Soybean drought-resistant transcription factor GmTGA15 and application thereof

Abstract

The invention discloses a soybean drought-resistant transcription factor gene GmTGA15 and an application of the soybean drought-resistant transcription factor gene GmTGA15. The GmTGA15 gene (Glyma. 11G236300.4) is obtained by cloning, and the encoded protein of the GmTGA15 gene is positioned in a cell nucleus and has transcriptional activation activity. A plant expression vector pCAMBIA3301-GmTGA15-GFP is constructed, after soybean hairy roots are transformed, through 20% PEG6000 drought stress treatment, the proline content of transgenic plants is increased by 14% (Plt; 0.05), and the chlorophyll retention rate is increased by 23% (Plt; 0.01), the malondialdehyde content is reduced by 22% (Plt; the invention provides efficient gene resources for stress resistance of crops, and has both theoretical value and application potential.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a drought-resistance-related transcription factor gene GmTGA15 cloned from soybean Williams 82 (the gene improves soybean drought resistance), and performs functional verification on the gene to prove its application in soybean drought resistance. Background Art

[0002] As an important oil crop in the world, soybean yield is severely restricted by abiotic stresses such as drought and salinity. Transcription factors play a core role in plant stress resistance regulation, among which TGA (TGACG motif-binding factor) belongs to the bZIP family and regulates downstream gene expression by binding to cis-acting elements. Although studies have shown that TGA genes in crops such as Arabidopsis and rice are involved in stress resistance response, the functional research of related genes in soybean is still insufficient.

[0003] In the prior art, soybean drought resistance gene resources are scarce and molecular breeding methods are limited. The present invention clones the GmTGA15 gene from soybean and reveals that it enhances drought resistance by regulating osmotic protective substances and antioxidant pathways, filling the technical gap in this field.

[0004] Provide a new soybean drought-tolerant transcription factor gene GmTGA15, predict its molecular characteristics and stress resistance function, and develop its application in genetic improvement of plant drought resistance. Summary of the invention

[0005] In view of this, the present invention aims to provide a soybean drought-resistant transcription factor GmTGA15 gene and its application, aiming to improve the adaptability of soybean to drought resistance and reduce the economic losses caused by drought to soybean.

[0006] The inventors have continuously innovated and reformed through long-term exploration and attempts, as well as multiple experiments and efforts, to solve the above technical problems. The technical solution provided by the present invention is to provide a soybean drought-resistant transcription factor GmTGA15 gene, which contains a 1089bp coding sequence and encodes 362 amino acids. The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0007] The present invention deeply understands the role of soybean drought-resistant transcription factor GmTGA15 gene from multiple aspects through gene cloning and sequence analysis, recombinant vector construction, drought-resistant function verification and application of transgenic soybean hairy roots.

[0008] 1. Gene cloning and sequence analysis

[0009] Cloning method: Soybean variety Williams 82 seedlings were used as materials, total RNA was extracted by Trizol method, and cDNA was obtained by reverse transcription. Specific primers were designed and PrimeSTARMAX polymerase was used to amplify the GmTGA15 sequence.

[0010] Sequence characteristics: The GmTGA15 genome sequence is 5872bp long, containing 8 exons and 7 introns; the open reading frame (ORF) is 1089bp, encoding 362 amino acids.

[0011] Protein characteristics: ProtParam analysis showed that the GmTGA15 protein had a molecular weight of 41.01 kDa and a theoretical isoelectric point of 6.28. It was a hydrophilic protein without a transmembrane region and a signal peptide. SMART predicted that it contained a typical bZIP domain.

[0012] 2. Construction of recombinant vector

[0013] Vector design: The GmTGA15 gene was inserted into the BamHI site of the pCAMBIA3301-GFP vector to construct the plant expression vector pCAMBIA3301-GmTGA15-GFP.

[0014] Agrobacterium transformation: The recombinant vector was introduced into Agrobacterium K599, and positive clones were screened and verified by PCR screening of bacterial liquid.

[0015] 3. Verification and application of drought resistance function of transgenic soybean hairy roots

[0016] Preparation of transgenic hairy roots: The soybean cotyledonary nodes were infected by Agrobacterium-mediated method to obtain the transgenic GmTGA15 hairy root line.

[0017] Drought resistance analysis: after 20% PEG6000 drought stress treatment, the proline content of the transgenic plants increased by 14%, the chlorophyll retention rate increased by 23%, and the malondialdehyde content decreased by 22%. The present invention provides a highly efficient gene resource for crop stress resistance, which has both theoretical value and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0019] Figure 1 : Electrophoresis of GmTGA15 gene cloning (M: DL2000 Marker; 1-2: PCR amplification product, about 1089 bp).

