an oxidative stress related pathway. GmCIPK6 GmCIPK6 The application proves that the gene positively regulates salt tolerance of soybeans by regulating an antioxidant enzyme system, and provides an effective gene resource for breeding new salt-tolerant soybean varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of the GmCIPK6 gene in regulating soybean salt tolerance. Background Technology

[0002] Soybeans Glycine max [L.) Merr.] Soybeans are an important economic crop used for grain, oil, and feed, playing a vital role in national economic development. However, my country's soybean yield is too low, while the demand for high-quality protein and edible oil is increasing. Domestic soybean production is insufficient to meet this demand, with approximately 85% of soybeans imported annually. Therefore, increasing domestic soybean production is urgent. Soybeans are susceptible to various geographical environmental factors during their growth. Among these, soil salinization is a significant factor affecting crop yield. Therefore, improving soybean salt tolerance is crucial for increasing soybean yield. Currently, the most effective breeding method is to identify and clarify the functions of soybean salt tolerance-related genes, and then use molecular breeding techniques to cultivate new salt-tolerant soybean varieties (lines). The results of this invention provide an important theoretical basis and effective gene resources for cultivating new soybean varieties (lines) with ideal salt tolerance.

[0003] Currently, researchers both domestically and internationally have identified numerous gene loci associated with soybean salt tolerance using various population and technological approaches. However, research on the functional verification and molecular mechanisms of action of candidate genes associated with these loci remains relatively lagging. This study aims to investigate a soybean protein kinase gene closely related to calcium signal transduction. GmCIPK6 Its role in regulating soybean salt tolerance. First, on... GmCIPK6 The gene was cloned and subjected to bioinformatics analysis. Next, transgenic soybean materials with gene overexpression and knockout were created using soybean genetic transformation technology, and their salt tolerance was identified; simultaneously, transcriptomic data from the transgenic materials were analyzed. Finally, the salt tolerance function was clarified. GmCIPK6 Genes enhance the salt tolerance of soybeans by regulating antioxidant enzymes in the body. This invention can provide important genetic resources for breeding new soybean varieties (lines) with ideal salt tolerance and has broad application potential for enhancing soybean salt tolerance. Summary of the Invention

[0004] The purpose of this invention is to investigate soybean protein kinase genes. GmCIPK6 The salt tolerance regulatory function of this gene was clarified by the inventors through cloning the gene and creating transgenic soybean materials with overexpression and knockout. Combined with physiological and transcriptomic analysis, the salt tolerance regulatory function was further defined. GmCIPK6 By regulating the antioxidant enzyme system to positively enhance the salt tolerance of soybeans, we can provide effective genetic resources and theoretical basis for breeding new salt-tolerant soybean varieties.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides overexpression GmCIPK6 Applications of genes, wherein the application is any of the following: A1) Application in improving the salt tolerance of soybeans; A2) Application in the preparation of salt-tolerant soybeans; GmCIPK6 The nucleotide sequence of the gene is shown in SEQ ID NO.1. GmCIPK6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0006] Furthermore, the application involves increasing the salt tolerance of soybeans by enhancing the activity of catalase, peroxidase, and ascorbate peroxidase in soybeans.

[0007] The second aspect of the present invention provides overexpression GmCIPK6 Applications of gene-related biomaterials, wherein the application is any of the following: B1) Application in improving the salt tolerance of soybeans; B2) Application in the preparation of salt-tolerant soybeans; The biomaterial is any one of the following C1) to C3): C1) Expression cassettes containing nucleic acid molecules with nucleotide sequences as shown in SEO ID NO. 1; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 1; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEO ID NO. 1, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium.

[0008] Furthermore, the application involves increasing the salt tolerance of soybeans by enhancing the activity of catalase, peroxidase, and ascorbate peroxidase in soybeans.

[0009] A third aspect of the present invention provides a method for cultivating salt-tolerant soybeans, the method comprising overexpressing in soybeans... GmCIPK6 Genes were used to obtain soybeans with improved salt tolerance. GmCIPK6 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0010] In the method described, overexpression is performed in soybeans. GmCIPK6 Genes are produced by using genetic engineering technology to make soybeans contain... GmCIPK6 Gene expression levels increase.

[0011] In the method described above, genetic engineering technology is used to induce genetic modification in soybeans. GmCIPK6 Increased gene expression is achieved by constructing expression vectors to enhance the expression levels in the soybean genome. GmCIPK6 The expression level of the gene increases, and the expression vector contains nucleotides with the sequence shown in SEQ ID NO:1.

