CN110643618A - Jatropha MYB transcription factor JcMYB16 gene and its application in improving plant drought resistance - Google Patents

Abstract

The present invention provides a Jatropha japonica MYB transcription factor JcMYB16 gene, the nucleotide sequence of which is SEQ ID NO.1, and the nucleotide sequence of the gene open reading frame is SEQ ID NO.2. Overexpression of the JcMYB16 gene does not affect the Plant growth and development can significantly improve drought stress resistance. The gene can be used for the cultivation of drought-tolerant varieties of Jatropha japonica and cereal crops such as rice and wheat, as well as for the cultivation of drought-tolerant varieties of crops such as barley, sorghum, corn, Arabidopsis, tomato, tobacco, soybean, and potato.

Description

Jatropha MYB transcription factor JcMYB16 gene and its role in improving plant drought resistance Applications

technical field

The invention belongs to the field of plant biotechnology, and relates to a Jatropha jatropha MYB transcription factor JcMYB16 gene and its application in plants.

Background technique

Plant physiological water shortage caused by drought stress seriously affects plant growth and development and crop yield. Statistics show that my country's arable land area is only 121 million hectares, while the desertification and salinization land has 260 million hectares, and 13 million hectares of abandoned land caused by human factors. Therefore, drought stress has become a bottleneck restricting the growth and development of crops in my country. An important way to solve this problem is to vigorously strengthen the mining of stress tolerance-related genes and their development and utilization in molecular breeding. At present, the research on the model plant Arabidopsis has preliminarily understood the signaling pathways of plants in response to drought stress signals, and identified some genes of drought resistance pathways. Due to the specificity of species evolution and the limitation of related genes in specific species, it is difficult to screen more drought resistance-related genes in a single species. However, due to the diversity of species, many plants have good drought-resistant characteristics, so mining drought-resistant genetic resources from plants with drought-resistant characteristics has important agricultural value for future application in the cultivation of drought-tolerant varieties of crops in my country. application prospects. The introduction of jatropha provides us with an ideal solution, which can not only improve the environment, improve the soil, maintain water and soil, but also generate greater economic benefits as biomass energy. Jatropha curcas L. is a perennial deciduous shrub/small tree of the genus Jatropha of Euphorbiaceae, which has the characteristics of fast reproduction, strong stress tolerance, especially drought tolerance.

MYB transcription factors are one of the many types of transcription factors. They are characterized by a conserved MYB domain consisting of more than 50 amino acids at the N-terminus of their proteins, and contain 1-3 tandem, incompletely repeated MYB structures. Domains (R1, R2 and R3). According to the number of MYB domains, MYB transcription factors can be divided into four categories, namely 1R-MYB, 2R-MYB (R2R3-MYB), 3R-MYB (R1R2R3-MYB) and 4R-MYB (four R1/R2) . MYB transcription factors play an important role in regulating plant growth and development, physiological and biochemical processes and stress resistance. In addition, MYB transcription factors also play a key role in the response of plants to drought stress. For example, the Arabidopsis thaliana AtMYB2 transcription factor and rd22BP1 bind to the promoter region of the drought stress-responsive gene rd22, respectively, and then jointly activate the expression of rd22, thereby achieving drought resistance. More and more studies have shown that MYB protein is involved in plant response to abiotic stress. regulation of the response. In conclusion, although many MYB proteins have been cloned and functionally analyzed, genes of the Jatropha MYB family in response to drought stress are still rarely reported. Therefore, cloning the Jatropha jatropha MYB family involved in the regulation of drought stress and researching its functions will provide a new genetic resource for the cultivation of drought-resistant crop varieties, provide a molecular theoretical basis for the cultivation of drought-resistant plants, and help my country's arid land to be effectively used. Development and utilization, and then promote the sustainable development of my country's national economy and the effective protection of the ecological environment.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Jatropha jatropha MYB transcription factor JcMYB16 gene and its application in plants. The gene can improve the drought resistance of plants and has important value for molecular breeding of plants to cope with adversity stress.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

The nucleotide sequence of a Jatropha jatropha MYB transcription factor JcMYB16 gene is SEQ ID NO.1, and the nucleotide sequence of the gene open reading frame is SEQ ID NO.2.

A polypeptide whose amino acid sequence is encoded by SEQ ID NO.3.

A recombinant construct comprising the nucleotide sequence shown in SEQ ID NO. 1, and the vector used for the construct is pMD18-T vector for cloning or pCAMBIA1301 vector for expression.

The transgenic Agrobacterium of Jatropha MYB transcription factor JcMYB16 gene is EHA105 or GV3101.

Application of the nucleotide sequence of Jatropha jatropha MYB transcription factor JcMYB16 gene in improving plant drought resistance.

The application of the nucleotide sequence of Jatropha jatropha MYB transcription factor JcMYB16 gene in improving the drought resistance of plants, the plants being monocotyledonous or dicotyledonous.

The monocotyledonous plant is rice, and the dicotyledonous plant is Arabidopsis jatropha.

