CN106497935A - Overexpression GhJAZ1 genes amplification Genes For Plant Tolerance low temperature stress - Google Patents

Abstract

The invention belongs to field of plant genetic.Specifically related to overexpression GhJAZ1 genes amplifications Genes For Plant Tolerance low temperature stress.From Cotton Gossypii, clone obtains JAZ (JASMONATE-ZIM DOMAIN) family protein gene GhJAZ1, the cDNA sequence such as SEQ ID NO of the GhJAZ1 genes：Shown in 1, its protein sequence such as SEQ ID NO for encoding：Shown in 2.The present invention builds the overexpression vector of the gene, obtains transfer-gen plant by genetic transforming method, and checking shows that the gene of present invention clone can improve toleration of the Cotton Gossypii to low temperature stress.

Description

Overexpression of GhJAZ1 gene enhances plant resistance to low temperature stress

technical field

The invention belongs to the technical field of plant genetic engineering. It specifically relates to the application of a GhJAZ1 gene in enhancing plant resistance to low temperature stress. The JAZ (JASMONATE-ZIM DOMAIN) family protein gene GhJAZ1 cloned from cotton, functional verification showed that overexpression of GhJAZ1 gene can improve the tolerance of cotton to low temperature stress. The cloned GhJAZ1 gene of the present invention can be applied to enhance the tolerance of plants to low temperature stress through genetic transformation.

Background technique

Low temperature stress includes cold stress (0-20°C) and freezing stress (<0°C), which will seriously affect the growth and yield of plants, and even lead to plant death in severe cases. At the same time, low temperature is also an important factor that limits the spatial distribution of plants. should affect the factor. When plants are in a cold stress environment, the normal metabolism of plants is blocked. In addition, cold stress can also induce plants to produce osmotic stress, increased active oxygen and other stress responses (Chinnusamy et al.2007). Some temperature-loving crops, such as corn, tomato, soybean, cotton, banana, etc., will cause serious damage if they grow in an environment lower than 10-15 degrees Celsius, showing symptoms such as leaf wilting, yellowing, and necrosis. Low temperature can also seriously affect the reproductive growth of plants. For example, if rice is exposed to low temperature stress during the flowering stage, it will lead to flower abortion (Wen et al. 2002).

Cotton is native to the tropics and subtropics, and is a temperature-loving crop. Each growth period has certain temperature requirements. The lowest temperature that cotton can withstand during seed germination and seedling growth is 12°C and 15.5°C respectively; budding requirements The lowest temperature is 19-20°C; if the ambient temperature is lower than 21°C at the flowering and boll stage, reducing sugars cannot be transformed into cellulose, which will seriously affect fiber development. The optimum temperature for the elongation of cotton pollen tubes is 28-32°C. Once the temperature is lower than 19°C or higher than 45°C, the pollen tubes cannot elongate at all (Kakani et al.2005). The most suitable temperature for the growth of cotton in the whole growth period is 20-30 ℃, if it is lower than this temperature range, cotton will not grow normally. However, with the change of cotton planting methods in my country, more and more cotton planting provinces and regions adopt direct seeding cotton planting methods. Under this cotton planting method, the probability of cotton seedlings encountering low temperature increases, which has caused serious damage to cotton planting in my country. Serious impact. For example, Xinjiang, my country's largest cotton-growing province and high-quality commercial cotton base, often encounters low-temperature weather disasters such as cold waves during the period from sowing to seedling growth. The surface temperature is far lower than the minimum growth temperature of cotton seedlings, even below 0°C, seriously affecting plants Growth and development and yield formation (Xin Huihui et al.2014). Therefore, cultivating high-yielding, multi-resistant cotton varieties to effectively cope with various adversities in the process of cotton growth and development is an important task of current cotton breeding.

