Application of GmFLZ protein or coding gene thereof in drought tolerance of plants

Abstract

The invention discloses application of GmFLZ protein or a coding gene thereof in drought tolerance of plants, in particular to application in drought tolerance of soybeans. The soybean GmFLZ protein or the coding gene thereof provided by the invention has a soybean drought-enduring function. According to the invention, the GmFLZ gene is introduced into soybean roots through an agrobacterium transformation method, so that a GmFLZ gene overexpressed soybean complex plant is obtained, and the drought tolerance of soybeans is remarkably improved.

Description

Technical Field

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

[0002] FLZ belongs to the FCS-like zinc finger protein family (FLZ), characterized by a C2C2-type zinc finger domain. These proteins are plant-specific regulatory proteins with a conserved FLZ domain (also known as DUF581) and play key roles 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. Research has shown that FLZ proteins participate in plant stress responses through multiple mechanisms. As scaffold proteins of the SnRK1 kinase complex, they interact with kinase subunits to help plants sense environmental changes and respond to stress conditions by regulating metabolic pathways (promoting catabolism and inhibiting anabolism). FLZ proteins also respond to various abiotic stresses. For example, the loss of AtFLZ6 and AtFLZ10 in Arabidopsis alters plant sensitivity to ABA and osmotic stress tolerance, while heterologous expression of wheat FLZ genes significantly enhances plant resistance to salt stress. However, the role of the FLZ protein family in drought stress is unknown, and further research is needed to identify specific FLZ proteins that contribute to plant drought tolerance. Summary of the Invention

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

[0004] The technical problem that the present invention also solves is to provide a protein encoding GmFLZ GZF Application of genes in plant drought tolerance.

[0005] The technical problem that the present invention also solves is to provide a GZF Application of gene expression cassettes in plant drought tolerance.

[0006] The technical problem that the present invention also solves is to provide a GZF Application of gene recombinant vectors in plant drought tolerance.

[0007] The technical problem that the present invention also solves is to provide a GZF Application of gene-recombinant cells in plant drought tolerance.

[0008] The technical problem to be solved by the present invention is to provide a GZF Application of gene-recombinant bacteria in plant drought tolerance.

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

[0010] The present invention also includes the coding GmFLZ protein GZF Application of genes in plant drought tolerance, the GZF The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] The present invention also includes GZF Application of gene expression cassette in plant drought tolerance, the GZF The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] The present invention also includes GZF Application of recombinant vector of gene in plant drought resistance, said GZF The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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

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

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

[0016] Wherein, the recombinant bacteria is first GZF The gene is introduced into a vector to obtain a recombinant vector, and then the recombinant vector is introduced into a host bacterium to obtain the gene.

[0017] The plants include but are not limited to soybeans.

[0018] Beneficial effects: Compared with the prior art, the outstanding effect of the present invention is that the GmFLZ protein or its encoding gene provided by the present invention has drought resistance function in plants. The present invention transforms the GmFLZ protein or its encoding gene into a plant through the Agrobacterium transformation method. GZFThe gene was introduced into soybean roots, and soybean complex plants with overexpression of the GmFLZ gene were obtained, which significantly improved the drought resistance of soybean. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the relative expression level of GmFLZ in transgenic hairy roots; Figure 2 The phenotype of the complex before and after 12 hours of 10% PEG6000 drought stress; GFP control: empty pGD-GFP transgenic hairy root complex; GmFLZ: overexpression GZF transgenic hairy root complex; Figure 3 To simulate drought stress, rewatering was performed for 1 day and overexpression GZF Phenotype of transgenic hairy root complex; GFP control: empty pGD-C-EGFP transgenic hairy root complex; GmFLZ: overexpression GZF transgenic hairy root complex; Figure 4 To simulate drought stress, rewatering was performed for 2 days to examine the phenotype of the transgenic hairy root complex overexpressing GmFLZ; GFP control: empty pGD-C-EGFP transgenic hairy root complex; GmFLZ: overexpressed GZF transgenic hairy root complex; Figure 5 The figure shows the phenotypic comparison of the hairy root complex after drought stress in 10% PEG6000+90% Hoagland's nutrient solution and rehydration for 2 weeks; GFP control: empty pGD-C-EGFP transgenic hairy root complex; GmFLZ-OE-K599: GmFLZ overexpressing transgenic hairy root complex. DETAILED DESCRIPTION