Described SEQ ID NO.1 is:

TTAGTGGTGGTTAAACTTTCAAAATCTCGATAGTGCAAGTATTTGAATTTTTAGGTTATGTTTCTTTTATGGCCTTTCGCACTGTTAGTGGAATTGGAGTGTGAAAATGGAATCTGGGGTTAGAGATTATTTTTCTTCTCTAATCAGGAGTGAGAAACCTTGAATTACTGGCCGTTCTACTCTCAATTAATGGCTTTCTGTTCCCAGGAATTGCCCTTCAAATCTGCTGTTGATAACATAGGTCAGCAGTTCACAAGAGTCCAATTTTTTTGAATTGTGGAGCAAATTACCATTGAATTCTTCACATACTCCGTTGGGATTTTGTGATTTTTATTGGAGGTGATCTCTGGTTCAGTTCTCTTGGCTTTTTTTGGGTCCATCTGCTTGTTTCTCGTCTTCCTTACCTCTACACCCTGACACATCCTGCTGCTTTCCCATAATTCTGTTCTGCCATCACTCCAACTGTTGGTCATAACCAAATATAGAAATTATTCAGATCCAAATTTCAAGAAACCGTTCTAGAATTTTTTTTTTTTTTCAAGATTGGCTTATTAATTTAGCCTAGATTCTAGAGCTAGGTTTTTCCTTTCTTTGCTAGTGTAAGATTCAAACCAGTCTAGATGATTGCGGATGAAGCAGACTGCAGCTCTGTGTGGACTAGGGAGCAGGATAAGGCATTTGAGGATGCCCTTGCAACATATCCTGAGGATGCTGTAGATCGGTGGGAGAAAATTGCTGCTGATGTTCCTGGGAAAACCTTAGAAGAGCTTAAACTTCACTATGAACTTCTGGTTGAAGATTTGAATCAGATTGAAGCTGGCTGTGTGCCTCTGCCTAACTACTCTTCTATGGAGGGTTCAATAAGCCAAGCTGGCGATGAAGGAACTACTAAGAAGGGTGGTCAAATGGGGCACCATAACAGTGAGTCTACTCATGGAAATAAGGCTTCAAGGTCAGATCAAGAACGCCGTAAAGGAATCGCTTGGACAGAGGATGAGCA CAGGTTATTTCTTCTTGGTTTGGACAAATATGGGAAAGGTGACTGGCGAAGTATTTCCAGAAACTTTGTTGTGACAAGGACACCTACGCAAGTGGCAAGCCATGCACAAAAATATTTCATTCGTTTGAACTCGATGAACAAAGATAGGAGGCGTTCCAGCATTCATGATATCACCAGTGTTGGCAATGGAGATATTTCAGCGCCACAAGGACCAATAACTGGTCAAACAAATGGTTCTGCTGCAGGAGGTTCCTCTGGTAAAGCTGCTAAACAACCCCCTCAACACCCTACTGGACCTCCAGGAGTTGGTGTTTATGGTCCTCCGACTATAGGGCAACCTATAGGAGGTCCCCTTGTCTCAGCAGTTGGCACCCCTGTGAATCTTCCTGCCCCTGCACACATGGCTTATGGCGTTAGAGCTCCTGTACCAGGAACAGTACCGGGAGCTGTGGTTCCTGGTGCACCAATGATGAACATGGGTCCTATGGCATATCCAATGCCACCGACAACTGCTCATAGGTGATATACATGGTTTAGCTGCAAAATGTACAAAGACAGAAGGCTACTTGCTTGTATTTCTGGTGGGTCAGTGGCTTCTCCATTTTAGCCTGAATAAAACTGCTTATTTGCAAGCAAAAATTGTCTGATGTCATTTGTTTATTCTGGTAGCAATATCAAATAAACCAATAGGTAGAGAAACTACATGCATTTGTATAGGCAGCAGCTGTGGAAAATATGGCAGCAGTTATGGGTAGGACACATTTTGGTACTTTTTTTTTGGTTTTACATTACAATGTTTAGTCTCAGTAGCAGTCAGTTAATGGTATTTTACTTTTAATGACCAAATTTGTAAAGAATCCATTTATACGTTTTACTATTTTGAGTAGTAGATGTTGGCACGGATTGTGCAAAGCCTTTGTAAAAAAAAGTACCAAAATGTGTCCTACCCATAACTGCTGCCATATTTTCCACAGCTGCTGCCTATACAAATGCATGTAGT TTCTCTACCTATTGGTTTATTTGATATTGCTACCAGAATAAACAAATGACATCAGACAATTTTTTGCTTGCAAATAAGCAGTTTTATTCAGGCTAAAATGGAGAAGCCACTGACCCACCAGAAATACAAGCAAGTAGCCTTCTGTCTTTGTACATTTTGCAGCTAAACCATGTATATCACCTATGAGCAGTTGTCGGTGGCATTGGATATGCCATAGGACCCATGTTCATCAT

Described SEQ ID NO.2 is:

ATGATTGCGGATGAAGCAGACTGCAGCTCTGTGTGGACTAGGGAGCAGGATAAGGCATTTGAGGATGCCCTTGCAACATATCCTGAGGATGCTGTAGATCGGTGGGAGAAAATTGCTGCTGATGTTCCTGGGAAAACCTTAGAAGAGCTTAAACTTCACTATGAACTTCTGGTTGAAGATTTGAATCAGATTGAAGCTGGCTGTGTGCCTCTGCCTAACTACTCTTCTATGGAGGGTTCAATAAGCCAAGCTGGCGATGAAGGAACTACTAAGAAGGGTGGTCAAATGGGGCACCATAACAGTGAGTCTACTCATGGAAATAAGGCTTCAAGGTCAGATCAAGAACGCCGTAAAGGAATCGCTTGGACAGAGGATGAGCACAGGTTATTTCTTCTTGGTTTGGACAAATATGGGAAAGGTGACTGGCGAAGTATTTCCAGAAACTTTGTTGTGACAAGGACACCTACGCAAGTGGCAAGCCATGCACAAAAATATTTCATTCGTTTGAACTCGATGAACAAAGATAGGAGGCGTTCCAGCATTCATGATATCACCAGTGTTGGCAATGGAGATATTTCAGCGCCACAAGGACCAATAACTGGTCAAACAAATGGTTCTGCTGCAGGAGGTTCCTCTGGTAAAGCTGCTAAACAACCCCCTCAACACCCTACTGGACCTCCAGGAGTTGGTGTTTATGGTCCTCCGACTATAGGGCAACCTATAGGAGGTCCCCTTGTCTCAGCAGTTGGCACCCCTGTGAATCTTCCTGCCCCTGCACACATGGCTTATGGCGTTAGAGCTCCTGTACCAGGAACAGTACCGGGAGCTGTGGTTCCTGGTGCACCAATGATGAACATGGGTCCTATGGCATATCCAATGCCACCGACAACTGCTCATAGGTGA

