Identification and application of sorghum drought-enduring gene SbMYC2

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an effect of a sorghum bHLH transcription factor SbMYC2 in the drought stress tolerance process of plants. The gene is highly conservative in a plurality of species and has a typical bHLH structure, and a transcription self-activation experiment shows that SbMYC2 has transcription activation activity. In the application, the inventor constructs a SbMYC2 overexpressed sorghum plant, and finds that the gene can enhance the tolerance of the transgenic plant to drought stress. Therefore, as the gene participating in drought tolerance of sorghum, the SbMYC2 is expected to provide an important theoretical basis for genetic engineering breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and discloses a sorghum bHLH family gene SbMYC2 and an application thereof. Background Art

[0002] Drought is the main cause of crop yield reduction among global abiotic factors, and it seriously threatens global food security. The damage of drought stress to crops mainly includes reduced chlorophyll content, decreased photosynthetic efficiency, damaged membrane system, and reduced activity of multiple enzymes in plants. Sorghum is an important economic crop, which is widely planted in arid and semi-arid areas due to its drought tolerance. However, the germination and filling stages of sorghum are extremely susceptible to drought stress. Long-term drought can reduce the germination rate of sorghum, resulting in a sharp decline in grain yield and quality. Therefore, the identification and discovery of sorghum drought-tolerant genes will help increase the yield of sorghum and other grass crops.

[0003] The effects of drought stress on plants can be divided into two categories: osmotic stress and oxidative stress. Osmotic stress signals can induce the accumulation of abscisic acid (ABA), leading to an adaptive response in plants. Oxidative stress is caused by the accumulation of excessive reactive oxygen species (ROS) in cells, which damages membrane systems, DNA, and proteins. ROS can also act as a second messenger to regulate the expression of stress response genes. In order to remove excess ROS, plants have evolved ROS scavenging enzyme systems, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate oxidase (APX), which are important enzyme systems for reducing oxidative damage caused by drought stress. Therefore, the activity of these antioxidant enzymes in plants is also considered to be an important indicator of plant drought tolerance. In addition, malondialdehyde (MDA) is one of the products of cell membrane lipid peroxidation, and its content can also be used to indicate the degree of damage to plant cell membranes.

[0004] Transcription factors are the main regulatory factors involved in various life processes of plants. When plants are subjected to drought stress, they can bind to the promoters of downstream target genes through a variety of transcription factors to regulate the expression of genes related to drought stress response. bHLH transcription factors are one of the largest transcription factor families known in plants. In recent years, MYC2 has been reported to be involved in plant response to abiotic stress in many species, but there are relatively few reports on MYC2's involvement in drought resistance. At present, the MYC2 gene in sorghum has not been identified, and whether it can participate in sorghum's response to drought stress needs further study. Summary of the invention

[0005] Purpose of the invention: The present invention aims to provide a new use of the SbMYC2 gene in plant drought resistance, thereby promoting the cultivation of new drought-resistant sorghum varieties.

[0006] The invention provides a sorghum SbMYC2 transcription factor. The SbMYC2 transcription factor gene is a SbMYC2 gene, a nucleotide sequence of which is shown in Seq ID No: 1, and an amino acid sequence of which is shown in Seq ID No: 2.

[0007] To achieve the above object, the technical solution adopted by the present invention comprises the following steps:

[0008] Step 1: Extraction of total RNA from sorghum

[0009] Three-week-old sorghum seedlings were selected and total RNA of sorghum was extracted using RNA isolator kit (Vazyme).

[0010] Step 2: Cloning of SbMYC2 gene and construction of expression vector

[0011] Take 1 μg of sorghum total RNA from step 1 for reverse transcription, synthesize sorghum cDNA, and amplify it using PCR to obtain the full length of SbMYC2 ORF. Amplify the recombinant fragment of SbMYC2 with pGBKT7 and pGreen-35S: GFP homologous sequences, and obtain pGBKT7-SbMYC2, pGBKT7-SbMYC2-N, pGBKT7-SbMYC2-C and pGreen-35S: GFP-SbMYC2 recombinant vectors through homologous recombination.

[0012] Step 3: Yeast transcriptional activation assay

[0013] The constructed pGBKT7-SbMYC2, pGBKT7-SbMYC2-N, pGBKT7-SbMYC2-C and empty vector pGBKT7 were transformed into yeast competent cells, respectively. The yeast colonies of positive clones were selected, diluted in series, and grown on the defective medium of SD / -Trp and SD / -Trp-His+5mM 3AT to observe the growth of yeast.

