Use of gmflz protein or its encoding gene in plant drought tolerance

Abstract

This invention discloses the application of GmFLZ protein or its encoding gene in plant drought resistance, particularly in soybean. The soybean GmFLZ protein or its encoding gene provided by this invention possesses soybean drought resistance function. This invention utilizes Agrobacterium-mediated transformation to... GmFLZ Genes were introduced into soybean roots, resulting in... GmFLZ Overexpression of the soybean complex significantly improved the drought resistance of soybeans.

Description

Technical Field

[0001] This invention belongs to the field of agricultural science and technology, specifically relating to the application of GmFLZ protein or its encoding gene in plant drought resistance. Background Technology

[0002] FLZ belongs to the FCS-like zinc finger protein family. Characterized by a C2C2 type zinc finger domain, it is a class of plant-specific regulatory proteins with a conserved FLZ domain (also known as DUF581), playing a crucial role in plant growth, development, and stress responses. Members of the FLZ gene family have been shown to be essential for various biological processes, including seed dormancy, plant development, senescence, and stress responses. The FLZ domain consists of 70 amino acid residues and possesses a conserved CX2CX17-19FCSX2C motif. Studies have shown that FLZ proteins participate in plant stress responses through multiple mechanisms. As a scaffold protein of the SnRK1 kinase complex, they interact with kinase subunits to help plants sense changes in the external environment and cope with stress conditions by regulating metabolic pathways (promoting catabolism and inhibiting anabolism). Secondly, FLZ also responds to various abiotic stresses. For example, the loss of AtFLZ6 and AtFLZ10 in Arabidopsis thaliana leads to changes in plant sensitivity to ABA and tolerance to osmotic stress, while heterologous expression of the wheat FLZ gene significantly enhances plant resistance to salt stress. However, the role of the FLZ protein family in drought stress remains unknown; therefore, further research is needed to identify which specific FLZ proteins possess the function of drought tolerance in plants. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of GmFLZ protein in plant drought resistance.

[0004] Another technical problem that this invention also aims to solve is to provide a protein encoding GmFLZ. GmFLZ Application of genes in plant drought resistance.

[0005] Another technical problem that this invention can solve is to provide a product containing... GmFLZ Application of gene expression cassettes in plant drought tolerance.

[0006] Another technical problem that this invention can solve is to provide a product containing... GmFLZ Application of gene recombination vectors in plant drought resistance.

[0007] Another technical problem that this invention can solve is to provide a product containing... GmFLZ Application of gene recombination cells in plant drought resistance.

[0008] The final technical problem to be solved by this invention is to provide a product containing... GmFLZ Application of recombinant bacteria in plant drought resistance.

[0009] Technical solution: In order to solve the above technical problems, the present invention provides the application of GmFLZ protein in plant drought resistance, wherein the amino acid sequence of the GmFLZ protein is shown in SEQ ID NO.2.

[0010] This invention also includes encoding the GmFLZ protein. GmFLZ The application of genes in plant drought resistance, the aforementioned GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] The present invention also includes containing GmFLZ The application of gene expression cassettes in plant drought tolerance, the aforementioned GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] The present invention also includes containing GmFLZ The application of recombinant gene vectors in plant drought resistance, the aforementioned GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0013] The recombinant vector is obtained by introducing the GmFLZ gene into the pGD-C-EGFP vector.

[0014] The present invention also includes containing GmFLZ The application of recombinant cells in plant drought resistance, the aforementioned GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] The present invention also includes containing GmFLZ The application of recombinant bacteria in plant drought resistance, the aforementioned GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] The recombinant bacteria are first prepared by... GmFLZ A recombinant vector is obtained by introducing a gene into a gene-introducing vector, and then the recombinant vector is introduced into a host bacterium.

[0017] The plant mentioned includes, but is not limited to, soybeans.

