CN110643618B - Jatropha MYB transcription factor JcMYB16 gene and its application in improving plant drought resistance - Google Patents

Abstract

The invention provides a Jatropha MYB class transcription factor JcMYB16 gene, its gene nucleotide sequence is SEQ ID NO.1, and the nucleotide sequence of its gene open reading frame is SEQ ID NO.2. Overexpression of JcMYB16 gene does not affect Plant growth and development can significantly improve drought stress resistance. The gene can be used for the cultivation of drought-resistant varieties of cereal crops such as jatropha, rice, and wheat, and can also be applied to 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 application in improving plant drought resistance

technical field

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

Background technique

Physiological water shortage in plants caused by drought stress seriously affects plant growth and crop yield. Statistics show that the arable land area in my country is only 121 million hectares at present, while the desertification and salinization land has 260 million hectares, and the abandoned land caused by human factors is 13 million hectares. 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 genes related to stress tolerance and the development and utilization research in molecular breeding. At present, the research on the model plant Arabidopsis has preliminarily understood the signaling pathways of plants responding 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 out more genes related to drought resistance in a single species. However, due to the diversity of species, many plants have good drought-tolerant characteristics, so excavating drought-resistant gene resources from plants with drought-tolerant characteristics has important agricultural values and Application prospects. The introduction of jatropha provides us with an ideal solution. It 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 in the Euphorbiaceae family. It has the characteristics of fast reproduction, strong stress tolerance, especially drought tolerance.

MYB transcription factors are one of many transcription factors, which are characterized by a conserved MYB domain consisting of more than 50 amino acids at the N-terminus of the protein, 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 process of plant response to drought stress. For example, the Arabidopsis AtMYB2 transcription factor and rd22BP1 respectively bind to the promoter region of the drought stress response gene rd22, and then jointly activate the expression of rd22 to achieve drought resistance. More and more studies have shown that MYB proteins are involved in plant response to abiotic stress Response regulation. In conclusion, although many MYB proteins have been cloned and functionally analyzed, the drought stress-responsive genes of the Jatropha MYB family are still rarely reported. Therefore, cloning the Jatropha MYB family involved in drought stress regulation genes and studying their functions will provide a new gene resource for the breeding 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 protected. Development and utilization, and then promote the sustainable development of my country's national economy and the effective protection of the ecological environment.

Contents of the invention

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

To achieve the above object, the technical scheme adopted in the present invention is as follows:

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

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

A kind of recombinant construct, recombinant construct comprises the nucleotide sequence shown in SEQ ID NO.1, and the vector that described construct is used is the pMD18-T vector that is used for cloning or the pCAMBIA1301 vector that is used for expressing.

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

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

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

The monocotyledon is rice, and the dicotyledon is Arabidopsis jatropha.

Said SEQ ID NO.1 is:

TTAGTGGTGGTTAAACTTTCAAAATCTCGATAGTGCAAGTTTGAATTTTTAGGTTATGTTTCTTTTATGGCCTTTCGCACTGTTAGTGGAATTGGAGTGTGAAAATGGAATCTGGGGTTAGAGATTATTTTTCTTCTCTAATCAGGAGTGAGAAACCTTGAATTACTGGCCGTTCTACTCTCAATTAATGGCTTTCTGTTCCCAGGAATTGCCCTTCAA ATCTGCTGTTGATAACATAGGTCAGCAGTTCACAAGAGTCCAATTTTTTTGAATTGTGGAGCAAATTACCATTGAATTCTTCATACTCCGTTGGGATTTTGTGATTTTTATTGGAGGTGATCTCTGGTTCAGTTTCTTGGCTTTTTTTGGGTCCATCTGCTTGTTTCTCGTCTTTCCTTACCTCTACACCCTGACACATCCTGCTGCTTTCCCATAATTCCTGTT CTGCCATCACTCCAACTGTTGGTCATAACCAAATAGAAATTATTCAGATCCAAATTTCAAGAAACCGTTCTAGAATTTTTTTTTTTTTTCAAGATTGGCTTATTAATTTAGCCTAGATTCTAGAGCTAGGTTTTTCCTTTCTTTGCTAGTGTAAGATTCAAACCAGTCTAGATGATTGCGGATGAAGCAGACTGCAGCTCTGTGTGGACTAGGGAGCAGGATAAGGCATTTGA GGATGCCCTTGCAACATATCCTGAGGATGCTGTAGATCGGTGGGAGAAAATTGCTGCTGATGTTCCTGGGAAAACCTTAGAAGAGCTTAAACTTCACTATGAACTTCTGGTTGAAGATTTGAATCAGATTGAAGCTGGCTGTGTGCCTCTGCCTAACTACTTCTTCTATGGAGGGTTCAATAAGCCAAGCTGGCGATGAAGGAACTACTAAGAAGGGTG GTCAAATGGGGCACCATAACAGTGAGTCTACTCATGGAAATAAGGCTTCAAGGTCAAGATCAAGAACGCCGTAAAGGAATCGCTTGGACAGAGGATGAGCACAGGTTATTTCTTCTTGGTTTGGACAAATATGGGAAAGGTGACTGGCGAAGTATTTCCAGAAACTTTGTTGTGACAAGGACACCTACGCAAGTGGCAAGCCATGCACAAAAATATTTCATTC GTTTGAACTCGATGAACAAAGATAGGAGGCGTTCCAGCATTCATGATATCACCAGTGTTGGCAATGGAGATATTTCAGCGCCACAAGGACCAATAACTGGTCAAACAAATGGTTCTGCTGCAGGAGGTTCCTCTGGTAAAGCTGCTAAACAACCCCCCTCAACACCCTACTGGACCTCAGGAGTTGGTGTTTATGGTCCTCCGACTATAGGGCAACCTATAG GAGGTCCCCCTTGTCTCCAGCAGTTGGCACCCCTGTGAATCTTCCTGCCCCTGCACACATGGCTTATGGCGTTAGAGCTCCTGTACCAGGAACAGTACCGGGAGCTGTGGTTCCTGGTGCACCAATGATGAACATGGGTCCTATGGCATATCCAATGCCACCGACAACTGCTCATAGGTGATATACATGGTTTAGCTGCAAAATGTACAAAGACAGAAGGCTACTT GCTTGTATTTCTGGTGGGTCAGTGGCTTCTCCATTTTAGCCTGAATAAAACTGCTTATTTGCAAGCAAAAATTGTCTGATGTCATTTGTTTATTCTGGTAGCAATATCAAATAAACCAATAGGTAGAGAAACTACATGCATTTGTATAGGCAGCAGCTGTGGAAAATATGGCAGCAGTTATGGGTAGGACACATTTTGGTACTTTTTTTTTGGTTTTCATTACAC ATGTTTAGTCTCAGTAGCAGCAGTCAGTTAATGGTATTTTACTTTTAATGACCAAATTTGTAAAGAATCCATTTATACGTTTTACTATTTTGAGTAGTAGATGTTGGCACGGATTGTGCAAAAGCCTTTGTAAAAAAAGTACCAAATGTGTCCTACCCATAACTGCTGCCATATTTTCCACAGCTGCTGCCTATACAAATGCATGTAGTTTCTCTACCTATTGGTTTATTTGATA TTGCTACCAGAATAAACAAATGACATCAGACAATTTTTGCTTGCAAATAAGCAGTTTTATTCAGGCTAAAATGGAGAAGCCACTGACCCACCAGAAATACAAGCAAGTAGCCTTCTGTCTTTGTACATTTTGCAGCTAAACCATGTATATCACCTATGAGCAGTTGTCGGTGGCATTGGATATGCCATAGGACCCATGTTCATCAT

Said SEQ ID NO.2 is:

ATGATTGCGGATGAAGCAGACTGCAGCTCTGTGTGGACTAGGGAGCAGGATAAGGCATTTGAGGATGCCCTTGCAACATATCCTGAGGATGCTGTAGATCGGTGGGAGAAAATTGCTGCTGATGTTCCTGGGAAAACCTTAGAAGAGCTTAAACTTCACTATGAACTTCTGGTTGAAGATTTGAATCAGATTGAAGCTGGCTGTGTGCCTCTGCCTAA CTACTTCTTCTATGGAGGGTTCAATAAGCCAAGCTGGCGATGAAGGAACTACTAAGAAGGGTGGTCAAATGGGGCACCATAACAGTGAGTCTACTCATGGAAATAAGGCTTCAAGGTCAGATCAAGAACGCCGTAAAGGAATCGCTTGGACAGAGGATGAGCACAGGTTATTTCTTCTTGGTTTGGACAAATATGGGAAAGGTGACTGGCGAAGTATTTC CAGAAACTTTGTTGTGACAAGGACACCTACGCAAGTGGCAAGCCATGCACAAAAATATTTCATTCGTTTGAACTCGATGAACAAAGATAGGAGGCGTTCCCAGCATTCATGATATCACCAGTGTTGGCAATGGAGATATTTCAGGCCACAAGGACCAATAACTGGTCAAACAAATGGTTCTGCTGCAGGAGGTTCCTCTGGTAAAGCTGCTAAACAACCCCCTC ACACCCTACTGGACCTCCAGGAGTTGGTGTTTATGGTCCTCCGACTATAGGGCAACCTATAGGAGGTCCCTTGTCTCCAGCAGTTGGCACCCCTGTGAATCTTCCTGCCCCTGCACACATGGCTTATGGCGTTAGAGCTCCTGTACCAGGAACAGTACCGGGAGCTGTGGTTCCTGGTGCACCAATGATGAACATGGGTCCTATGGCATATCCAATGCCACC GACAACTGCTCATAGGTGA

Said SEQ ID NO.3 is:

MIADEADCSSVWTREQDKAFEDALATYPEDAVDRWEKIAADVPGKTLEELKLHYELLVEDLNQIEAGCVPLPNYSSMEGSISQAGDEGTTKKGGQMGHHNSESTHGNKASRSDQERRKGIAWTEDEHRLFLLGLDKYGKGDWRSISRNFVVTRTPTQVASHAQKYFIRLNSMNKDRRRSSI HDITSVGNGDISAPQGPITGQTNGSAAGGSSGKAAKQPPQHPTGPPGVGVYGPPTIGQPIGGPLVSAVGTPVNLPAPAHMAYGVRAPVPGTVPGAVVPGAPMMNMGPMAYPMPPTTAHR*

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

Description of drawings

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

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

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

Fig. 4 shows the phenotype observation of overexpression JcMYB16 gene in rice;

Fig. 5 shows the phenotype observation of the overexpression JcMYB16 gene under the drought stress condition of rice;

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

Fig. 7 shows the relative conductivity determination result of overexpression JcMYB16 gene under rice drought stress condition;

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

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

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

Figure 11 shows the phenotype observation of overexpressing JcMYB16 gene under the drought stress condition of Arabidopsis;

Figure 12 shows the phenotype observation of overexpression JcMYB16 gene under the condition of Arabidopsis drought stress (300mM mannitol);

Figure 13 shows the main root length of overexpressing JcMYB16 gene under normal growth conditions and drought stress conditions of Arabidopsis;

Fig. 14 shows the proline content determination result of the overexpression JcMYB16 gene under the drought stress condition of Arabidopsis;

Figure 15 represents the malondialdehyde (MDA) assay result of overexpression JcMYB16 gene under Arabidopsis drought stress condition;

Fig. 16 shows the relative conductivity determination result of overexpression JcMYB16 gene under the drought stress condition of Arabidopsis;