[0014] Step 4: Drought phenotyping experiment

[0015] Three-week-old wild-type and SbMYC2 transgenic overexpressing sorghum seedlings were selected and subjected to two weeks of drought treatment. The phenotypes were observed, and sorghum leaves before and after treatment were taken to determine the SOD and POD enzyme activities, as well as the MDA content.

[0016] The present invention finds that the sorghum transcription factor SbMYC2 has transcription activation activity, and confirms that its transcription activation domain is at the N-terminus of the protein.

[0017] The SbMYC2 gene of the present invention is a gene that has not been reported in sorghum, and its role in the drought resistance of sorghum has not been reported. The present invention has found that overexpression of the SbMYC2 gene can significantly enhance the drought resistance of sorghum. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Homologous sequence alignment of MYC2 genes in different species. The amino acid sequences of MYC2 from sorghum (Sorghum bicolor), Arabidopsis thaliana, rice (Oryza sativa), corn (Zea mays), peony (Paeonia suffruticosa) and soybean (Glycine max) were used for homologous sequence alignment; the part marked by the black line is the more conserved bHLH domain.

[0019] Figure 2 Subcellular localization of SbMYC2 in tobacco leaves. Agrobacterium transformed with pGreen-35S:GFP-SbMYC2 and pGreen-35S:GFP were injected into tobacco leaves, respectively. Fluorescence was observed using a laser confocal microscope. Scale bar: 50 μm.

[0020] Figure 3 Verification of yeast transcriptional activation of SbMYC2. pGBKT7-SbMYC2, pGBKT7-SbMYC2-N, pGBKT7-SbMYC2-C and pGBKT7 were transformed into yeast competent cells, respectively, and the yeast growth was observed after gradient dilution and plating on different defective media. The numbers represent the dilution multiples. pBD represents pGBKT7.

[0021] Figure 4 Identification of SbMYC2 transgenic overexpression lines. (a): Genotype identification of each SbMYC2-OE line (b): Relative expression level of SbMYC2 in each SbMYC2-OE line.

[0022] Figure 5 Phenotypic and physiological measurements of SbMYC2 transgenic overexpressing strains before and after drought treatment. (a) Phenotypic measurements of wild type and SbMYC2-OE strains before and after drought treatment (bd) SOD and POD enzyme activities, and MDA content of wild type and SbMYC2-OE strains before and after drought treatment. Specific implementation method:

[0023] The following examples are convenient for better understanding of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The primers involved in the embodiments of the present invention are synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0024] Example 1

[0025] The sorghum SbMYC2 gene acquisition process of this embodiment is as follows:

[0026] 1) Extraction of total RNA from sorghum

[0027] 0.1 g of two-week-old sorghum leaves were weighed and total RNA was extracted using an RNA isolator kit (Vazyme), and the integrity and concentration of RNA were confirmed by UV spectrophotometer.

[0028] 2) Reverse transcription of RNA

[0029] 1 μg of the total RNA extracted in (1) was taken and reverse transcribed using a reverse transcription kit (Vazyme) to synthesize the first-strand cDNA.

[0030] Primers with homology arms of pGBKT7-SbMYC2, pGBKT7-SbMYC2-N and pGBKT7-SbMYC2-C were designed respectively, and PCR amplification experiments were performed using cDNA reverse transcribed from RNA as template, where:

[0031] The primers for constructing the pGBKT7-SbMYC2 vector are as follows:

[0032] F: 5'-GAGGACCTGCATATGATGAACCTGTGGACGGACGA-3'

[0033] R: 5'-CAGGTCGACGGATCCCTTACCTGCCCATGGCAGACC-3'

[0034] The primers for constructing the pGBKT7-SbMYC2-N vector are as follows:

[0035] F: 5'-GCATATGGCCATGGAGGCCATGAACCTGTGGACGGACG-3'

[0036] R: 5'-CAGGTCGACGGATCCCCGGGTCAGGGCGGCCACGAG-3'

[0037] The primers for constructing the pGBKT7-SbMYC2-C vector are as follows:

[0038] F: 5'-GCATATGGCCATGGAGGCCCCTCAGGCGCCGTCG-3'

[0039] R: 5'-CAGGTCGACGGATCCCCGGGTTACCTGCCCATGGCAGACC-3'

[0040] Primers with pGreen-35S: GFP homology arms were designed and PCR amplification experiments were performed using sorghum cDNA as a template. The primers for constructing the pGreen-35S: GFP-SbMYC2 vector are as follows:

[0041] F: 5'-GGAAGCCCAAGTACGGCGTCATGAACCTGTGGACGGACG-3'

[0042] R: 5'-ATCAGCTCCGGCGCCGGGCCTGCCCATGGCAGACC-3'

[0043] 3) Vector construction

[0044] The amplified DNA fragment was recombined with the linearized vector according to the operating instructions of the ClonExpress II One Step Cloning Kit (Vazyme).