Described SEQ ID NO.3 is:

MIADEADCSSVWTREQDKAFEDALATYPEDAVDRWEKIAADVPGKTLEELKLHYELLVEDLNQIEAGCVPLPNYSSMEGSISQAGDEGTTKKGGQMGHHNSESTHGNKASRSDQERRKGIAWTEDEHRLFLLGLDKYGKGDWRSISRNFVVTRTPTQVASHAQKYFIRLNSMNKDRRRSSIHDITSVGNGDISAPQGPITGQTNGSAAGGSSGKAAKQPPQHPTGPPGVGVYGPPTIGQPIGGPLVSAVGTPVNLPAPAHMAYGVRAPVPGTVPGAVVPGAPMMNMGPMAYPMPPTTAHR*

Compared with the prior art, the present invention has the advantages that: the present invention provides a Jatropha jatropha MYB transcription factor JcMYB16 gene and its application in improving the drought resistance of plants. Overexpression of the JcMYB16 gene does not affect the growth and development of plants, and can Significantly improves drought stress resistance.

Description of drawings

Figure 1 shows the expression of JcMYB16 in different tissues of Jatropha;

Figure 2 shows the expression of JcMYB16 in Jatropha under drought stress;

Figure 3 shows the expression level of JcMYB16 in transgenic rice under normal growth conditions;

Figure 4 shows the phenotypic observation of overexpressed JcMYB16 gene in rice;

Figure 5 shows the phenotypic observation of overexpressing JcMYB16 gene under drought stress in rice;

Figure 6 shows the statistical results of the survival rate of overexpressed JcMYB16 gene under rice drought stress;

Figure 7 shows the relative conductivity measurement results of overexpressing JcMYB16 gene under drought stress in rice;

Figure 8 shows the results of proline content determination of overexpressed JcMYB16 gene in rice under drought stress conditions;

Figure 9 shows the expression level of JcMYB16 in transgenic Arabidopsis under normal growth conditions;

Figure 10 shows the phenotypic observation of overexpressing JcMYB16 gene in Arabidopsis;

Figure 11 shows the phenotypic observation of overexpressed JcMYB16 gene under drought stress in Arabidopsis;

Figure 12 shows the phenotypic observation of overexpressing JcMYB16 gene under drought stress (300 mM mannitol) conditions in Arabidopsis;

Figure 13 shows the taproot length of overexpressed JcMYB16 gene under normal growth conditions and drought stress conditions in Arabidopsis;

Figure 14 shows the results of proline content determination of overexpressed JcMYB16 gene under drought stress in Arabidopsis;

Figure 15 shows the results of the malondialdehyde (MDA) assay of overexpressed JcMYB16 gene under drought stress in Arabidopsis;

Figure 16 shows the relative conductivity measurement results of overexpressing JcMYB16 gene under drought stress in Arabidopsis;

Figure 17 shows the expression levels of abiotic stress response genes in wild-type and overexpressing JcMYB16 gene Arabidopsis plants under drought stress conditions;

Detailed ways

The present invention intends to further analyze the upstream and downstream genes of the JcMYB16 gene and the related regulatory signaling pathways involved, and to analyze whether the JcMYB16 gene has a broader role in drought resistance. By analyzing the role of this gene in the research on the regulation mechanism of drought stress in rice and Arabidopsis and breeding of drought-resistant varieties, in order to enrich the molecular mechanism accumulation data of Jatropha and rice and other cereal crops in response to drought stress. The design and breeding of drought-resistant molecular modules for cereal crops such as rice and wheat provides new genetic resources.

Example 1

A rice transgenic line acquisition and phenotype, drought stress analysis experiment

1 Materials and methods

1.1 Plant materials and planting methods

The rice variety tested was the japonica rice variety Zhonghua 11, or rice (Oryza sativa L.) cv. Zhonghua 11, which was preserved in the Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University. Before planting, the newly harvested seeds were firstly sterilized with 5% sodium hypochlorite for 40min or 1/1000 of carbendazim for 12h, and then soaked with 0.1mol/L _HNO3 (1mL 63% concentrated _HNO3 added 100mL water) 16h, rinsed with tap water, soaked the sterilized seeds at 28°C for 1 day, and then spread the seeds evenly in a petri dish, where a layer of filter paper was placed in the petri dish and moistened with water; covered with a lid, then Put them into cultivation at 32 °C, and change the water two to three times a day. After seeing that most of the seeds have broken shells and roots, they are transferred to cultivation at 30 °C. The seeds that have been stored for more than half a year should be dried for 2 days before sowing, and then soaked after disinfection. Disinfection method: soak the seeds in warm soup at 55°C for 30 minutes, and when the rice buds grow to 4.8-5.2mm long, sow them on the screen cloth (cultivated in rice nutrient solution), After the 3-leaf stage, the rice seedlings were moved into the soil and the corresponding labels were made for phenotypic observation and subsequent experimental analysis. The rice was fertilized every 8 or 10 days.

1.2 Reagents and carriers used

The Escherichia coli used in this experiment is DH5α, and the Agrobacterium is EHA105. These strains are preserved by the Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, and the plant expression vector is pCAMBIA1301; among them, the restriction endonuclease and cloning vector pMD18-T , T4 DNA ligase, Taq DNA polymerase were purchased from TaKaBa Biological Company; DNA recovery kits were products of Magen Biological Company; Hygromycin (Hyg), Kanamycin (Kan) and Amikacin (Amp) were purchased from All primers in the experiment were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.