[0012] The beneficial effects of this invention are: First cloned and validated soybean GmCIPK6 This gene can positively regulate soybean salt tolerance, filling the gaps in the study of soybean salt tolerance mechanisms by the GmCIPKs family of genes, and providing a novel functional gene for molecular breeding of soybean salt tolerance. GmCIPK6 By regulating the antioxidant enzyme system (enhancing the activities of CAT, POD, and APX), the H2O2 catalysis and oxidative stress pathway were mediated, elucidating the mechanism of salt tolerance at the molecular level and providing theoretical support for the breeding of salt-tolerant soybean varieties. Successfully obtained... GmCIPK6 Overexpression and knockout soybean lines produce stable and heritable materials that can be directly applied to the molecular breeding of salt-tolerant soybean varieties (lines), shortening the breeding cycle and improving breeding efficiency. GmCIPK6 It can significantly enhance the salt tolerance of soybeans, reduce the damage of salt stress to plants, and adapt to the planting needs of saline-alkali land. It provides efficient gene resources and technical solutions for salt-tolerant soybean breeding, and helps to improve the yield and stress resistance of domestic soybeans. Attached Figure Description

[0013] Figure 1 For soybeans GmCIPK6 Results of PCR amplification of the coding region of the gene.

[0014] Figure 2 For the structural prediction of soybean GmCIPK6 protein: A is the predicted secondary structure of soybean GmCIPK6 protein; B is the predicted tertiary structure of soybean GmCIPK6 protein.

[0015] Figure 3 for GmCIPK6 Analysis of the starter region control elements.

[0016] Figure 4 For overexpression GmCIPK6 PCR identification results of soybean plants.

[0017] Figure 5 for GmCIPK6 Relative expression levels in wild-type and overexpression transgenic soybean lines: WT represents wild-type soybean, OE-1~OE-4 represent... GmCIPK6 Overexpression transgenic soybean lines.

[0018] Figure 6 To knock out GmCIPK6 Sequence analysis of soybean edited forms.

[0019] Figure 7 for GmCIPK6 Identification of salt tolerance function of genes in soybean: A represents overexpression / knockout GmCIPK6 Identification of soybean salt tolerance phenotypes; B represents overexpression under salt stress. GmCIPK6 GO analysis of soybean and wild-type soybean; C is transcriptome analysis of antioxidant enzyme genes in soybean under salt stress; D is overexpression / knockout. GmCIPK6 Identification of CAT enzyme activity in soybean; E indicates overexpression / knockout GmCIPK6 Identification of POD enzyme activity in soybean; F indicates overexpression / knockout GmCIPK6 Identification of APX enzyme activity in soybeans. Detailed Implementation

[0020] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0021] Explanation of the sequence list SEQ ID NO.1:ATGAGCTCATCCTCAGAGAAGAAACCTTCTTCTTCTTCTTCTTCTTCTTCCTTCAGTGTTGAAGGTATTTGGTTCCCCTTTGACGGCTATGAGAAGTGACGAGGCTCTCAGAGA TGGCTGGGTTAAGAAAATCGAGTGTCCTTTCTGCCATCGCGAGTTTCAGAGTTTGCAAGCTCTAGGGGGACACCAAAATGCGCATAGGAGAGAAAGACAAATGGCTAGGTTTGCCCAATTT GAATATATGTGTCTGAATCAAAGCAACCAAATGTTCCAATCGGTTACACCTCTTGTGGTTGAGCATGGAGCAGCACCAACCTCTGTTTTCGGTGGTGCAACTCGGTTATGGACAGCACCTGAGTCATCACAGCTACCTGTGGCGGAGCCACCAGGTATGCTTATCCCTATTGTACAAGCACCAGTAGTAGGGGATGATGATAATATCGACCTAGAACTGAGACTAGGTAATTCTTCTAAGTAA SEQ ID NO.2: MSSSSEKKPSSSSSSSSPSVLKVFGSPLTAMRSDEALRDGWVKKIECPFCHREFQSLQALGGHQNAHRRERQMARFAQFEYMCLNQSNQMFQSVTPLVVEHGAAPTSVFGGATRLWTAPESSQLPVAEPPGMLIPIVQAPVVGDDDNIDLELRLGNSSK SEQ ID NO.3: 5'-ATGAGTGAGAAAGAGAAAGGC-3' SEQ ID NO.4: 5'-TCAAGCACTAGCAGGTGCGGAAT-3' SEQ ID NO.5: 5'-ATACAGTCTCAGAAGACCAAAGG-3' SEQ ID NO.6: 5'-GAAGTCGGAGACTTTGAGG-3' SEQ ID NO.7: GGACAGTCGACGTTGCTGCACGG SEQ ID NO.8: ACGCGCGGAACCTGAATACAGGG Example 1 GmCIPK6 Gene cloning and bioinformatics analysis The cultivated soybean variety Williams82 was selected as the gene cloning material, and total RNA was extracted from soybean leaves. The extracted RNA was analyzed, and the RNA that passed the analysis was reverse transcribed into cDNA using a Cisco reverse transcription kit (AG0305-B). GmCIPK6 Cloning primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 were designed for the gene coding region. The target gene was amplified by PCR using TOYOBO KOD-Plus-Neo high-fidelity enzyme. The product was detected by agarose gel electrophoresis, yielding a 1293bp fragment of the target gene. Figure 1 The product was purified using a gel extraction kit (DR511-250T) from Coollabis. The PCR reaction system and procedure are shown below: PCR reaction system (20 μL):