Jasmonic acid and its derivatives (JAs) are endogenous growth regulator substances in plants, which play a vital role in maintaining plant development, growth, stress resistance and other biological processes. In the model plant Arabidopsis thaliana, JAs affect the development of stamens and villi, vegetative growth, cell cycle regulation, senescence, in addition to affecting the response of plants to external biotic and abiotic stresses. JAZs (JASMONATE ZIM-DOMAIN Protein) is an important regulator of the jasmonic acid signaling pathway and regulates the jasmonic acid signaling pathway. Under normal circumstances, the content of jasmonic acid in plants is very low, and JAZs binds to transcription factors (such as MYC2) to inhibit the jasmonic acid signaling pathway; when plants are stressed by biotic and abiotic stresses, the content of jasmonic acid in plants increases significantly, In the presence of JA-Ile, JAZ protein binds to COI1, and then SCF ^COI1 is recognized by E3 ubiquitin ligase to ubiquitinate JAZ protein, and finally it enters the 26s protease degradation pathway for degradation, and the transcription factors inhibited by JAZs are degraded release, thereby initiating the jasmonate-responsive gene response (Pauwels and Goossens 2011).

JAZs can also combine with regulators in other metabolic pathways, so that the jasmonic acid metabolic pathway and other metabolic pathways cooperate and regulate each other, thus playing a vital role in biological processes such as plant development, growth, and stress resistance (Sun Cheng 2013). Arabidopsis JAZ proteins (JAZ1, JAZ8 and JAZ11) can interact with MYB21 and MYB24 to inhibit the transcriptional function of MYB21 and MYB24, thereby inhibiting the anther development process regulated by jasmonic acid and affecting plant fertility (Song et al.2011). DELLAs proteins (RGA, GAI, RGL, RGL1, RGL2) are inhibitory regulators of the gibberellin signaling pathway, and JAZ1 can interact with the DELLA protein RGA in vivo, interfering with the interaction between DELLAs and PIF3 transcription factors to regulate gibberellin signaling Similarly, DELLA protein RGA can compete with MYC2 to bind JAZ to regulate the jasmonic acid signaling pathway (Hou et al.2010).

Soybean JAZ family protein GsJAZ2 is a nuclear localized protein that is induced by salt, alkali, low temperature and drought. Overexpression of GsJAZ2 in Arabidopsis can enhance the tolerance of transgenic plants to salt and alkali, and it is a marker gene for some stress responses It was found that their expression levels were significantly enhanced in overexpression materials (Zhu et al.2012). Jasmonic acid can enhance the tolerance of Arabidopsis to low temperature by regulating the ICE-CBFs signaling pathway, JAZ1 and JAZ4 can interact with ICE1 protein to inhibit the transcriptional function of ICE1, overexpression of JAZ1 or JAZ4 inhibits the tolerance of Arabidopsis to freezing injury Receptivity (Hu et al. 2013). Studies on Arabidopsis TIFY10s (AtTIFY10a and AtTIFY10b) mutants found that under alkaline environmental stress, the mutants exhibited lower germination rates than the wild type. Overexpression of soybean gene GsTIFY10a in alfalfa can enhance the tolerance of transgenic plants to alkali stress, mainly by promoting plant growth, increasing malate dehydrogenase activity, citric acid content and proline content, and reducing malondialdehyde content. The detection of transcript levels of some stress-responsive genes found that malate dehydrogenase (NADP-ME), pyrophosphatase (H ⁺ -PPase), and carboxylic acid synthase (P5CS) synthetic genes were up-regulated in GsTIFY10a transgenic plants. In addition, overexpression of GsTIFY10a in alfalfa can also increase the content of endogenous jasmonic acid (Dan Zhu et al.2014).

Contents of the invention

Based on the comparison of homologous sequences of Arabidopsis thaliana and the screening of differential expression in cotton under adversity, the present invention isolates and clones a cotton JAZ family gene involved in the regulation of the jasmonic acid signaling pathway. We name this gene GhJAZ1 gene, and transform upland cotton YZ1 , to obtain transgenic upland cotton to verify its function in cotton resistance to abiotic stress, to provide a theoretical basis for how jasmonic acid signaling participates in plant resistance to abiotic stress, and to provide a reference for cotton to increase yield under stress.

Technical scheme of the present invention is as follows:

(1) Through the STIFDB (Stress Responsive Transcription Factor Database) database and Genevestigator technology, the regulatory factors of Arabidopsis thaliana that may be involved in the response to abiotic stress stress were screened out. Source cotton EST sequence. The upland cotton YZ1 seedlings were subjected to stress treatments such as salt, cold, and ABA, and the RNA of roots and leaves was extracted. Real-time PCR technology was used to analyze the expression of the selected cotton genes. According to the induced expression of the genes, the purpose of the research was finally determined. Gene.