1.3 RNA extraction, cDNA synthesis and RT-PCR amplification of the target gene

For RNA extraction, Magen's plant RNA extraction kit was used. Using 1 μg of RNA as a template, the first-strand cDNA was synthesized according to the instructions of the cDNA synthesis kit (TaKaRa). Specific primers were designed according to the full-length sequence of the OsHT1 gene cDNA, and the specific primer sequences are shown in Table 1. RT-PCR reaction system (20 μL): 10× PCR reaction buffer 2 μL, dNTP (2.5 mmol/L) 1 μL, primers (10 pm/μL) 1 μL each, Taq polymerase (5 U/μL) 0.2 μL, template cDNA 2 μL, ddH ₂ O 12.8 μL. PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 33 cycles; extension at 72°C for 10 min, and storage at 4°C.

Table 1 Primer sequences for RT-PCR amplification of target genes

Table1 The sequences of primers used in RT-PCR of objective genes

1.4 JcMYB16 gene expression pattern analysis

Tissue-specific expression analysis was as follows: Jatropha japonica roots, stem phloem, leaves, flowers, and seeds 35 days after pollination at the 6-leaf stage were selected for expression pattern analysis. The analysis of the expression pattern of drought stress is as follows: the drought treatment started at the six-leaf stage of Jatropha japonica (eight weeks after germination), the control group was watered with 1/2 MS medium, and the experimental group was directly stopped watering. The leaves were subjected to RNA extraction and expression analysis. Quantitative PCR (qRT-PCR) was used to detect the expression of JcMYB16 gene. Primers used for qRT-PCR analysis were designed based on the full-length sequences of the genes as quantitative PCR primers (Table 2).

Table 2 Primer sequences for quantitative qRT-PCR

Table 2 The sequences of primers used in quantitative qRT-PCR

1.5 Construction of JcMYB16 gene plant expression vector

The PCR product of the target gene was purified according to the operation of Takara Agarose Gel DNA Purification Kit. After double restriction endonucleases KpnI and Sal I digested the pMD18-T plasmid or pCAMBIA1301 plasmid with the target gene fragment, the target gene was connected to the plant expression vector pCAMBIA1301 with T4 DNA ligase _{. The} reaction system is as follows: T4 DNA ligase (5U/μL) 1μL, 10×buffer 1μL, target gene fragment 5μL, pCAMBIA1301 vector 3μL; reaction conditions: 16°C, 2h. The ligation product was transformed into competent E.coli DH5α, coated on LB plate (50 mg/L Kan) and cultured, and incubated overnight at 37°C by inversion to form a single colony.

1.6 Identification of JcMYB16 gene plant expression vector

The single clone formed after the transformation of E.coli DH5α with the JcMYB16 gene plant expression vector plasmid was selected, and the plasmid was extracted for PCR identification. The positive plasmids were transformed into competent Agrobacterium EAH105, coated on LB plates (50 mg/L Kan, 50 mg/L Rif) and cultured, inverted at 28°C for 2 d, and positive clones were selected to extract plasmids and verified by enzyme digestion.

1.7 Agrobacterium infection and acquisition of transgenic seedlings

The rice callus was used as the experimental material. Agrobacterium EHA105 was transformed with a plant expression vector (plasmid) of the JcMYB16 gene by freeze-thaw method. Pick out Agrobacterium (plant overexpression vector containing JcMYB16 gene) single clones and culture at 28°C overnight, take 10mL of culture medium, 3000rpm, 20min, centrifuge to collect bacterial pellets, and resuspend them in AAI solution (AA medium, 30g/ L sucrose, 70 g/L glucose, 200 μmol/L acetosyringone, pH 5.2), OD ₆₀₀ = 1.0, and then the suspension was shaken and cultured at 28° C. on a shaker for 3-5 h. Pick out the rice callus that has grown to a certain size, put it into the Agrobacterium suspension for 30min; then take out the callus, place it on sterilized filter paper and drain it for 50min; place the callus on a co-culture medium (2N6 , 10g/L glucose, 200μmol/L acetosyringone, PH 5.5), and cultivated in the dark at 28°C for 3d. Three days later, wash 6 times with 500 mg/L cefmetin sterilized water, 5 times with 100 mL sterilized water (including 16 μL Tween), and then once with sterile water. The drained rice calli were transferred to screening medium (2N6, 500 mg/L cefmetin, 50 mg/L hygromycin), and cultured in the dark at 28°C for 30 days. Transfer the newly grown resistant callus into differentiation medium (MS+30g/L sucrose+30g/L sorbitol+2mg/L6-BA (KT for Flower Dance)+0.8% agar+1.0mg/L NAA+250mg/L cephalosporin+50mg/L hygromycin+2g/L hydrolyzed casein, pH 5.8). Pick the rice callus with green shoots and transfer it into a conical flask containing rooting medium (MS/2+30g/L sucrose+250mg/L cephalosporin+50mg/L hygromycin), and put it into a constant temperature incubator for 28 ℃ light culture for 15d. Ready to transplant. Firstly, the positive transgenic plants were preliminarily screened by hygromycin resistance gene and GUS activity analysis, and then the expression of the target gene in wild-type and transgenic plants was detected by qRT-PCR technology, and the positive plants in the initial screening were further screened again. .

1.8 Detection of expression of JcMYB16 gene in wild-type and transgenic rice

The 14-day wild-type and transgenic rice leaves were taken for RNA extraction, and 1 μg of RNA was used as a template to synthesize the first-strand cDNA according to the instructions of the cDNA synthesis kit (TaKaRa). Specific quantitative PCR primers were designed with the cDNA of JcMYB16 gene (Table 2), and the expression of JcMYB16 in wild-type and transgenic lines was detected by qRT-PCR.