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

Detailed ways

In the following, the present invention intends to further analyze the upstream and downstream genes of the JcMYB16 gene and related regulatory signaling pathways involved in it, and analyze whether the JcMYB16 gene has a broader drought resistance effect. By analyzing the role of this gene in the research on the regulation mechanism of drought stress in rice and Arabidopsis thaliana and the cultivation of drought-resistant varieties, it is hoped to enrich the molecular mechanism accumulation data of cereal crops such as Jatropha and rice in response to drought stress. The design and breeding of drought-resistant molecular modules of cereal crops such as rice and wheat provide 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 used in the test is the japonica rice variety Zhonghua 11, namely Oryza sativa L. cv. Zhonghua 11, which is kept in the Key Laboratory of Plant Genetics and Molecular Breeding of Zhoukou Normal University. Before planting, the newly harvested seeds were first disinfected with 5% sodium hypochlorite for 40 minutes or 1/1000 carbendazim for 12 hours, and then soaked in 0.1mol/L HNO ₃ (1 mL of 63% concentrated HNO ₃ added to 100 mL of water) for 16 hours After rinsing with tap water, soak the sterilized seeds at 28°C for 1 day, spread the seeds evenly in a petri dish, place a layer of filter paper in the petri dish, and moisten it with water; put the lid on, and put Cultivate at 32°C, change the water two to three times a day, and transfer to 30°C for cultivation after seeing that most of the seeds have broken shells and rooted out. The seeds stored for more than half a year should be sun-dried for 2 days before sowing, and then soaked after disinfection. Disinfection method: Soak the seeds in warm water at 55°C for 30 minutes. When the rice buds grow to 4.8-5.2mm long, they should be sown on screen cloth (cultivated in rice nutrient solution). After the 3-leaf stage, the rice seedlings were moved into the soil and labeled accordingly for phenotypic observation and subsequent experimental analysis. The rice is fertilized every 8 or 10 days.

1.2 Reagents and carriers used

The Escherichia coli used in this experiment was DH5α, and the Agrobacterium was EHA105. These strains were preserved by the Key Laboratory of Plant Genetics and Molecular Breeding of Zhoukou Normal University, and the plant expression vector was pCAMBIA1301; among them, the restriction enzyme and the cloning vector pMD18-T , T4 DNA ligase, and Taq DNA polymerase were purchased from TaKaBa Biological Company; the DNA recovery kit was a product of Magen Biological Company; All primers were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. from Beijing Dingguo Biotechnology Co., Ltd.

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

For RNA extraction, a plant RNA extraction kit from Magen was used, and the reference method was a small amount of RNA extraction from plant tissues, which is relatively easy to extract. Using 1 μg of RNA as a template, the first-strand cDNA was synthesized according to the operating instructions of the cDNA synthesis kit (TaKaRa). Specific primers were designed according to the full-length sequence of OsHT1 gene cDNA, and the sequences of specific primers are shown in Table 1. RT-PCR reaction system (20 μL): 2 μL of 10×PCR reaction buffer, 1 μL of dNTP (2.5 mmol/L), 1 μL of each primer (10 pm/μL), 0.2 μL of Taq polymerase (5U/μL), 2 μL of template cDNA, 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, 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 Analysis of JcMYB16 gene expression pattern

Tissue-specific expression analysis was as follows: Jatropha Jatropha roots, stem phloem, leaves, flowers, and seeds 35 days after pollination were selected for expression pattern analysis at the 6-leaf stage. The analysis of the expression pattern of drought stress is as follows: the drought treatment started at the six-leaf stage of Jatropha Jatropha (eight weeks after germination), the control group was irrigated with 1/2MS medium, and the experimental group directly stopped watering. Leaves were subjected to RNA extraction and expression analysis. Quantitative PCR (qRT-PCR) was used for the detection of JcMYB16 gene expression. The primers used for qRT-PCR analysis were designed according to the full-length sequence of the gene 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 the pMD18-T plasmid or pCAMBIA1301 plasmid with restriction endonuclease KpnI and Sal I double-digested and ligated with the target gene fragment, use T ₄ DNA ligase to connect the target gene to the plant expression vector pCAMBIA1301, and the reaction system is as follows: T ₄ DNA ligase (5U/μL) 1 μL, 10× buffer 1 μL, target gene fragment 5 μL, pCAMBIA1301 vector 3 μL; reaction conditions: 16°C, 2h. The ligation product was transformed into competent E.coli DH5α, spread on LB plates (50mg/L Kan) for culture, and cultured overnight at 37°C to form a single colony.