The present invention clones a functional gene GhJAZ1 related to cotton stress resistance from upland cotton, and its cDNA sequence is the sequence shown in the 1st-792th base in the sequence table SEQ NO: 1, wherein the sequence 1-792th base The sequence shown in the base is the coding region of the gene, and the encoded protein sequence is shown in SEQ NO:2. Analysis of the gene structure shows that GhJAZ1 contains 4 introns, and its ORF is 792bp long, encoding a 263 amino acid JAZ family protein. Homologous comparison analysis of amino acid sequences revealed that GhJAZ1 has classic ZIM domain and Jas domain, and is most homologous to Arabidopsis JAZ1 (TIFY10a), with a homology degree of 46%. Using ClustalX 1.83 and MEGA5.2 software to analyze the similarity between GhJAZ1 gene protein sequence and 12 AtJAZ protein sequences, GhJAZ1 was clustered together with AtJAZ1 and AtJAZ2 (see Figure 1).

The isolated gene GhJAZ1 of the present invention is derived from cotton, and there is no report on the influence of the gene on adversity stress, hormones and cotton growth and development. Through different stress treatments and hormone treatments of wild-type cotton YZ1 (that is, Yuzao No. 1 upland cotton), the expression of the gene was detected, and it was found that the gene was affected by PEG-simulated drought, salt (NaCl), low temperature (4°C), and abscission. Acid (ABA), jasmonic acid (JA) and active oxygen (H ₂ O ₂ ) treatment induced expression (see Figure 2). The present invention respectively constructs the overexpression vector pK2GW7.0 of the gene and the interference expression vector pHELLSGATE4 (see Fig. 3D in Fig. 3, the plasmid vector is also called pHellsgate4-JAZ1, and its construction process is shown in Fig. 3), we will use this The two vectors were respectively transformed into Yuzao No. 1 upland cotton (YZ1) to obtain overexpression and interference transgenic positive lines of the gene. The expression levels were detected and two overexpression pure lines with appropriate expression levels were selected (numbered as J39, J92) and 2 pure lines of interference (numbered JR3, JR3) and followed up verification tests, the test results are shown in Figure 4.

Through the expression analysis of GhJAZ1 under low temperature treatment, we believe that GhJAZ1 gene may play a certain role in cotton response to low temperature stress. First, we used the method of hydroponics to identify phenotypes under low temperature stress. When the seedlings grew to the stage of two leaves and one heart, they were treated with low temperature at 4°C. It was found that the plant phenotypes of different materials had changed greatly after 3 hours of treatment. The main manifestation is that the leaves of the two interfering lines have been severely wilted, and the leaves of the control wild-type YZ1 also began to wilt, but the plants of the two overexpression lines did not show obvious phenotypes. After 15 hours of treatment, the leaves of the overexpression plants also began to wilt, while the intervention plants and the control wild-type YZ1 were completely wilted during the same period. After 25 hours of treatment, the treated materials were restored to normal temperature. After 50 hours of restoration, it was found that the intervention and the control had more severe leaf deadness, but the overexpression plants had less dead leaf area (see Figure 5A).

In order to more realistically simulate the impact of low temperature on cotton plants under natural conditions, we used an artificial climate chamber to simulate cold stress (8°C), and planted cotton seedlings in soil filled with nutrients (seedling substrate: vermiculite=3: 1) In the nutrient pot, cold stress treatment was carried out when the cotton seedlings grew to two leaves and one heart. Two days after the treatment, we found that most of the plants of the interference line and the control wild-type YZ1 showed wilting leaves, and only a few plants of the overexpression material showed slight wilting of leaves. Then we carried out the room temperature recovery experiment on the treated materials. After recovery for one day, we found that most of the plants of the interference line and the control wild-type YZ1 showed severe leaf death, which was the same as the result of the previous hydroponic treatment experiment (see Figure 5B). After the treatment, the relative conductivity and chlorophyll content of the leaves were measured, and it was found that the leaves of the overexpression material had a lower ion exudation rate and higher chlorophyll content than the interference and control lines, which indicated that the leaves of the overexpression GhJAZ1 material were less damaged , showing a stronger tolerance to low temperature (see Figure 5C).