[0045] 4) Construction of relevant expression vectors

[0046] The amplified DNA fragments were subjected to homologous recombination according to the operating instructions of the ClonExpress II One Step Cloning Kit (Vazyme).

[0047] 5) Transformation of vector into E. coli

[0048] The recombinant product was transformed into competent E. coli (WEIDI), spread on plates, and cultured in a 37°C incubator overnight.

[0049] 6) Conduct colony PRC identification

[0050] PCR identification and vector sequencing were performed to obtain the recombinant vectors pGBKT7-SbMYC2, pGBKT7-SbMYC2-N, pGBKT7-SbMYC2-C and pGreen-35S:GFP-SbMYC2.

[0051] Example 2

[0052] The sorghum SbMYC2 protein function analysis process of this example is as follows:

[0053] 1) Comparison of sorghum SbMYC2 homologous sequences with MYC2 in other species

[0054] The CDS of SbMYC2 is 2124 bp long and the protein sequence is 707 amino acids long. The amino acid sequences of MYC2 from sorghum (Sorghumbicolor), Arabidopsis thaliana, rice (Oryza sativa), corn (Zea mays), peony (Paeonia suffruticosa) and soybean (Glycine max) were compared. The results showed that sorghum SbMYC2 has a high similarity with MYC2 from other species and belongs to the bHLH transcription factor family ( Figure 1 ).

[0055] 2) Subcellular localization of sorghum SbMYC2 protein

[0056] The inventor transformed pGreen-35S:GFP and the constructed pGreen-35S:GFP-SbMYC2 plasmids into Agrobacterium GV3101-pSOUP competent cells (WEIDI) according to the product instructions. The transformed Agrobacterium was spread on a plate of LB+Rif+Gen+Kan (triple antibody) and cultured at 28°C for two days. 2mL of LB liquid culture medium with triple antibodies was added to a 10mL bacterial tube, and the monoclonal colonies on the plate were selected in the tube and cultured overnight at 28°C. The next day, 10mL of LB liquid culture medium with triple antibodies was added to a 50mL bacterial tube, and the overnight cultured bacterial solution was transferred to a 50mL tube at a ratio of 1 / 100, and cultured overnight at 28°C. On the third day, first centrifuge at 5000rpm, collect the bacteria, and remove the supernatant. The collected Agrobacterium was resuspended with 10mM MgCl2 and the OD was adjusted to 0.6. Add acetosyringone to 200 μM and let the resuspension stand in the dark for 3 hours. Then, inject it into the tobacco leaves from the back through a syringe. The tobacco leaves were cultured in the dark for 1 day, and then transferred to the light for one day. Finally, for the fluorescence observation stage, the infected tobacco leaves were first cut off, immersed in DAPI solution, stained for 30 minutes, and then the fluorescence of the tobacco epidermal cells was observed under laser confocal microscope and photographed. The experimental results showed that SbMYC2 is a protein localized in the cell nucleus ( Figure 2 ).

[0057] 3) Yeast transcriptional activation experiment of sorghum SbMYC2 protein

[0058] pGBKT7 and the constructed pGBKT7-SbMYC2, pGBKT7-SbMYC2-N and pGBKT7-SbMYC2-C were transformed into yeast (AH109) competent cells respectively, according to the traditional yeast transformation method. The transformed yeast was spread on the SD / -Trp plate and cultured in a 30°C incubator for 3 days. The grown yeast monoclonal colonies were dissolved in sterile water and diluted in a ten-fold gradient. 10 μL of the mixed yeast dilution was respectively aspirated and spotted on the SD / -Trp and SD / -Trp-His+5mM 3AT yeast defective medium plates. The plates were cultured in a 30°C incubator for 3 days, and the growth of the yeast was observed. The results showed that SbMYC2 has a transcriptional activation domain, which is located at the N-terminus of the protein ( Figure 3 ).

[0059] Example 3

[0060] The functional verification of SbMYC2 is as follows:

[0061] 1) Obtaining SbMYC2 transgenic overexpression lines in sorghum

[0062] The successfully constructed pGreen-35S:GFP-SbMYC vector was transformed into sorghum Tx430 line using Agrobacterium AGL1-pSOUP-mediated genetic transformation method.