[0018] Beneficial effects: Compared with the prior art, the outstanding effect of this invention is that the GmFLZ protein or its encoding gene provided by this invention has drought resistance function in plants. This invention uses Agrobacterium-mediated transformation to... GmFLZGenes were introduced into soybean roots, resulting in soybean complex plants overexpressing the GmFLZ gene, which significantly improved the drought resistance of soybeans. Attached Figure Description

[0019] Figure 1 The relative expression level of GmFLZ in transgenic hairy roots;

[0020] Figure 2 Phenotypes of the complex before and after 12 hours of drought stress with 10% PEG6000; GFP control: hairy root complex with empty pGD-GFP transgenic vector; GmFLZ: overexpression GmFLZ Transgenic hairy root complex;

[0021] Figure 3 To simulate drought stress, after one day of rehydration treatment, overexpression was observed. GmFLZ Phenotype of transgenic hairy root complex; GFP control: empty pGD-C-EGFP transgenic hairy root complex; GmFLZ: overexpression GmFLZ Transgenic hairy root complex;

[0022] Figure 4 To simulate the phenotype of the GmFLZ-overexpressing transgenic hairy root complex after 2 days of rehydration following drought stress; GFP control: empty pGD-C-EGFP transgenic hairy root complex; GmFLZ: overexpressing GmFLZ Transgenic hairy root complex;

[0023] Figure 5 Phenotypic comparison after 2 weeks of rehydration treatment following drought stress with 10% PEG6000 + 90% Hoagland's nutrient solution; GFP control: empty pGD-C-EGFP transgenic hairy root complex; GmFLZ-OE-K599: GmFLZ overexpressing transgenic hairy root complex. Detailed Implementation

[0024] The embodiments of the present invention will be described in detail below with reference to the examples. The following examples are only for illustrating the present invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0025] Example 1 Genetic transformation of soybean hairy roots

[0026] 1. Construction of vectors for genetic transformation experiments of soybean hairy roots:

[0027] RNA was extracted from soybean variety Heinong 44 (from the Heilongjiang Academy of Agricultural Sciences), and cDNA was obtained by reverse transcription. The cDNA was then used as a template for PCR amplification. GmFLZGene fragments were used in a 50 μL PCR reaction, consisting of 25 μL PCR Mix, 0.5 μL cDNA template, 2 μL each of forward and reverse primers, and water to make up the total 50 μL. The reaction conditions were: 98℃ for 10 s; 58℃ for 5 s; 72℃ for 5 s / kb, for 35 cycles. Restriction enzyme sites and homologous arms of XbaI and SmaI were added to the forward and reverse primers, respectively. The forward and reverse primer pairs are: 5'-ACGATAGCCGGATCATCTAGAATGGTGGGTCTTAGTGTAGTCCTG-3 (SEQ ID NO.3) and 5'-ATCGAATTCCTGCAGCCCGGGAAAATAAGCACCACCCCCTCG-3 (SEQ ID NO.4). The amplified fragment is ligated into the pGD-C-EGFP vector (which was double-digested with XbaI and SmaI at 37℃ for 1h) using homologous recombination. This vector was provided by the authors of the article, Xu K, Nagy PD. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus. PLoS Biol. 2016, 14(10):e2000128.) to obtain the GmFLZ-GFP overexpression vector. 1 μg of the above GmFLZ-GFP overexpression vector was mixed with 50 μL of Agrobacterium K599 competent cells, and then incubated in ice bath, liquid nitrogen, 37℃ water bath, and ice bath for 5 min in sequence. Immediately afterward, antibiotic-free LB liquid medium was added, and after recovery at 28℃ for 2 hours, the mixture was plated onto Kan+Rif resistant LB medium and grown for 48 hours. Positive Agrobacterium strains were obtained by screening.