1.9 Phenotypic analysis of JcMYB16 gene in wild-type and transgenic rice

After germination of wild-type and JcMYB16 transgenic rice plants, seedlings were grown in Rice International Yoshida nutrient solution and phenotypic analysis was performed 12 days later.

2.0 Experimental analysis of drought stress in JcMYB16 transgenic rice

After germination of wild-type and JcMYB16 transgenic rice plants, 14-day-old seedlings with consistent growth were selected for salt stress treatment experiments. The method is as follows: seedlings of wild-type and transgenic plants grown for 14 days were put into Yoshida nutrient solution containing 300 mM mannitol, and phenotypic analysis was performed after 6 days; then the seedlings that had been under drought stress for 6 days were grown in Yoshida nutrient solution, and phenotypic analysis was performed after 10 days. Type analysis and statistics of survival rate. And select the aboveground leaves of drought stress treatment for 3 days to detect physiological indicators such as electrical conductivity, proline and MDA content. Each experiment contained three biological replicates.

2.2 The full length of the gene corresponding to the JcMYB16 sequence

The full-length sequence of the gene corresponding to the JcMYB16 sequence is 2 232 bp, including the complete reading frame sequence with a length of 903 bp, and the sequence is as follows SEQ ID NO.1;

The NCBI ORF-finder is used to predict that the encoded protein is 300 amino acids, and the sequence is as follows SEQ ID NO.3;

According to sequence analysis, JcMYB16 is a novel drought stress-related gene of Jatropha japonica with unknown function, and no homologous gene with known function has been found yet.

Example 2

An Arabidopsis thaliana transgenic line acquisition and phenotype, drought stress analysis experiment

1 Materials and methods

1.1 Plant materials and planting methods

The Arabidopsis thaliana used in this experiment is the Columbia ecotype, which is preserved in the Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University. The Arabidopsis thaliana seed disinfection method is as follows: wash once with sterile water, sterilize with 70% alcohol for 5 minutes, wash with sterile water once, disinfect with 1% sodium hypochlorite for 15 minutes, and wash with sterile water for 5 times. Arabidopsis seeds were treated with a low temperature of 4°C for 2 days before planting to make them germinate consistently. Sterilized planted seeds were transplanted into the planting medium after germination on 1/2MS (1% sucrose, 8% agar, pH 5.7) medium for 7-10 days. Seeds that do not need to be sterilized can be sown directly after being treated at low temperature. The planting medium was large-grained vermiculite, covered with film for about 5 days during germination. During the growth stage, water the nutrient solution every 3-5 days. The culture conditions between growths were: 22±2°C with a photoperiod of 16 hours light/8 hours dark.

Drought stress analysis experiment of overexpression plants

1.2 Strains, vectors and reagents

Escherichia coli used in this experiment was DH5α, and Agrobacterium was GV3101. The plant expression vector is pCAMBIA1301. The above strains and vectors are kept in our laboratory. The pMD18-T vector, restriction endonuclease, T4 ligase, nucleic acid recovery kit, etc. were all purchased from Dalian Biotechnology Company (Takara). Ampicillin (Amp) and hygromycin (Hyg) were purchased from Beijing Dingguo Biotechnology Co., Ltd. All primers in the experiment were synthesized by Beijing Aoke Dingsheng Biological Company.

In this experiment, the stress resistance of each transgenic line was tested mainly by drought stress on mature seedlings, 150 mM NaCl stress on seedlings, and 300 mM mannitol (Man) stress on seedlings. The specific steps are as follows: sterilized Arabidopsis thaliana seeds are treated at low temperature for 2 days, sown in a medium of 1/2MS+1% sucrose for germination, and cultured vertically for 4 days. Transplant into the corresponding stress medium (minimum medium is still 1/2MS) for vertical cultivation for 5-7 days. Observe and record growth and survival rate, etc.

1.3 RNA extraction, cDNA synthesis and RT-PCR amplification of the target gene

For RNA extraction, Magen's plant RNA extraction kit was used. Using 1 μg of RNA as a template, the first-strand cDNA was synthesized according to the instructions of the cDNA synthesis kit (TaKaRa). Specific primers were designed according to the full-length sequence of the OsHT1 gene cDNA, and the specific primer sequences are shown in Table 3. RT-PCR reaction system (20 μL): 10× PCR reaction buffer 2 μL, dNTP (2.5 mmol/L) 1 μL, primers (10 pm/μL) 1 μL each, Taq polymerase (5 U/μL) 0.2 μL, template cDNA 2 μL, ddH ₂ O 12.8 μL. PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 33 cycles; extension at 72°C for 10 min, and storage at 4°C.

Table 3 Primer sequences for RT-PCR amplification of target genes

Table3 The sequences of primers used in RT-PCR of objective genes

1.4 Construction of JcMYB16 gene plant expression vector

Extract and sequence the correctly cloned plasmid, and perform double digestion with the added restriction site, as follows: use Kpn I and SalI double restriction digestion. 0.5 μl of each enzyme was added to the 10 μl system, other conditions were carried out according to the instructions provided by Takara, and the digestion time was 2 hours. The digested fragment was recovered and ligated overnight with the pCAMBIA1301 vector that was also double digested. The ligation products were directly transformed into DH5α competent cells, and the resistant clones were selected for PCR detection using Kan. The positive clones were expanded and cultured, and plasmids were extracted. Double-enzyme digestion verification of the extracted plasmid: In addition to the restriction enzyme restriction site added by PCR, the existing restriction enzyme restriction sites EcoR I and Hind III in the vector are also selected for combined restriction restriction digestion verification, and the direction of connection into the expression vector is judged. is it right or not. The correctly ligated plasmid was transformed into Agrobacterium GV3101 competent, and resistant clones were picked for PCR detection after 1 day. The positive clones were extracted with plasmids and verified by rotation, that is, the plasmids were extracted into E. coli for double-enzyme digestion verification. Correct clones detected at each step will be used for the next transformation and stored at -80°C for later use.