Concrete operation steps of the present invention are as follows:

1) Using the cDNA of wild-type cotton YZ1 leaves as a template, design primers to amplify the full length of the GhJAZ1 gene to obtain the target DNA fragment. The nucleotide sequence and CDS sequence of the full length are shown in SEQ ID NO: 1 (1-792bp), The primer sequences for amplifying the gene are as follows:

GhJAZ1-F: 5'-GAGCAAATAGTTGGGATTCTGG-3'

GhJAZ1-R: 5'-AACTCGGCTGGGACTACTAC-3'

2) According to the obtained gene sequence, design primers with BP-LR reaction adapters for constructing expression vectors, and obtain PCR products containing complete CDS with BP-LR reaction adapter bases at both ends through PCR amplification. The PCR product was connected to the pDONR ^TM 221 vector by BP reaction (BP enzyme was purchased from Invitrogen, USA, and reacted at room temperature for 4 hours, see Figure 3C, and the pDONR ^TM 221 vector was obtained from CSIRO Plant Industry, Australia), and the GhJAZ1 The gene was connected to the plant expression vector pK2GW7.0 (LR enzyme was purchased from Invitrogen, the United States, and reacted at room temperature for 4 hours, Figure 3A, and the vector pK2GW7.0 was from Ghent University, Belgium) (pK2GW7.0-GhJAZ1, Figure 3B, provided by the application self-prepared, Wuhan). At the same time, primers were designed for non-conserved region interference, and the selected interference fragment was connected to the interference carrier pHELLSGATE 4 (gifted by CSIRO Plant Industry, Australia) through BP reaction, and then transformed into E. coli competent cell TOP10 by heat shock. The positive monoclonal extracted plasmids were detected by single enzyme digestion with XhoI and XbaI respectively, so as to obtain the GhJAZ1 interference expression vector (pHELLSGATE 4-GhJAZ1, prepared by the applicant) in the correct connection direction. Using the Agrobacterium-mediated transgenic method, the vector pHELLSGATE 4-GhJAZ1 was transformed into the hypocotyl of upland cotton YZ1 (from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences, Anyang, Henan), and the overexpression and interference were obtained through tissue culture. Transgenic plants expressing GhJAZ1.

The primer sequences used are as follows:

OE-GhJAZ1-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCGAGCAAATAGTTGGGATTCTGG-3'

OE-GhJAZ1-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAACTCGGCTGGGACTACTAC-3'

RNAi-GhJAZ1-F: 5'-GGGGACAAGTTTGTACAAAAAAAGCAGGCTTGGGTTCTTCAGTGGAGCCT-3'

RNAi-GhJAZ1-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGATGCTGTGGAGTTACTTATCTGGT-3'

M13-F: 5'-GGGGACAAGTTTGTACAAAAAAAGCAGGCTTCCAAAAATGATGATCGGAGA-3'

35S-F: 5'-CCACTATCCTTCGCAAGACCCT-3'

Further specific steps are described below:

1) Obtain transgene-positive homozygous plants by means of kanamycin screening, copy number identification and segregation ratio statistics, and detect the expression level of the transgenic plants by RT-PCR.

2) Treat wild-type cotton variety YZ1 seedlings at the two-leaf one-heart stage with PEG-simulated drought, salt (NaCl), low temperature (4°C), abscisic acid (ABA), jasmonic acid (JA) and active oxygen (H2O2), and detect Responses of GhJAZ1 to different stress treatments.

3) GhJAZ1 overexpression and interference plants were cultured in hydroponics and vegetative pots at the seedling stage to identify low temperature phenotypes, observe their phenotypes, and measure the expression of related indicators and genes under low temperature stress treatment.

Advantages of the invention

Cotton is an important economic crop. During its growth and development, it is often subjected to various adverse environmental stresses, such as drought, high salinity, low temperature, etc. Among these factors, low temperature is one of the important factors affecting the normal growth and development of cotton. The optimum temperature for the growth of cotton in the whole growth period is 20-30°C, if it is lower than this range, the cotton will not grow normally. With the change of cotton planting methods in my country, more and more cotton planting provinces and regions adopt the direct cotton planting method. Under this cotton planting method, the probability of cotton seedlings encountering low temperature increases, which has caused serious damage to cotton planting in my country. influences. At present, there is no research report on the molecular mechanism of cotton response to low temperature stress. We hope that through this study, we will aim to construct a gene regulatory network that responds to low temperature in plants, and further enrich the molecular mechanism that regulates cotton cold resistance.