[0022] PCR reaction program (35 cycles):

[0023] Furthermore, the physicochemical properties, secondary structure, and tertiary structure of the GmCIPK6 protein were analyzed using the online websites SOPMA (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) and Expasy (https: / / www.expasy.org / resources / swiss-model). Bioinformatics analysis showed that the theoretical relative molecular mass of the GmCIPK6 protein is 48.36 kDa, the theoretical isoelectric point is 9.10, the average hydrophilicity is -0.351 (negative values ​​represent hydrophilicity), and the instability index is 32.63 < 40, indicating that the GmCIPK6 protein is a stable hydrophilic protein. The secondary structure results showed that random coil structures accounted for the largest proportion (46.28%) of the GmCIPK6 protein, followed by α-helices (36.05%) and extended chains (17.67%). Figure 2 ).

[0024] To further explore GmCIPK6 Possible regulatory functions of genes, selection GmCIPK6 The 2000bp upstream of the ATG start codon was used as the upstream promoter region of the gene. The sequence was entered into the PlantCARE website (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) for analysis of the cis-regulatory elements in the promoter region. Figure 3 The results showed that the promoter region of this gene contains multiple cis-regulatory elements, most of which are light-responsive elements. It also contains various hormone-responsive elements, such as gibberellin-responsive elements, as well as cis-regulatory elements related to environmental and developmental responses, such as defense and stress-responsive elements. These cis-regulatory elements are significantly associated with plant growth and development under stress. Therefore, this study hypothesizes that... GmCIPK6 Genes may be involved in biological responses that influence salt tolerance in soybeans.

[0025] Example 2 (transfer) GmCIPK6 Obtaining gene overexpression soybeans To clarify the salt tolerance function of this gene, Agrobacterium-mediated stable genetic transformation of soybean was used to obtain regenerated plants, and transgenic molecular identification was performed.

[0026] First, an overexpression vector was constructed. The plant expression vector pCAMBIA3301 plasmid was double-digested with restriction endonucleases Nco I and Spe I. The digested products were then ligated with purified PCR products using a ligase. The ligation product was transformed into *E. coli* DH5α competent cells and cultured at 37°C for 12-16 hours. Single clones were picked and cultured for identification using colony PCR. Sequencing was performed on colony cultures with the correct PCR band size. The sequencing results confirmed the successful construction of the recombinant plasmid pCAMBIA3301-GmCIPK6. Based on this, the recombinant plasmid was transformed into *Agrobacterium* EHA105 competent cells. Single clones were picked and cultured for identification using colony PCR (PCR procedure and reaction system were consistent with Example 1), and correct colony cultures were retained.

[0027] After obtaining positive Agrobacterium tumefaciens bacterial suspension, a soybean genetic transformation experiment was conducted. Specifically, plump, spotless, and uniformly sized Dongnong 50 seeds were selected, sterilized, and soaked in sterile water to allow them to absorb water and germinate. After 16 hours, they were washed 2-3 times with sterile water. The washed seeds were divided in half along the cotyledons, the seed coat was removed, and the hypocotyl was retained for about 3-4 mm. Six to eight incisions were made along the cotyledon node. Simultaneously, Agrobacterium tumefaciens suspension pCAMBIA3301-GmCIPK6 was activated and prepared. This suspension and the cut soybeans were placed in an Erlenmeyer flask and cultured for 30 minutes. After infection, excess bacterial suspension was discarded, and the soybeans were placed on a co-culture medium and cultured in the dark for 3-5 days. Subsequently, they were transferred to recovery medium and cultured normally for one week. After two rounds of selection, they were transferred to stem-growing medium. The elongation medium was changed every two weeks, and the culture was repeated 3-4 times. When the stem length was about 3-4 cm, the stems were transferred from the callus tissue to the rooting medium. After about 10-14 days, when the root length and plant condition are good, the seedlings are hardened off at room temperature. After 3-4 days of hardening off, the regenerated seedlings are transplanted into the soil. After they are growing well, positive identification is performed.