1.5 Transgenic JcMYB16 plant expression vector into Arabidopsis thaliana

The method of transforming Arabidopsis in this experiment is the pollen tube channel method, also known as the inflorescence dip method. The specific steps are as follows: select the Arabidopsis thaliana plants with buds to be released (about 4 weeks), remove the flower buds that have opened, and infect the night before Water adequately. The Agrobacterium containing the target vector was shaken to OD ₆₀₀ =1.8, and the cells were collected by centrifugation at 5000 g for 5 min. Add infiltration medium (1/2MS without vitamins + 1ml 1000×Gamborg's Vitamins (inositol 100mg/mL, niacin 1mg/mL, B6 1mg/mL, B1 10mg/mL) + 6-BA 10μg/mL, pH 5.7 ) to resuspend Agrobacterium at a 1:1 ratio and add the surfactant Silwet to a final concentration of 0.02%. After the preparation is completed, dip-dye is carried out, and the dip-dye time is 2min. During this period, the dip-dye is continuously stirred to fully combine with the inflorescence. After dip-dyeing, the plants were dried for a short time, and the plants were placed in a dark box for 24 hours of dark cultivation. During this period, newspapers were used to shade and maintain a certain humidity in the dark box. After dark cultivation, normal cultivation was continued, and seeds were collected for screening.

1.6 Screening of JcMYB16 transgenic plants and detection of target gene expression

The collected Arabidopsis seeds were sterilized and sown on 1/2MS+hygromycin (Hyg) 30 μg/mL medium, and positive plants were obtained one week later. Positive plants are characterized by greener leaves and developed roots. These positive plants are the T1 generation lines, each individual plant is a line, and the seeds are individually numbered and collected. The seeds of the T1 generation line were sterilized and screened by hygromycin, and the separation ratio was calculated. Lines with a segregation ratio of about 3:1 (positive plants:negative plants) were selected for transplantation, and each line had 12 individual plants (T2 generation). Then the T2 generation single plant was harvested, and then sterilized and hygromycin screened. It was observed whether the seeds of the T2 generation individual plants were separated, and the single plant that no longer separated was a homozygous plant, and the seeds were harvested, stored separately, and sown to obtain the seeds of the T3 generation plants. The T3 generation plants obtained in this way have more seeds for subsequent phenotypic analysis.

The fourth leaf of T3 generation plants grown for 20 days was used for RNA extraction. The extraction method was Trizol method. Extracted RNA was tested for concentration and integrity using a Nanodrop 2000 spectrophotometer and agarose gel electrophoresis. RNA of better quality was used for reverse transcription, and the amount of reverse transcription RNA was 2 μg, and the kit was purchased from Premega Company. The quality of reverse transcribed cDNA was detected by PCR using JcActin gene, and the concentration was adjusted to be consistent. Semi-quantitative PCR was used to detect the expression levels of each target gene in each Arabidopsis line, and 3 overexpressing lines were selected for subsequent phenotype analysis.

1.7 Experimental analysis of drought stress in JcMYB16 transgenic plants

The drought stress treatment conditions were as follows: first, wild-type and JcMYB16 transgenic Arabidopsis seeds were sterilized, and then Arabidopsis thaliana were seeded on-demand in square sterile petri dishes containing 1/2 MS medium, cultured vertically for 4 d, and then wild-type plants with consistent growth were selected. Type and transgenic Arabidopsis seedlings were transferred to 1/2MS and 1/2MS medium containing 300mM mannitol and continued to grow vertically for 7 days for phenotypic analysis, electrical conductivity, proline and MDA content detection. In addition, under the drought stress in the nutrient soil, the Arabidopsis thaliana directly stopped watering for 3 weeks, and the phenotype was observed after 2 weeks, and then the survival rate was counted after rehydration for 7 days.

1.8 RNA extraction and qRT-PCR

The RNA required in this experiment was carried out with the plant RNA extraction kit from Magen company; the first-strand cDNA synthesis was carried out with the first-strand cDNA synthesis kit from TAKARA company. LightCycler 480 and TB GreenTM Premix Ex TaqII were used for qRT-PCR analysis. The 2 ^-ΔΔCT method was used to calculate the relative gene expression, and AtActin2 was used as the reference gene in Arabidopsis thaliana. Quantitative PCR primers are shown in Table 4.