Using STIFDB (Stress Responsive Transcription Factor Database) database and Genevestigator technology to screen Arabidopsis abiotic stress-responsive genes, and then Blast cotton EST database to find target genes, the feasibility and scientific feasibility of this research strategy has been confirmed (Zhu et al. al., 2010).

The GhJAZ1 gene cloned by the present invention utilizes the STIFDB (Stress Responsive Transcription Factor Database) database and the Genevestigator technology to screen Arabidopsis thaliana by abiotic stress response genes, and then the target gene found in the Blast cotton EST database. This screening target gene research The feasibility and scientificity of the strategy have been proven. Jasmonic acid (JA), as a growth regulator in plants, is widely involved in adversity stress and other stress responses. Arabidopsis, rice, banana, wheat and other crops have reported that jasmonic acid participates in the response of plants to low temperature stress, but there are few studies on jasmonic acid in cotton in terms of abiotic stress. Arabidopsis JAZ1 is a negative regulator on the jasmonic acid signaling pathway, inhibiting the transmission of jasmonic acid signaling. Cotton GhJAZ1 is the most homologous gene to Arabidopsis JAZ1, and it is likely to be involved in the jasmonic acid signaling pathway. Overexpressing the GhJAZ1 gene in cotton can effectively improve the tolerance of transgenic plants at the seedling stage to low temperature stress, so the genetic engineering technology can be used to improve the ability of crops to withstand low temperature stress.

Description of drawings

Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of the isolated and cloned GhJAZ1 gene of the present invention (the sequence length is 792bp), and it is also the coding region of the gene (CDS, the sequence shown by 1--792 bases), encoding 263 amino acids.

Sequence listing SEQ ID NO: 2 is the protein sequence encoded by the isolated and cloned GhJAZ1 gene of the present invention.

Figure 1: is the result of the cluster analysis of the amino acid sequence encoded by GhJAZ1 and the Arabidopsis JAZ family members in the Tair database using ClustalX software and MEGA5.2 software (publicly available software). Cluster analysis showed that the amino acid sequence encoded by GhJAZ1 had the highest homology with those encoded by AtJAZ1 and AtJAZ2 in Arabidopsis.

Figure 2: Analysis of the expression pattern of the GhJAZ1 gene in response to stress treatment using Real-time PCR. Description of reference numerals: Fig. 2A is the expression analysis of GhJAZ1 in leaves and roots under 15% PEG treatment (wherein: the left figure in Fig. 2A is the expression situation of GhJAZ1 in leaves, and the right figure in Fig. 2A is GhJAZ1 in roots 2B is the expression analysis of GhJAZ1 in leaves and roots under 200mM NaCl treatment (wherein: the left figure in Figure 2B is the expression of GhJAZ1 in leaves, and the right figure in Figure 2B is the expression of GhJAZ1 in roots expression); Figure 2C is the expression analysis of GhJAZ1 in leaves and roots under 4°C cold treatment (wherein: the left figure in Figure 2C is the expression of GhJAZ1 in leaves, and the right figure in Figure 2C is the expression of GhJAZ1 in roots situation); Figure 2D is the expression analysis of GhJAZ1 in leaves and roots treated with abscisic acid (ABA) (wherein: the left figure in Figure 2D is the expression of GhJAZ1 in leaves, and the right figure in Figure 2D is the expression of GhJAZ1 in roots 2E is 100uM hydrogen peroxide (H2O2) and jasmonic acid (MeJA) the expression analysis of GhJAZ1 in the leaf under the treatment (wherein: the left picture in Fig. 2E is GhJAZ1 in 100uM hydrogen peroxide (H2O2) treatment cotton The expression situation in the leaves of the seedlings, the right panel in Fig. 2E is the expression situation of GhJAZ1 in the leaves of cotton seedlings treated with jasmonic acid (MeJA)). The results of expression analysis showed that the expression of GhJAZ1 gene was up-regulated induced by PEG, NaCl, ABA, H2O2 and MeJA, and its expression was first down-regulated and then up-regulated by 4°C low temperature treatment.