[0028] T0 generation overexpression was performed using specific primers with nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6. GmCIPK6 Soybean genome PCR identification successfully yielded positive plants for the target band. Figure 4 Subsequently, T1 generation plants were identified by sowing, and after individual harvesting, they were sown again, and the high expression level of T2 generation plants was identified using quantitative real-time PCR. Figure 5 The T2 generation lines with high expression were propagated, and the harvested T3 generation plants could be used for subsequent identification of soybean salt tolerance.

[0029] Example 3 GmCIPK6 Obtaining gene knockout mutants based on GmCIPK6Gene knockout targets were designed and constructed into a CRISPR / Cas9 vector. The gene was then targeted for knockout using Agrobacterium-mediated soybean genetic transformation. This invention designed two knockout targets: target sequence 1 is GGACAGTCGACGTTGCTGCACGG (SEQ ID NO. 7), and knockout sequence 2 is ACGCGCGGAACCTGAATACAGGG (SEQ ID NO. 8). Genomic DNA was extracted from the knockout plants and sequenced. Figure 6 The mutant plants were obtained. The obtained T2 generation plants underwent effective editing, resulting in premature sequence termination, which can be used for subsequent identification of salt tolerance in the plants.

[0030] Example 4 Overexpression / Knockout GmCIPK6 Salt tolerance assessment of soybeans The overexpression obtained above GmCIPK6 plant ( GmCIPK6-OE-1 ; GmCIPK6-OE-2 ) and knocking out plants ( GmCIPK6-KO1 ; GmCIPK6-KO3 Salt stress treatment was conducted and its salt tolerance was assessed. The results showed that ( Figure 7 In soybean A), under salt stress treatment, compared with wild-type soybean, overexpression of [a specific gene] was observed. GmCIPK6 Soybean leaves suffered less damage; knockout GmCIPK6 Soybean leaves suffered greater damage, which preliminarily indicates GmCIPK6 The gene positively regulates soybean salt tolerance. Further transcriptomic and GO enrichment analyses were performed. Figure 7 (B) was found to be overexpressed under salt stress. GmCIPK6 Differential genes in soybean compared to wild type are significantly enriched in the hydrogen peroxide (H2O2) catalytic pathway and oxidative stress-related pathways. Based on this, this study examined some antioxidant enzymes regulating oxidative stress and the hydrogen peroxide (H2O2) catalytic pathway, and found that ( Figure 7 C, D, E, and F in soybeans, compared to wild-type soybeans, showed overexpression under salt stress. GmCIPK6 The contents of antioxidant enzymes CAT, POD, and APX in soybean leaves were significantly increased, while those knocked out... GmCIPK6 The contents of CAT, POD and APX in soybean leaves decreased significantly. Figure 7 (D, E, F in the text), thereby demonstrating that the present invention confirms GmCIPK6 Genes can enhance soybean salt tolerance by regulating the activity of antioxidant enzymes in the body in response to hydrogen peroxide (H2O2) catalytic pathway and oxidative stress pathway.

Claims

1. Overexpression GmCIPK6 The application of genes is characterized by, The application is any one of the following: A1) Application in improving the salt tolerance of soybeans; A2) Application in the preparation of salt-tolerant soybeans; GmCIPK6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application involves increasing the salt tolerance of soybeans by enhancing the activity of catalase, peroxidase, and ascorbate peroxidase in soybeans.

3. The application according to claim 2, characterized in that, The GmCIPK6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. Overexpression GmCIPK6 The application of gene-related biomaterials is characterized by, The application is any of the following: B1) Application in improving the salt tolerance of soybeans; B2) Application in the preparation of salt-tolerant soybeans; The biomaterial is any one of the following C1) to C3): C1) Expression cassettes containing nucleic acid molecules with nucleotide sequences as shown in SEO ID NO. 1; C2) Recombinant vectors containing nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 1; C3) A recombinant microorganism containing a nucleic acid molecule with a nucleotide sequence as shown in SEO ID NO. 1, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium; GmCIPK6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The application involves increasing the salt tolerance of soybeans by enhancing the activity of catalase, peroxidase, and ascorbate peroxidase in soybeans.

6. The application according to claim 5, characterized in that, The GmCIPK6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

7. A method for cultivating salt-tolerant soybeans, characterized in that, The method involves overexpression in soybeans GmCIPK6 Genes were used to obtain soybeans with improved salt tolerance. GmCIPK6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, The overexpression in soybeans GmCIPK6 Genes are produced by using genetic engineering technology to make soybeans contain... GmCIPK6 Gene expression levels increase.

9. The method according to claim 8, characterized in that, The use of genetic engineering technology to make soybeans... GmCIPK6 The increased gene expression was achieved by introducing an expression vector into soybeans that integrates the nucleic acid molecule shown in SEQ ID NO. 1.

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