Table 4 Primer sequences for quantitative qRT-PCR

Table 4 The sequences of primers used in quantitative qRT-PCR

The function of Jatropha jatropha MYB family gene JcMYB16 in plant response to drought stress was cloned and studied for the first time, that is, the present invention can provide a gene that can increase plant drought resistance. Firstly, tissue-specific expression analysis of JcMYB16 gene was carried out, and qRT-PCR results showed that JcMYB16 gene was highly expressed in Jatropha JcMYB16, the recombinant vector was transferred into Agrobacterium for further transformation. Using rice callus and Arabidopsis thaliana as receptors, the constructed vector was transferred into rice callus and Arabidopsis thaliana respectively by Agrobacterium-mediated method to obtain JcMYB16 transgenic homozygous lines; Phenotypic and drought stress analyses were performed. Phenotypic analysis showed that overexpression of the JcMYB16 gene did not affect plant growth and development. Analysis of drought stress experiments showed that overexpression of the JcMYB16 gene increased drought stress resistance in transgenic rice and Arabidopsis. In addition, we have grown 4 generations of transgenic lines, and JcMYB16 transgenic rice and transgenic Arabidopsis can significantly improve drought stress resistance from the T1 generation, indicating that the JcMYB16 transgenic plants we obtained can be stably inherited. The JcMYB16 gene is a key regulator of drought stress in rice and Arabidopsis, indicating that genetic engineering technology can be used to improve the resistance of rice and jatropha to drought stress. Bioengineering technology can be used to purposefully regulate the drought stress characteristics of plants. Through molecular breeding methods It is very important to cultivate new varieties of drought-tolerant rice or Jatropha so as to promote large-scale planting of rice or Jatropha in arid areas. The gene can be used for the cultivation of drought-tolerant varieties of Jatropha japonica and cereal crops such as rice and wheat, as well as for the cultivation of drought-tolerant varieties of crops such as barley, sorghum, corn, Arabidopsis, tomato, tobacco, soybean, and potato. The amino acid sequence of the protein encoded by JcMYB16, or functionally similar proteins in other species, has no less than 30% homology with the amino acid sequence shown in Seq ID No.3. The application of protein in cultivating stress-tolerant varieties of plants. Stress tolerance is to increase the drought resistance of cereal crops, thereby increasing the yield per unit area in drought and saline-alkali land.

The above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced without Any modifications or partial substitutions that depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

SEQUENCE LISTING

<110> Zhoukou Normal University

<120> Jatropha MYB transcription factor JcMYB16 gene and its application in improving plant drought resistance