Figure 3: The relevant vector diagrams used in the construction of overexpression vectors and interference expression vectors. Explanation of reference numerals: FIG. 3A is a schematic diagram of the pK2GW7.0 vector used in the overexpression vector constructed in the present invention. Fig. 3B is a schematic diagram of the constructed target gene overexpression vector pK2GW7.0-GhJAZ1. Fig. 3C is a schematic diagram of the intermediate vector pDONR ^TM 221 in the BP reaction. Fig. 3D is a schematic diagram of the pHELLSGATE4 (or called pHellsgate4-JAZ1) vector used in the interfering expression vector constructed in the present invention.

Figure 4: Analysis of GhJAZ1 expression in leaves of overexpression and interference expression GhJAZ1 transgenic lines detected by RT-PCR method. Explanation of reference numerals: the upper swimming lane of Figure 4 is the detection of GhJAZ1 gene expression, the lower swimming lane of Figure 4 is the detection of the expression of cotton internal reference gene UB7; J39 and J92 are GhJAZ1 overexpression materials, YZ1 is the control material, JR3 and JR13 It is the GhJAZ1 interference strain.

Figure 5: Overexpression of GhJAZ1 gene can improve the tolerance of seedling upland cotton to low temperature stress. Explanation of reference numerals: Fig. 5A is the phenotypic identification of transgenic lines treated at 4°C at the seedling stage. The growth environment of the seedlings is the commonly used Hoagland nutrient solution. The pictures were taken after 25 hours of treatment and 5 days after recovery. From the left, the first two are the overexpression lines J39 and J92, the third is the transgenic receptor YZ1 (ie, the control, CK), and the last two are the interference expression lines JR3 and J92. JR13. Figure 5B shows the phenotype of large transgenic cotton seedlings treated at 8°C for 25 days. The growth environment of the seedlings is cultivated in conventional nutrient soil. Phenotype, the first two from the left are the overexpression lines J39 and J92, the third is the transgenic receptor YZ1 (ie, the control, CK), and the last two are the interference expression lines JR3 and JR13. Fig. 5C is the measurement results of relative ion exudation rate and total chlorophyll content after two days of low temperature treatment at 8°C and recovery for one day.

detailed description

The following examples define the present invention and describe the methods of cloning a DNA fragment containing the complete coding segment of the GhJAZ1 gene and verifying the function of the GhJAZ1 gene. From the following descriptions and these examples, those skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention so that it can be applied to different uses and conditions.

Example 1 Isolation, cloning and expression pattern analysis of GhJAZ1 gene

A. RNA extraction and cDNA acquisition

The ORF, protein sequence and conserved domain of GhJAZ1 gene were obtained from NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Total RNA was extracted from Yuzao No. 1 upland cotton (YZ1) plant leaves, and the method of extracting total RNA was referred to the method reported by Zhu et al (2005). 2ug RNA samples were taken, and reverse transcriptase Superscript III (purchased from Invitrogen Company, U.S.) reverse-transcribed it to synthesize cDNA, and the reaction conditions were: 65° C. for 5 minutes, 50° C. for 60 minutes, and 70° C. for 10 minutes.

B. Obtaining the full-length sequence of GhJAZ1 gene

According to the ORF sequence of the GhJAZ1 gene obtained from the query, the primers GhJAZ1-F (5'-GAGCAAATAGTTGGGATTCTGG-3') and GhJAZ1-R (5'-AACTCGGCTGGGACTACTAC-3') were designed to amplify the gene, and PCR was performed using the cDNA of cotton YZ1 leaves as a template Amplification, the amplified PCR product was connected to the pGEM-T vector (purchased from Promega, the United States), positive clones were screened and sequenced, and the PCR reaction conditions were: 94°C pre-denaturation for 5min; 94°C for 30sec, 57°C for 30sec, 72°C for 1min, 28 cycles; 72°C for 5min. Its CDS sequence is the sequence shown in 1-792bp in SEQ ID NO:1, the amino acid of the expressed protein is shown in the sequence corresponding to 1-792 bases in SEQ ID NO:1, and the protein sequence is shown in SEQ ID NO:2 sequence shown.