1.6 Identification of JcMYB16 gene plant expression vector

Pick the single clone formed after transforming the JcMYB16 gene plant expression vector plasmid into E.coli DH5α, and extract the plasmid for PCR identification. The positive plasmid was transformed into competent Agrobacterium EAH105, spread on LB plates (50mg/L Kan, 50mg/LRif) for culture, and cultured upside down at 28°C for 2 days, and the positive clones were selected to extract the plasmid and carry out enzyme digestion verification.

1.7 Agrobacterium infection and acquisition of transgenic seedlings

Rice callus was used as the experimental material. JcMYB16 gene plant expression vector (plasmid) was transformed into Agrobacterium EHA105 by freeze-thaw method. Pick out single clones of Agrobacterium (plant overexpression vector containing JcMYB16 gene) and culture them overnight at 28°C, take 10mL culture solution, 3000rpm, 20min, centrifuge to collect the bacterial pellet, resuspend in AAI solution (AA culture solution, 30g/ L sucrose, 70 g/L glucose, 200 μmol/L acetosyringone, pH 5.2), OD ₆₀₀ =1.0, and then shake the suspension on a shaker at 28° C. for 3-5 hours. Pick out the rice callus that has grown to a certain size, put it into the Agrobacterium suspension and dip it for 30 minutes; then take out the callus, put it on sterile filter paper and drain it for 50 minutes; place the callus in a co-culture medium (2N6 , 10g/L glucose, 200μmol/L acetosyringone, pH 5.5), cultured in the dark at 28°C for 3d. Three days later, wash with 500 mg/L cefotaxime sterilized water 6 times, 100 mL sterilized water (containing 16 μL Tween) 5 times, and then wash once with sterile water. The drained rice callus was transferred to a selection medium (2N6, 500 mg/L cefotaxime, 50 mg/L hygromycin), and cultured in the dark at 28°C for 30 days. The newly grown resistant callus was transferred to the 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, pH5.8). Pick the paddy rice callus that appears green bud and move into the triangular flask that rooting medium (MS/2+30g/L sucrose+250mg/L cephalosporin+50mg/L hygromycin) is housed, put into constant temperature incubator 28 ℃ light culture 15d. Ready to transplant. First, the positive transgenic plants were initially 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 screened for the first time were further screened .

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

14d 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 (Table 2) were designed using JcMYB16 gene cDNA, and the expression of JcMYB16 in wild-type and transgenic lines was detected by qRT-PCR.

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

After germination of wild-type and JcMYB16 transgenic rice plants, the seedlings were planted in the rice international Yoshida nutrient solution, and phenotype analysis was performed 12 days later.

2.0 Experimental Analysis of Drought Stress in JcMYB16 Gene Transgenic Rice

After germination of wild-type and JcMYB16 transgenic rice plants, 14d seedlings with consistent growth were selected for salt stress treatment experiment. The method is as follows: the wild-type and transgenic plant seedlings grown for 14 days were put into Yoshida nutrient solution containing 300mM mannitol, and phenotype analysis was carried out after 6 days; Type analysis and statistical survival rate. The aboveground leaves of the drought stress treatment for 3 days were selected 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 232bp, which includes a complete reading frame sequence length of 903bp, and the sequence is as follows SEQ ID NO.1;

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 new drought stress-related gene with unknown function, and no homologous gene with known function has been found.

Example 2

Obtaining a transgenic line of Arabidopsis thaliana and analyzing its phenotype and drought stress

1 Materials and methods

1.1 Plant materials and planting methods

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

Drought stress analysis experiment of overexpression plants

1.2 Strains, vectors and reagents

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

This experiment mainly uses seedling drought stress, seedling 150mM NaCl stress, 300mM mannitol (Man) stress to test the stress resistance of each transgenic line. The specific steps are as follows: the sterilized Arabidopsis seeds were treated at low temperature for 2 days, sowed in the medium of 1/2MS+1% sucrose for germination, and cultured vertically for 4 days. Transplant to the corresponding stress medium (basic medium is still 1/2MS) and culture vertically for 5-7 days. Observe and record the growth and survival rate.