[0020] Figure 2: Schematic diagram of the construction of pCAMBIA3301-GmTGA15-GFP vector (marking the BamHI restriction site and GFP tag).

[0021] Figure 3 : PCR of bacterial solution to verify Agrobacterium transformation (M: DL2000 Marker; 1-4: positive clone amplification bands).

[0022] Figure 4 : Comparison of physiological parameters under 20% PEG6000 treatment (P<0.05, P<0.01). DETAILED DESCRIPTION

[0023] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0024] Nucleotide sequence of GmTGA15 gene (SEQ ID NO.1)

[0025]

[0026] Amino acid sequence of GmTGA15 gene (SEQ ID NO.2)

[0027] MNSASPQFVSPRSMSVYDPIHQISMWGEGFKSNGNLSAAMPLIDEADMKFDSQSEDASHGILGEPNKYDQEASKPTDKIQRRLAQNREAARKSRLRKKAYVQQLESSRLKLMQLEQELERARQQGMYIGGGLDSNHLGFAGSVNSGITTFEMEYGHWVNEQNRQITELRNALNAHIGDVEL RILVDGMMSHYAEMFRMKSAAAKADVFYVMSGMWKTTAERFFLWIGGFHPSELLKVLGPLIEPLTEQQRLNIYNLGQSCQAEDALSQGMDKLRQTLADSVAAGQFMEGTYIPQMTSAMEKLEDLVSFVKQADHLRQETLEQMSRILTIRQAARCLLALGEYFQRLRALSSLWSNRPREPA*

[0028] Example 1: Cloning of GmTGA15 gene

[0029] 1. Material Preparation

[0030] Plant material: Soybean variety Williams 82, grown in an artificial climate chamber (16 h light / 8 h dark, temperature 25°C).

[0031] Reagents: RNAiso Plus (TaKaRa), PrimeSTAR MAX DNA polymerase (TaKaRa), pMD18-T cloning vector (TaKaRa).

[0032] 2. RNA Extraction and Reverse Transcription

[0033] RNA extraction: 100 mg of soybean seedling root tissue was taken, ground with liquid nitrogen, and 1 mL of RNAiso Plus was added to extract total RNA according to the instructions. Removal of DNA contamination: RNA was treated with DNase I (TaKaRa) for 30 minutes, and the concentration was measured after purification (NanoDrop2000).

[0034] Reverse transcription: All-in-One First-Strand cDNA Synthesis Kit (GeneCopoeia) was used to synthesize cDNA from 1 μg RNA and stored at -20°C for future use.

[0035] 3. PCR amplification of GmTGA15 gene

[0036] Primer design:

[0037] Forward primer GmTGA15-F: 5′-ATGAATTCAGCATCCCCGCA-3′ Reverse primer GmTGA15-R: 5′-CTAAGCAGGTTCCCGGGGTCTAT-3′

[0038] Reaction system (50 μL):

[0039] PrimeSTARMAXPremix 25μL Forward primer (10 μM) 1μL Reverse primer (10 μM) 1μL cDNA template 1μL <![CDATA[ddH 2 The]]> 22μL

[0040] PCR procedure:

[0041] 98℃ pre-denaturation 2 minutes 98℃ denaturation 10 seconds Annealing at 53.6℃ 15 seconds → 35 cycles 72℃ extension 70 seconds Final extension at 72°C 5 minutes

[0042] Electrophoresis verification: Take 5 μL of PCR product and run 1% agarose gel electrophoresis (120V, 20 minutes), and the 1089 bp target band can be seen ( Figure 1 ).

[0043] 4. Cloning and Sequencing

[0044] Gel recovery: After gel excision, use the AxyPrep DNA Gel Recovery Kit to purify the target fragment.

[0045] Ligation and transformation: The fragment was ligated to the pMD18-T vector, transformed into E. coli DH5α, and plated on LB plates containing Amp (100 μg / mL).

[0046] Positive clone screening: Pick a single colony for bacterial solution PCR verification and send it to a sequencing company to verify the sequence consistency (consistent with the GenBank accession number).

[0047] Example 2: Construction of plant expression vector

[0048] 1. Vector and enzyme digestion

[0049] Vector selection: Use the plant expression vector pCAMBIA3301 (containing GFP tag and bar resistance gene).

[0050] Enzyme digestion reaction (20 μL system): react at 37°C for 3 hours, and recover the linearized vector by gel.

