Use of gmbtb protein or gmbtb gene in plant drought tolerance

Abstract

The application discloses a GmBTB protein or GmBTB Gene in plant drought resistance, the amino acid sequence of the GmBTB protein is shown as SEQ ID NO.2, the nucleotide sequence of the GmBTB Gene is shown as SEQ ID NO.1. The application also discloses a method for obtaining drought-resistant plants and a method for identifying drought-resistant plants. The application introduces the GmBTB Gene into the roots of soybeans by a method of agrobacterium transformation, and obtains GmBTB Soybean complex plants with overexpression, and significantly improves 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 GmBTB protein or GmBTB gene in plant drought resistance. Background Technology

[0002] Drought stress is one of the main abiotic stresses affecting soybean growth and yield. Affected by adverse climate factors such as El Niño, high temperature and drought have occurred frequently in recent years, especially before June to September each year. This has caused significant damage to key periods of soybean growth, such as emergence and flowering, resulting in missing seedlings, broken rows, and severe flower and pod drop, which seriously affects soybean yield and income.

[0003] The plant-specific BTB-TAZ(BT) domain protein subfamily, comprising five members from BT1 to BT5, belongs to the BTB protein family. These members contain an N-terminal BTB domain, a transcription linker zinc finger domain, and a C-terminal calmodulin-binding domain. The BTB protein family responds to abiotic stresses, such as drought, cold, heat, and salt stress, by regulating multiple aspects of plant growth and adaptation. Currently, the BTB protein family has been identified in various plant species and participates in various physiological and developmental processes, such as plant growth and development, hormone signaling, and stress responses. The BTB domain, as a key module in protein-protein interactions, can mediate the formation of protein complexes and regulate target protein functions. For example, the Arabidopsis thaliana BR biosynthesis gene AtDWARF4 significantly enhances the drought tolerance of transgenic Brassica napus through a BTB-mediated protein interaction network. Overexpression of the sweet potato IbBT4 gene in Arabidopsis thaliana enhances its drought resistance. Furthermore, it was found that the IbBT4 gene improves drought resistance by enhancing the BR signaling pathway and proline biosynthesis, and further activating the ROS scavenging system in transgenic Arabidopsis. Current research has demonstrated that overexpression of the BTB gene in soybean increases resistance to Phytophthora sojae, but its effect on drought stress remains unclear. Therefore, further screening and exploration of the BTB gene's function in improving soybean drought resistance are needed. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of GmBTB protein or GmBTB gene in plant drought resistance.

[0005] Another technical problem to be solved by this invention is to provide the application of expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria containing the GmBTB gene in plant drought resistance.

[0006] Another technical problem that this invention aims to solve is to provide a method for obtaining drought-resistant plants.

[0007] The final technical problem to be solved by this invention is to provide a method for identifying whether plants obtained by the method have drought resistance.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides the application of GmBTB protein or GmBTB gene in plant drought resistance, wherein the amino acid sequence of GmBTB protein is shown in SEQ ID NO.2, and the nucleotide sequence of GmBTB gene is shown in SEQ ID NO.1.

[0009] The present invention also includes the application of expression cassettes, recombinant vectors or recombinant cells containing the GmBTB gene in plant drought resistance, wherein the nucleotide sequence of the GmBTB gene is shown in SEQ ID NO.1.

[0010] The recombinant vector is obtained by introducing the GmBTB gene into the pGD-C-EGFP vector.

[0011] The present invention also includes the application of recombinant bacteria containing the GmBTB gene in plant drought resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] The recombinant bacteria are obtained by introducing a recombinant vector containing the GmBTB gene into a host bacterium.

[0013] The present invention also includes a method for obtaining drought-resistant plants, the method comprising: upregulating the expression or activity of GmBTB protein, wherein the amino acid sequence of GmBTB protein is shown in SEQ ID NO.2.

[0014] The method for upregulating the expression or activity of the GmBTB protein is to overexpress the GmBTB gene in the genome of a plant, and the nucleotide sequence of the GmBTB gene is shown in SEQ ID NO.1.

[0015] The plant mentioned includes, but is not limited to, soybeans.

[0016] The present invention also includes a method for identifying whether the plants obtained by the method have drought resistance, by identifying whether the plants contain GmBTB protein or GmBTB gene.

[0017] The identification method involves RT-qPCR detection.

[0018] Beneficial effects: Compared with the prior art, the outstanding effect of this invention is that it provides a soybean GmBTB protein and GmBTB gene with plant drought resistance function. This invention introduces the GmBTB gene into soybean roots through Agrobacterium-mediated transformation, obtaining GmBTB-overexpressing soybean complex plants, which significantly improves the drought resistance of soybeans. Attached Figure Description

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

[0020] Figure 2 Phenotype of the complex after 24 hours of drought stress with 10% PEG6000; K599: control pGD-GFP transgenic hairy root complex; GmBTB: GmBTB overexpressing transgenic hairy root complex;

[0021] Figure 3 To simulate the phenotype of the GmBTB transgenic hairy root complex after 1 day of rehydration following drought stress; K599: empty pGD-GFP transgenic hairy root complex; GmBTB: GmBTB transgenic hairy root complex overexpressing. Figure 3 The left and right images in the picture are taken from different angles;

[0022] Figure 4 To simulate the phenotype of the GmBTB transgenic hairy root complex after 2 days of rehydration following drought stress; K599: control empty vector pGD-GFP transgenic hairy root complex; GmBTB: GmBTB transgenic hairy root complex overexpressing. Figure 4 The left and right images in the picture are taken from different angles;