[0020] The embodiments of the present invention will be described in detail below with reference to the examples. The following examples are intended to illustrate the present invention only. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0021] Example 1 Genetic transformation of soybean hairy roots 1. Vector construction for soybean hairy root genetic transformation experiment: RNA was extracted from soybean Heinong 44 (from Heilongjiang Academy of Agricultural Sciences) and reverse transcribed to obtain cDNA. PCR amplification was performed using the cDNA as a template using primers. GZFFor gene fragments, the PCR reaction system was 50 μL, including 25 μL of PCR Mix, 0.5 μL of cDNA template, 2 μL of forward and reverse primers, and 50 μL of water. Reaction conditions were: 98°C for 10 s, 58°C for 5 s, and 72°C for 5 s / kb, for 35 cycles. The forward and reverse primers were enriched with XbaI and SmaI restriction enzyme sites and homology arms, respectively. The forward and reverse primer pairs are: 5'- ACGATAGCCGGATCATCTAGAATGGTGGGTCTTAGTGTAGTCCTG-3 (SEQID NO.3) and 5'- ATCGAATTCCTGCAGCCCGGGAAAATAAGCACCACCCCCTCG-3 (SEQ ID NO.4). The amplified fragment was ligated into the pGD-C-EGFP vector (the vector was provided by the authors of the article, Xu K, Nagy PD. Enrichment of Phosphatidylethanolamine inViral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPaseby a Positive-Strand RNA Virus. PLoS Biol. 2016, 14(10):e2000128.) by homologous recombination to obtain the GmFLZ-GFP overexpression vector. 1 μg of the above-mentioned GmFLZ-GFP overexpression vector was mixed with 50 μL of Agrobacterium K599 competent strain, and the mixture was placed in an ice bath, liquid nitrogen bath, 37°C water bath, and ice bath for 5 min. Antibiotic-free LB liquid medium was immediately added, and the mixture was restored at 28°C for 2 hours. After that, the mixture was spread on Kan+Rif-resistant LB medium and grown for 48 hours to screen for positive Agrobacterium.

[0022] Soybean seedlings germinated in vermiculite for 4-5 days were cut longitudinally along the hypocotyl with a blade, and K599 Agrobacterium carrying GmFLZ-GFP was collected and smeared on the wound (successfully transformed Agrobacterium was smeared on the culture medium for culture, and then the bacteria were collected and smeared on the wound); the inoculated seedlings were gently inserted into the vermiculite and placed at 25°C and a 16:8h photoperiod for rooting culture; 3-4 days after infection, the seedlings were pulled out to check for adventitious roots (if they grew, they should be cleaned up), and the adventitious roots were checked and cleaned every two days; after the wound swelled and callus tissue grew, the seedlings were transferred to water for rooting culture (tap water), and the water was changed every other day.

[0023] Two weeks after infection, root samples were taken from the chimeras and qRT-PCR was used to detect whether the expression level of GmFLZ-GFP in the transgenic hairy roots was increased. The primer pair was 5'-ATCCACAACAAAACCCTCCCC -3' (SEQ ID NO. 5) and 5'-ACCTGCAATCCACGCTACAA -3' (SEQ ID NO. 6). The results showed that the expression level of GmFLZ-GFP was increased compared with that in the plants transformed with the pGD-C-EGFP vector ( Figure 1 ), therefore, it is a positive chimeric plant GZF -OE-K599.

[0024] Example 2 Evaluation of Drought Tolerance of Chimera Plants Drought was simulated by 10% and 15% PEG6000, respectively. The plants transformed with pGD-C-EGFP vector (control group) and GZF -OE-K599 were treated with 6 plants each. After 12 hours of treatment, all soybean plants treated with 15% PEG6000 died. Under 10% PEG6000 treatment, all leaves of the GFP control group wilted. GZF - The OE-K599 strain has slightly curled leaves at the edges and less wrinkling, showing stronger drought tolerance. Figure 2 .

[0025] After 12 hours of drought treatment, the leaves of the control group were rehydrated. The specific steps were as follows: the leaves were placed in water and the water was changed every two days for two weeks. After 1 day of treatment, the leaves of the control group were still wilting and losing their green color, while the leaves of the GZF -OE-K599 strain leaves began to unfold, and the plant survival rate was 100% (see Figure 3 ), after 2 days, the control group showed no relief trend and the plant survival rate was 0. GZF - The survival rate of OE-K599 plants remained 100% (see Figure 4 The experiment was repeated 3 times. These results showed that overexpression in hairy roots GZF Can improve the drought tolerance of soybean plants.

[0026] Plants transformed with pGD-C-EGFP vector (control group) and overexpression GZF The chimeric transgenic soybean plants were treated with 10% PEG6000 + 90% Hoagland's nutrient solution and simulated drought stress for 12 hours. The plants were then placed in water and the water was changed every two days. The results were observed after 2 weeks of treatment. Figure 5 , overexpression GZFThe chimeric transgenic soybean plants had a 100% survival rate, with leaves continuing to grow and the second triptery leaf fully expanded, while the GFP control had a 50% survival rate, with only the stem remaining green.

Claims

1. Application of GmFLZ protein in plant drought tolerance, characterized in that: The amino acid sequence of the GmFLZ protein is shown in SEQ ID NO.

2.

2. Encoding GmFLZ protein GZF The application of the gene in plant drought tolerance is characterized in that, described GZF The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. Contains GZF The application of the gene expression cassette in plant drought tolerance is characterized in that: described GZF The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. Contains GZF The application of a recombinant vector of a gene in plant drought resistance is characterized in that: described GZF The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that The recombinant vector is obtained by introducing the GmFLZ gene into the pGD-C-EGFP vector.

6. Contains GZF The application of a recombinant cell containing a gene in plant drought resistance is characterized in that: described GZF The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. Contains GZF The application of gene-recombinant bacteria in plant drought resistance is characterized in that: described GZF The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The use according to claim 7, characterized in that The recombinant bacteria are first GZF The gene is introduced into a vector to obtain a recombinant vector, and then the recombinant vector is introduced into a host bacterium to obtain the gene.

9. The use according to claim 1, 2, 3, 4, 6 or 7, characterized in that The plant is soybean.

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