[0051] 10×KBuffer 2μL pCAMBIA3301 plasmid 1 μg B H 1μL HindIII 1μL <![CDATA[ddH 2 The]]> Up to 20 μL

[0052] 2. Connect the target gene to the vector

[0053] (1) Thaw Hi-Fusion Cloning Mix (2×), target fragment and linearized pPSN vector on ice;

[0054] (2) The ligation reaction system is shown in the following table:

[0055] Reaction liquid composition Volume (5μL system) Hi-Fusion Cloning Mix (2×) 2.5μL Purpose fragment 1μL Linearized pPSN-GFP vector 1.5μL

[0056] Reaction conditions: 50℃ for 30 minutes, ice bath for 5 minutes and then transform into E. coli DH5α. Figure 2 )

[0057] 3. Agrobacterium Transformation

[0058] (1) Take 200 μL of competent cells, add 0.5 μg of plasmid, and mix gently;

[0059] (2) Freeze in liquid nitrogen for 50 seconds, place on ice for 40 minutes, and heat shock at 42°C for 90 seconds;

[0060] (3) Recovery culture at 30°C for 4 h;

[0061] (4) Spread an appropriate amount of cells on the screening medium LB plate and blow dry the surface liquid in a clean bench;

[0062] (5) Place the plate in an incubator at 30°C for 2 days;

[0063] (6) Pick out a round, plump, white single colony and propagate it in LB liquid medium (containing Str resistance), place it in a shaker at 37°C and 200 rpm for 12-16 h;

[0064] (7) Identification and sequencing of positive clones: Take 1 μL of the culture medium as a template and perform PCR verification. The positive clones verified by PCR are sent to Shanghai Sangon Biotechnology Co., Ltd. for positive clone verification: PCR detection of 35S promoter (primers 35S-F / R) and bar gene (primers Bar-F / R). Figure 3 )

[0065] Example 3: Verification of drought resistance of transgenic soybean hairy roots

[0066] 1. Soybean Hairy Root Transformation

[0067] (1) Soybean Williams 82 seeds were planted in a mixed soil (nutrient soil: vermiculite = 3:1) in an artificial climate chamber (16 h light / 8 h dark, temperature 25°C);

[0068] (2) Five-day-old soybean seedlings with unexpanded cotyledons and good growth conditions were used for infection with Agrobacterium K599. The bacteria were collected and infected at the hypocotyl of the seedlings at the cotyledon node;

[0069] (3) After injection, cover with a transparent lid to keep it moist and leave it in the dark overnight;

[0070] (4) Cover the infected area with vermiculite to keep it moist, and pour BD solution every two days to keep the infected area moist;

[0071] (5) After 15 days, water the plants with a BD solution containing potassium nitrate to supplement nitrogen.

[0072] (6) After 25 days, when the hairy roots grow to 5-10 cm, the taproot is removed and the composite plant is buried in mixed soil and watered with BD solution containing potassium nitrate every three days;

[0073] (7) After 40 days, when the hairy roots have recovered sufficiently, they are treated with PEG6000 for one week and then washed for use in transgenic hairy root detection and related physiological experiments.

[0074] 2. Screening of transgenic plants

[0075] GFP fluorescence detection: Using a LUYOR portable fluorescent lamp for observation, the transgenic hairy roots exhibit green fluorescence.

[0076] PCR verification: Extract hairy root genomic DNA and amplify 35S promoter and bar gene by PCR.

[0077] 3. Drought stress treatment and physiological index determination

[0078] Treatment conditions: 20% PEG6000 solution was used to simulate drought and the transgenic and wild-type plants were treated for 7 days. Malondialdehyde, proline and chlorophyll in soybean leaves of transgenic and control plants were measured. Figure 4 )

[0079] Each experiment was repeated three times, which reduced the contingency of the single experimental results and made the results more convincing. The above disclosure is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights outside the present application. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present application still fall within the scope covered by the present application.

Claims

1. A soybean GmTGA15 gene, characterized in that: The gene contains a coding sequence of 1089 bp, encoding 362 amino acids, and the nucleotide sequence of the gene contains a coding sequence of 1089 bp as shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The use of the gene according to claim 1, characterized in that: By increasing the expression level of the GmTGA15 gene through transgenic technology, soybean's resistance to drought stress was significantly enhanced. The protein sequence it encodes has a bZIP conserved domain and transcriptional activation activity.

3. A plant expression vector pCAMBIA3301-GmTGA15-GFP according to claim 2, characterized in that: The invention comprises the GmTGA15 gene according to claim 1, which is inserted into the multiple cloning site of the pCAMBIA3301 vector through the BamH I and Hind III restriction sites, and fused with a GFP tag.

4. A method for improving drought resistance of soybean according to claim 3, characterized in that: The method comprises the following steps: constructing the plant expression vector according to claim 2; transforming soybean hairy roots by Agrobacterium K599-mediated method; screening positive transgenic plants and subjecting them to drought stress treatment.

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