[0023] Figure 5 Phenotypic comparison after 2 weeks of rehydration treatment following drought stress with 10% PEG6000 + 90% Hoagland nutrient solution; K599: control empty vector pGD-GFP transgenic hairy root complex; GmBTB-OE-K599: overexpression of GmBTB 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 (a gift from the Heilongjiang Academy of Agricultural Sciences), and cDNA was obtained by reverse transcription. Using the cDNA as a template, the GmBTB gene fragment was amplified by primer PCR. The PCR reaction volume was 50 μL, including 25 μL PCR Mix, 0.5 μL cDNA template, 2 μL each of forward and reverse primers, and water to make up to 50 μL. The reaction conditions were: 98℃ for 10 s; 58℃ for 5 s, 72℃ for 5 s / kb, for 35 cycles. XbaI and [other active ingredients] were added to the forward and reverse primers, respectively.

[0028] The restriction enzyme sites and homologous arms of SmaI are specified. The forward and reverse primer pairs are: 5'-ACGATAGCCGGATCATCTAGAATGAGTATGGAGGAACCTCAGTTCC-3' and 5'-ATCGAATTCCTGCAGCCCGGGAACAAGCTGCACTACAGCAATG-3'. 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 GmBTB-GFP overexpression vector. 1 μg of the above GmBTB-GFP overexpression vector was mixed with 50 μL of competent Agrobacterium K599 strain (laboratory-preserved), and the mixture was 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.

[0029] Soybean seedlings that have germinated in vermiculite for 4-5 days are longitudinally cut along the hypocotyl with a blade, and the seeds carrying the seedlings are collected.

[0030] Apply GmBTB-GFP K599 Agrobacterium to the wound (successfully transformed Agrobacterium is smeared on a culture medium for cultivation, and then the bacterial cells are collected and applied to the wound); gently insert the inoculated seedlings into vermiculite and place them at 25℃ with a photoperiod of 16:8h for rooting culture; after 3-4 days of infection, pull them out to check for adventitious roots (remove any that grow), and check and remove adventitious roots every two days; after the wound swells and callus tissue grows, transfer them to tap water for rooting culture, changing the water every other day.

[0031] Two weeks after infection, root samples from chimeric plants were collected, and qRT-PCR was used to detect whether the expression level of GmBTB-GFP in transgenic hairy roots was increased. The primer pairs used were 5'-ATGCTTCATGGGCCTTGGAA-3' and 5'-GCAGCAGCAAGAACACGAAA-3'. The results showed that compared with plants transformed with the pGD-C-EGFP vector, the expression level of GmBTB-GFP was increased ( Figure 1 Therefore, it is a positive chimeric plant, GmBTB-OE-K599. Example 2: Identification of drought tolerance in chimeric plants.

[0032] Drought was simulated using PEG6000 at concentrations of 10% and 15%, respectively. Six plants each were transformed with the pGD-C-EGFP vector (control group) and GmBTB-OE-K599. After 12 hours of treatment, all soybean plants treated with 15% PEG6000 died. Under 10% PEG6000 treatment, all leaves in the control group wilted; the GmBTB-OE-K599 line showed slightly curled leaf edges and less leaf wrinkling, exhibiting stronger drought resistance. (See attached results). Figure 2 .

[0033] Twenty-four hours after drought treatment, rehydration treatment was carried out. 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 of the control group were still wilted and chlorotic, while the leaves of the GmBTB-OE-K599 strain began to unfold, and the plant survival rate was 100% (see...). Figure 3 Two days later, the control group showed no signs of improvement, and the plant survival rate was 0 (see [reference]). Figure 4 The experiment was repeated three times. These results indicate that overexpression of GmBTB in hairy roots can improve the drought resistance of soybean plants.

[0034] Plants transformed with the pGD-C-EGFP vector (control group) and chimeric transgenic soybean plants overexpressing GmBTB were treated with 10% PEG6000 + 90% Hoagland nutrient solution to simulate drought stress for 12 hours, followed by rehydration for 2 weeks. Results were observed as follows: Figure 5 The chimeric transgenic soybean plants overexpressing GmBTB had a 100% survival rate, with leaves continuing to grow and the second trifoliate leaf fully unfolded. In contrast, the control group had a 67% survival rate, with only the stems remaining green.

Claims

1. GmBTB protein or GmBTB The application of genes in plant drought resistance is characterized by, The amino acid sequence of the GmBTB protein is shown in SEQ ID NO.

2. GmBTB The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

2. Contains GmBTB The application of gene expression cassettes, recombinant vectors, or recombinant cells in plant drought resistance is characterized by, The GmBTB The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

3. Contains GmBTB The application of recombinant bacteria in plant drought resistance is characterized by, The GmBTB The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is soybean.

4. The application according to claim 3, characterized in that, The recombinant bacteria are those containing... GmBTB The gene was obtained by introducing a recombinant vector into a host bacterium.

5. A method for obtaining drought-resistant plants, characterized in that, The method includes: upregulating the expression or activity of GmBTB protein, wherein the amino acid sequence of GmBTB protein is shown in SEQ ID NO.2, and the plant is soybean.

6. The method according to claim 5, characterized in that, The method for upregulating the expression or activity of the GmBTB protein is to overexpress it in the plant genome. GmBTB Genes, the ones mentioned GmBTB The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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