[0028] Soybean seedlings that have germinated in vermiculite for 4-5 days are longitudinally cut along the hypocotyl with a blade. At the same time, Agrobacterium K599 carrying GmFLZ-GFP is collected and applied to the wound (successfully transformed Agrobacterium is smeared on a culture medium for culture, and then the bacterial cells are collected and applied to the wound). The inoculated seedlings are gently inserted into vermiculite and placed under a photoperiod of 25℃ and 16:8h for rooting culture. After 3-4 days of infection, the seedlings are pulled out to check for adventitious roots (those that have grown should be removed). Adventitious roots are checked and removed every two days. After the wound swells and callus tissue grows, the seedlings are transferred to water for rooting culture (tap water is used), and the water is changed every other day.

[0029] Two weeks after infection, root samples from chimeric plants were collected, and qRT-PCR was used to detect whether the expression level of GmFLZ-GFP in transgenic hairy roots was increased. The primer pairs used were: 5'-ATCCCAACAAACCCTCCCC-3' (SEQ ID NO.5) and 5'-ACCTGCAATCCACGCTACAA-3' (SEQ ID NO.6). The results showed that compared with plants transformed with the pGD-C-EGFP vector, the expression level of GmFLZ-GFP was increased ( Figure 1 Therefore, it is a positive chimeric plant. GmFLZ -OE-K599.

[0030] Example 2: Identification of drought tolerance in chimeric plants

[0031] Drought was simulated using PEG6000 at concentrations of 10% and 15%, respectively. Plants transformed with the pGD-C-EGFP vector (control group) and... GmFLZ Six plants were prepared using OE-K599. After 12 hours of treatment, all soybean plants treated with 15% PEG6000 died. Under 10% PEG6000 treatment, all leaves in the GFP control group wilted. GmFLZ The -OE-K599 strain exhibits slightly curled leaf edges and less leaf wrinkling, demonstrating stronger drought tolerance. (See results below.) Figure 2 .

[0032] Twelve hours after drought treatment, a rehydration treatment was performed. The specific steps were: placing the plant in water, changing the water every two days, and continuing this treatment for two weeks. One day after treatment, the leaves in the control group were still wilted and chlorotic, while... GmFLZ -OE-K599 strain leaves begin to unfold, plant survival rate 100% (see...) Figure 3 Two days later, the control group showed no signs of improvement, and the plant survival rate was 0. GmFLZ The survival rate of OE-K599 plants remained at 100% (see below). Figure 4 The experiment was repeated three times. These results indicate that overexpression in hairy roots... GmFLZ It can improve the drought resistance of soybean plants.

[0033] Plants transformed with pGD-C-EGFP vector (control group) and plants overexpressing pGD-C-EGFP vector. GmFLZ Chimeric transgenic soybean plants were treated with a 10% PEG6000 + 90% Hoagland's solution to simulate drought stress for 12 hours, then transferred to water with the water changed every two days. The treatment was continued for two weeks, and the results were as follows: Figure 5 overexpression GmFLZThe chimeric transgenic soybean plants had a 100% survival rate, with leaves continuing to grow and the second trifoliate leaf fully unfolded, while the GFP control had a 50% survival rate, with only the stem remaining green.

Claims

1. The application of overexpression of GmFLZ protein in improving plant drought tolerance, characterized in that, The amino acid sequence of the GmFLZ protein is shown in SEQ ID NO.2, and the plant is soybean.

2. Overexpression of the protein encoding GmFLZ GmFLZ The application of genes in improving plant drought tolerance is characterized by, The GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

3. Contains GmFLZ The application of gene expression cassettes in improving plant drought tolerance is characterized by, The GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

4. Contains GmFLZ The application of gene recombinant vectors in improving plant drought tolerance is characterized by, The GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

5. The application according to claim 4, characterized in that, The recombinant vector was obtained by introducing the GmFLZ gene into the pGD-C-EGFP vector, and the plant was soybean.

6. Contains GmFLZ The application of recombinant bacteria in improving plant drought tolerance is characterized by, The GmFLZ The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

7. The application according to claim 6, characterized in that, The recombinant bacteria are first prepared by... GmFLZ A recombinant vector is obtained by introducing a gene into a gene-introducing vector, and then the recombinant vector is introduced into a host bacterium.

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