[0063] 2) Identification of SbMYC2 transgenic overexpressing lines in sorghum

[0064] Genomic DNA and total RNA were extracted from T0 transgenic sorghum. Genomic DNA was extracted according to the conventional CTAB method, and total RNA was extracted as described previously.

[0065] The primers for identification of transgenic sorghum genomic DNA are:

[0066] F: 5'-GTCCCTACAAATGCCATCATTG-3'

[0067] R: 5'-GTCGCAGCCCTTGTAGTAG-3'

[0068] Total RNA was extracted and the primer sequences for identifying the expression of SbMYC2 in transgenic lines by RT-qPCR were:

[0069] SbEIF4A primer (as internal reference primer)

[0070] F: 5'-CAACTTTGTCACCCGCGATGA-3'

[0071] R: 5'-TCCAGAAACCTTAGCAGCCCA-3'

[0072] SbMYC2-RT primer

[0073] F: 5'-GCCATCTTTTGGCAGTCCTC-3'

[0074] R: 5'-CGGAGATGAGGGAATTGAGC-3'

[0075] The identification results showed that the three strains (OE-9, OE-8 and OE-18) with SbMYC2 transgenic overexpression were all positive strains, and SbMYC2 was significantly upregulated in the three transgenic strains ( Figure 4 ).

[0076] 3) Drought phenotypes of sorghum SbMYC2 transgenic overexpressing lines

[0077] Three-week-old wild-type and SbMYC2 overexpression seedlings were selected for a two-week drought stress treatment experiment, and the phenotypes of each strain before and after the treatment were photographed.

[0078] 4) Detection of SOD and POD enzyme activities and MDA content in sorghum SbMYC2 transgenic overexpression lines before and after drought treatment

[0079] For the determination of the enzyme activities of SOD and POD and the content of MDA in each sorghum strain, it is first necessary to sample the leaves of sorghum before and after drought treatment. Then the relevant determination methods are carried out according to the instructions of the relevant product kits of Biyuntian. The results showed that the overexpression of SbMYC2 can significantly enhance the drought tolerance of sorghum, and the enzyme activities of SOD and POD in the transgenic overexpression lines are significantly increased, and the MDA content is significantly reduced. ( Figure 5 ).

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene SbMYC2 regulating drought tolerance in sorghum, characterized by The nucleotide sequence is shown in Seq ID No.

1.

2. A protein encoded by the SbMYC2 gene according to claim 1, the amino acid sequence of which is shown in Seq ID No. 2 in the sequence listing.

3. The protein SbMYC2 according to claim 2 is a factor with activation activity, characterized in that: The amino terminus of this transcription factor has a typical bHLH domain, and yeast transcription activation verification experiments showed that SbMYC2 is a bHLH-type transcription factor with transcription activation activity.

4. The use of the sorghum SbMYC2 protein in regulating sorghum tolerance to drought stress according to claim 2, characterized in that: The SbMYC2 protein sequence is highly similar in multiple higher plants.

5. The use of the sorghum gene SbMYC2 in regulating sorghum tolerance to drought stress according to claim 1, characterized in that: Overexpression of the sorghum gene SbMYC2 in sorghum can enhance the plant's tolerance to drought stress.

6. The yeast transcription activation verification experiment according to claim 3, characterized in that: The specific steps are as follows: Firstly, the full-length CDS sequence, N-terminus and C-terminus of sorghum SbMYC2 gene were cloned into pGBKT7 vector, respectively, to construct recombinant vectors pGBKT7-SbMYC2, pGBKT7-SbMYC2-N and pGBKT7-SbMYC2-C; Subsequently, the pGBKT7-SbMYC2, pGBKT7-SbMYC2-N, and pGBKT7-SbMYC2-C vectors were transformed into yeast AH109 competent cells, respectively; Finally, yeast cells containing pGBKT7-SbMYC2, pGBKT7-SbMYC2-N, and pGBKT7-SbMYC2-C were plated on different defective media.

7. The method for constructing a recombinant overexpression vector according to claim 5, characterized in that: The recombinant vector is specifically constructed by the following method: Using the sorghum SbMYC2 gene sequence shown in Seq ID NO: 1 as a template, amplification primers containing vector homology arm fragments were designed, the amplified product was recovered, and then connected to pGreen-35S: GFP by homologous recombination to obtain the recombinant overexpression vector pGreen-35S: GFP-SbMYC2.

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