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

For RNA extraction, a plant RNA extraction kit from Magen was used, and the reference method was a small amount of RNA extraction from plant tissues, which is relatively easy to extract. Using 1 μg of RNA as a template, the first-strand cDNA was synthesized according to the operating instructions of the cDNA synthesis kit (TaKaRa). Specific primers were designed according to the full-length sequence of OsHT1 gene cDNA, and the sequences of specific primers are shown in Table 3. RT-PCR reaction system (20 μL): 2 μL of 10×PCR reaction buffer, 1 μL of dNTP (2.5 mmol/L), 1 μL of each primer (10 pm/μL), 0.2 μL of Taq polymerase (5U/μL), 2 μL of template cDNA, 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, 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 JcMYB16 gene plant expression vector construction

Extract the plasmid cloned correctly for sequencing, and perform double digestion with the added restriction site, as follows: double restriction with Kpn I and SalI. 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 product was directly transformed into DH5α competent, and Kan screening was used to pick out resistant clones for bacterial liquid PCR detection. The positive clones were expanded and cultured to extract plasmids. Double-enzyme digestion verification of the extracted plasmid: In addition to selecting the restriction sites added by PCR, the existing restriction sites EcoR I and Hind III in the vector were also selected for combined restriction restriction verification to determine the direction of connection into the expression vector is it right or not. The correctly connected plasmids were transformed into competent Agrobacterium GV3101, and resistant clones were picked one day later for bacterial liquid PCR detection. Plasmids were extracted from clones that were positive for detection, and then verified by rotation, that is, they were transformed into E. coli to extract plasmids for double enzyme digestion verification. Correct clones detected in each step will be used for the next step of transformation and stored at -80°C for future use.

1.5 JcMYB16 plant expression vector transgene into Arabidopsis

The method of transforming Arabidopsis thaliana in this experiment is the pollen tube passage method, also known as the inflorescence dipping method. The specific steps are as follows: select the Arabidopsis plants (about 4 weeks) that are about to bloom, remove the flower buds that have opened, and infect them the night before. Water well and set aside. The Agrobacterium containing the target vector was shaken to OD ₆₀₀ =1.8, and the bacteria were collected by centrifugation at 5000g for 5 minutes. Add infiltration medium (1/2MS without vitamins + 1ml 1000×Gamborg's Vitamins (inositol 100mg/mL, niacin 1mg/mL, B6 1mg/mL, B110mg/mL) + 6-BA 10μg/mL, pH 5.7) The Agrobacterium was resuspended at a ratio of 1:1, and the surfactant Silwet was added to make the final concentration 0.02%. After the preparation is completed, carry out dip-dyeing, and the dip-dyeing time is 2 minutes, during which the dipping solution is constantly stirred to fully combine with the inflorescences. After dipping and drying for a short time, the plants were placed in a dark box for dark cultivation for 24 hours. During this period, newspapers were used for shading and the dark box was kept at a certain humidity. After the dark cultivation, continue the normal cultivation, collect the seeds for screening.

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

The harvested Arabidopsis seeds were sterilized, 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 well-developed root systems. These positive plants are the T1 generation strains, and each single plant is a strain, and the seeds are numbered and collected separately. The seeds of the T1 generation strains were sterilized and screened with hygromycin, and the segregation ratio was counted. Select lines with a segregation ratio of about 3:1 (positive plants: negative plants) for transplantation, and each line has 12 individual plants (T2 generation). Then the T2 generation single plants were harvested, and then disinfected and screened with hygromycin. Observe whether the seeds of the individual plants of the T2 generation segregate, and the individual plants that no longer segregate are homozygous plants. The seeds are harvested separately, preserved, and sowed to obtain the seeds of the T3 generation plants. The T3 generation plants obtained in this way have more seeds and are used for subsequent phenotypic analysis.