<130> 191131

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 2232

<212> DNA

<213> Nucleotide sequence of JcMYB16 gene

<400> 1

ttagtggtgg ttaaactttc aaaatctcga tagtgcaagt atttgaattt ttaggttatg 60

tttcttttat ggcctttcgc actgttagtg gaattggagt gtgaaaatgg aatctggggt 120

tagagattat ttttcttctc taatcaggag tgagaaacct tgaattactg gccgttctac 180

tctcaattaa tggctttctg ttcccaggaa ttgcccttca aatctgctgt tgataacata 240

ggtcagcagt tcacaagagt ccaatttttt tgaattgtgg agcaaattac cattgaattc 300

ttcacatact ccgttgggat tttgtgattt ttattggagg tgatctctgg ttcagttctc 360

ttggcttttt ttgggtccat ctgcttgttt ctcgtcttcc ttacctctac accctgacac 420

atcctgctgc tttcccataa ttctgttctg ccatcactcc aactgttggt cataaccaaa 480

tatagaaatt attcagatcc aaatttcaag aaaccgttct agaatttttt ttttttttca 540

agattggctt attaatttag cctagattct agagctaggt ttttcctttc tttgctagtg 600

taagattcaa accagtctag atgattgcgg atgaagcaga ctgcagctct gtgtggacta 660

gggagcagga taaggcattt gaggatgccc ttgcaacata tcctgaggat gctgtagatc 720

ggtgggagaa aattgctgct gatgttcctg ggaaaacctt agaagagctt aaacttcact 780

atgaacttct ggttgaagat ttgaatcaga ttgaagctgg ctgtgtgcct ctgcctaact 840

actcttctat ggagggttca ataagccaag ctggcgatga aggaactact aagaagggtg 900

gtcaaatggg gcaccataac agtgagtcta ctcatggaaa taaggcttca aggtcagatc 960

aagaacgccg taaaggaatc gcttggacag aggatgagca caggttattt cttcttggtt 1020

tggacaaata tgggaaaggt gactggcgaa gtatttccag aaactttgtt gtgacaagga 1080

cacctacgca agtggcaagc catgcacaaa aatatttcat tcgtttgaac tcgatgaaca 1140

aagataggag gcgttccagc attcatgata tcaccagtgt tggcaatgga gatatttcag 1200

cgccacaagg accaataact ggtcaaacaa atggttctgc tgcaggaggt tcctctggta 1260

aagctgctaa acaaccccct caacacccta ctggacctcc aggagttggt gtttatggtc 1320

ctccgactat agggcaacct ataggaggtc cccttgtctc agcagttggc acccctgtga 1380

atcttcctgc ccctgcacac atggcttatg gcgttagagc tcctgtacca ggaacagtac 1440

cgggagctgt ggttcctggt gcaccaatga tgaacatggg tcctatggca tatccaatgc 1500

caccgacaac tgctcatagg tgatatacat ggtttagctg caaaatgtac aaagacagaa 1560

ggctacttgc ttgtatttct ggtgggtcag tggcttctcc attttagcct gaataaaact 1620

gcttatttgc aagcaaaaat tgtctgatgt catttgttta ttctggtagc aatatcaaat 1680

aaaccaatag gtagagaaac tacatgcatt tgtataggca gcagctgtgg aaaatatggc 1740

agcagttatg ggtaggacac attttggtac ttttttttttg gttttacatt acaatgttta 1800

gtctcagtag cagtcagtta atggtatttt acttttaatg accaaatttg taaagaatcc 1860

atttatacgt tttactattt tgagtagtag atgttggcac ggattgtgca aagcctttgt 1920

aaaaaaaagt accaaaatgt gtcctaccca taactgctgc catattttcc acagctgctg 1980

cctatacaaa tgcatgtagt ttctctacct attggtttat ttgatattgc taccagaata 2040

aacaaatgac atcagacaat ttttgcttgc aaataagcag ttttattcag gctaaaatgg 2100

agaagccact gacccaccag aaatacaagc aagtagcctt ctgtctttgt acattttgca 2160

gctaaaccat gtatatcacc tatgagcagt tgtcggtggc attggatatg ccataggacc 2220

catgttcatc at 2232

<210> 2

<211> 903

<212> DNA

<213> Nucleotide sequence of gene open reading frame

<400> 2

atgattgcgg atgaagcaga ctgcagctct gtgtggacta gggagcagga taaggcattt 60

gaggatgccc ttgcaacata tcctgaggat gctgtagatc ggtgggagaa aattgctgct 120

gatgttcctg ggaaaacctt agaagagctt aaacttcact atgaacttct ggttgaagat 180

ttgaatcaga ttgaagctgg ctgtgtgcct ctgcctaact actcttctat ggagggttca 240

ataagccaag ctggcgatga aggaactact aagaagggtg gtcaaatggg gcaccataac 300

agtgagtcta ctcatggaaa taaggcttca aggtcagatc aagaacgccg taaaggaatc 360

gcttggacag aggatgagca caggttattt cttcttggtt tggacaaata tgggaaaggt 420

gactggcgaa gtatttccag aaactttgtt gtgacaagga cacctacgca agtggcaagc 480

catgcacaaa aatatttcat tcgtttgaac tcgatgaaca aagataggag gcgttccagc 540

attcatgata tcaccagtgt tggcaatgga gatatttcag cgccacaagg accaataact 600

ggtcaaacaa atggttctgc tgcaggaggt tcctctggta aagctgctaa acaaccccct 660

caacacccta ctggacctcc aggagttggt gtttatggtc ctccgactat agggcaacct 720

ataggaggtc cccttgtctc agcagttggc acccctgtga atcttcctgc ccctgcacac 780

atggcttatg gcgttagagc tcctgtacca ggaacagtac cgggagctgt ggttcctggt 840

gcaccaatga tgaacatggg tcctatggca tatccaatgc caccgacaac tgctcatagg 900

tga 903

<210> 3

<211> 300

<212> PRT

<213> DNA-encoded amino acid sequence of the open reading frame of the gene

<400> 3

Met Ile Ala Asp Glu Ala Asp Cys Ser Ser Val Trp Thr Arg Glu Gln

1 5 10 15

Asp Lys Ala Phe Glu Asp Ala Leu Ala Thr Tyr Pro Glu Asp Ala Val

20 25 30

Asp Arg Trp Glu Lys Ile Ala Ala Asp Val Pro Gly Lys Thr Leu Glu

35 40 45

Glu Leu Lys Leu His Tyr Glu Leu Leu Val Glu Asp Leu Asn Gln Ile

50 55 60

Glu Ala Gly Cys Val Pro Leu Pro Asn Tyr Ser Ser Met Glu Gly Ser

65 70 75 80

Ile Ser Gln Ala Gly Asp Glu Gly Thr Thr Lys Lys Gly Gly Gln Met

85 90 95

Gly His His Asn Ser Glu Ser Thr His Gly Asn Lys Ala Ser Arg Ser

100 105 110

Asp Gln Glu Arg Arg Lys Gly Ile Ala Trp Thr Glu Asp Glu His Arg

115 120 125

Leu Phe Leu Leu Gly Leu Asp Lys Tyr Gly Lys Gly Asp Trp Arg Ser

130 135 140

Ile Ser Arg Asn Phe Val Val Thr Arg Thr Pro Thr Gln Val Ala Ser

145 150 155 160

His Ala Gln Lys Tyr Phe Ile Arg Leu Asn Ser Met Asn Lys Asp Arg

165 170 175

Arg Arg Ser Ser Ile His Asp Ile Thr Ser Val Gly Asn Gly Asp Ile

180 185 190

Ser Ala Pro Gln Gly Pro Ile Thr Gly Gln Thr Asn Gly Ser Ala Ala

195 200 205

Gly Gly Ser Ser Gly Lys Ala Ala Lys Gln Pro Pro Gln His Pro Thr

210 215 220

Gly Pro Pro Gly Val Gly Val Tyr Gly Pro Pro Thr Ile Gly Gln Pro

225 230 235 240

Ile Gly Gly Pro Leu Val Ser Ala Val Gly Thr Pro Val Asn Leu Pro

245 250 255

Ala Pro Ala His Met Ala Tyr Gly Val Arg Ala Pro Val Pro Gly Thr

260 265 270

Val Pro Gly Ala Val Val Pro Gly Ala Pro Met Met Asn Met Gly Pro

275 280 285

Met Ala Tyr Pro Met Pro Pro Thr Thr Ala His Arg

290 295 300

Claims (7)

1. MYB transcription factor of jatropha curcasJcMYB16A gene characterized by: the above-mentionedJcMYB16The nucleotide sequence of the gene is SEQ ID NO.1, and the nucleotide sequence of the gene open reading frame is SEQ ID NO. 2.

2. A polypeptide, characterized by: the amino acid sequence of the polypeptide is encoded by SEQ ID NO. 3.

3. A recombinant construct, characterized in that: the recombinant construct comprises a nucleotide sequence shown in SEQ ID NO.1, and the vector for the construct is a pMD18-T vector for cloning or a pCAMBIA1301 vector for expression.

4. The Jatropha curcas MYB transcription factor of claim 1JcMYB16The transgenic agrobacterium of the gene is EHA105 or GV 3101.

5. The Jatropha curcas MYB transcription factor of claim 1JcMYB16The application of the nucleotide sequence of the gene in improving the drought resistance of plants.

6. The Jatropha curcas MYB transcription factor of claim 5JcMYB16The application of the nucleotide sequence of the gene in improving the drought resistance of plants is characterized in that: the plant is a monocotyledon or a dicotyledon.

7. The Jatropha curcas MYB transcription factor of claim 6JcMYB16The application of the nucleotide sequence of the gene in improving the drought resistance of plants is characterized in that: the monocotyledon is rice, and the dicotyledon is riceThe plant is Jatropha curcas Arabidopsis thaliana.

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