The fourth leaf of T3 generation plants grown for 20 days was taken for RNA extraction. The extraction method is Trizol method. The concentration and integrity of the extracted RNA were detected by Nanodrop2000 spectrophotometer and agarose gel electrophoresis. RNA with good quality was used for reverse transcription, the amount of reverse-transcribed RNA was 2 μg, and the kit was purchased from Premega. 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 three overexpressed lines were selected for subsequent phenotypic analysis.

1.7 Experimental analysis of drought stress in JcMYB16 transgenic plants

The drought stress treatment conditions were as follows: firstly, the wild-type and JcMYB16 transgenic Arabidopsis seeds were sterilized, and then the Arabidopsis was seeded in a square sterile petri dish containing 1/2 MS medium, cultured vertically for 4 days, 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 to continue to grow vertically for 7 days, and the phenotype analysis, conductivity, proline and MDA content detection were performed. In addition, for the drought stress in the nutrient soil, the Arabidopsis thaliana stopped watering for 3 weeks, observed the phenotype after 2 weeks, and then rewatered for 7 days to count the survival rate.

1.8 RNA extraction and qRT-PCR

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

Table 4 Primer sequences for quantitative qRT-PCR

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

For the first time, the function of the Jatropha MYB family gene JcMYB16 in plant response to drought stress was cloned and studied, that is, the present invention can provide a gene capable of increasing plant drought resistance. First, the tissue-specific expression analysis of the JcMYB16 gene was carried out, and the qRT-PCR results showed that the JcMYB16 gene was highly expressed in Jatropha Jatropha roots; then the JcMYB16 gene was constructed into the plant expression vector pCAMBIA1301 driven by the 35S promoter, and the recombinant vector pCAMBIA1301- JcMYB16, transfer the recombinant vector into Agrobacterium for further transformation. Using rice callus and Arabidopsis thaliana as receptors, the constructed vectors were transformed into rice calli and Arabidopsis thaliana by the Agrobacterium-mediated method to obtain JcMYB16 transgenic homozygous lines; then JcMYB16 transgenic lines Lines were subjected to phenotypic and drought stress analysis. Phenotypic analysis showed that overexpression of JcMYB16 gene did not affect plant growth and development. Analysis of drought stress experiments showed that overexpression of JcMYB16 gene increased the drought stress resistance of transgenic rice and Arabidopsis. In addition, we have planted 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 regulatory gene for drought stress in rice and Arabidopsis thaliana, indicating that genetic engineering technology can be used to improve the resistance of rice and Jatropha to drought stress. Breeding new varieties of drought-tolerant rice or jatropha to promote the large-scale planting of rice or jatropha has very important application value. The gene can be used for the cultivation of drought-resistant varieties of cereal crops such as jatropha, rice, and wheat, and can also be applied to the cultivation of drought-tolerant varieties of crops such as barley, sorghum, corn, Arabidopsis, tomato, tobacco, soybean, and potato. The protein encoded by JcMYB16, or the functionally similar protein in other species, its amino acid sequence 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-tolerant is to increase the drought-resistant ability 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 modification or partial replacement departing from the spirit and scope of the present invention shall fall within the scope of the claims of the present invention.

SEQUENCE LISTING

<110> Zhoukou Teachers College

<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 ttatggagg 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 atttggtac tttttttttg 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 aattttgca 2160

gctaaaccat gtatatcacc tatgagcagt tgtcggtggc attggatatg ccataggacc 2220

catgttcatc at 2232

<210> 2

<211> 903

<212> DNA

<213> Nucleotide sequence of the open reading frame of the gene

<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 caggttatt 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 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 (2)

1. The application of the Jatropha Jatropha MYB class transcription factor JcMYB16 gene in improving the drought resistance of plants, characterized in that: the plant is a monocotyledonous plant or a dicotyledonous plant, and the nucleotide sequence of the JcMYB16 gene is SEQ ID NO.1. 2. the application of Jatropha MYB class transcription factor JcMYB16 gene as claimed in claim 1 in improving plant drought resistance, it is characterized in that: described monocot is rice, and described dicotyledon is Jatropha, Arabidopsis, The nucleotide sequence of the JcMYB16 gene is SEQ ID NO.1.

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