C. Tissue expression analysis and stress/hormone induced expression analysis of GhJAZ1 gene

Using Yuzao No. 1 upland cotton (YZ1) as material, extract PEG, NaCl, ABA and the RNA of each sample of root and leaf at different time points of cold treatment (extraction method refers to the above-mentioned method of the present invention), the selected PEG, NaCl and 4°C The time points of cold treatment are 0h, 1h, 4h, 8h, the time points of ABA treatment are 1h, 2h and 4h, the time points of H2O2 treatment are 0h, 1h, 3h and 5h, and the time points of MeJA treatment are 0h, 3h and 12h , select the corresponding time point with normal growth YZ1 as parallel control. cDNA was synthesized by reverse transcription using reverse transcriptase Superscript III (purchased from Invitrogen, USA). The reaction conditions were: 65°C for 5 minutes, 50°C for 60 minutes, and 70°C for 10 minutes. Using the cDNA synthesized by inversion above as a template, Real-time PCR was used to detect the expression level of GhJAZ1. The primers used were: GhJAZ1-QRT-F: (5'-ATCTGCTCAGCGACCCATTCC-3') and GhJAZ1-QRT-R: 5'- (GAGATTGAGCAGCCAAACCGA-3'). At the same time, the primers GhUbiquitin7-F: (5'-GAAGGCATTCCACCTGACCAAC-3') and GhUbiquitin7-R: (5'-CTTGACCTTCTTCTTCTTGTGCTTG-3') were used for specific amplification of cotton genes, which were used as internal controls for relative quantitative analysis. The model of quantitative PCR instrument was ABI7500, and the quantitative PCR reagents were purchased from Bio-Rad Company. The PCR reaction system (total volume 20 μl) includes: 10 μl diluted cDNA (equal to 20 ng initial total RNA), 10 μl 2×PCR Master Mix, 200 nM primers. The reaction conditions are: 95°C for 30 sec; 95°C for 5 sec, 58°C for 35 sec, 40 cycles. Real-time quantitative analysis by fluorescence detection was performed during the reaction. The results showed that the expression of the gene was induced by PEG-simulated drought, salt (NaCl), low temperature (4°C), abscisic acid (ABA), jasmonic acid (JA) and reactive oxygen species (H2O2) (see Figure 2).

Embodiment 2: Construction of GhJAZ1 gene plant overexpression vector and interference expression vector

A. Construction of overexpression vector

According to the obtained gene sequence, primers with BP-LR reaction adapters were designed to construct expression vectors. The primer sequences were OE-GhJAZ1-F (5'-GGGGACAAGTTTGTACAAAAAGCAGGCTGCGAGCAAATAGTTGGGATTCTGG-3') and OE-GhJAZ1-R (5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCAACTCGGCTGGGACTACTAC-3'), T-GhJAZ1 plasmid (purchased from Promega, USA) was used as a template for PCR amplification. 1 cycle; 72°C extension for 5 min, amplified by PCR to obtain a PCR product containing the complete ORF with linker bases of BP-LR reaction at both ends. The PCR product was ligated to the pDONR ^TM 221 vector by BP reaction (BP enzyme was purchased from Invitrogen, USA, and reacted at room temperature for 4 hours, the pDONR ^TM 221 vector map is shown in Figure 3C, and the pDONR ^TM 221 vector was obtained from CSIRO PlantIndustry, Australia) and then transformed into large intestine Bacillus competent cells TOP10, pick a single clone after 10-12 hours for positive PCR detection, the primers are M13-F universal primer (5'-GGGGACAAGTTTGTACAAAAAGCAGGCTTCCAAAAATGATGATCGGAGA-3') and OE-GhJAZ1-R (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAACTCGGCTGGGACTACTAC-3' ), positive monoclonal activation extracted plasmids. The PCR reaction conditions were: 94°C pre-denaturation for 5 minutes; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 28 cycles; 72°C for 5 min. Then use the LR reaction (Invitrogen) to connect the GhJAZ1 gene to the plant expression vector pK2GW7.0 (wherein: LR enzyme was purchased from Invitrogen Company, the United States; it was reacted at room temperature for 4 hours, and the vector map of pK2GW7.0 is shown in Figure 3A; the intermediate vector pK2GW7.0 was obtained from Ghent University, Belgium), using the reaction product to transform Escherichia coli competent cell TOP10. After 10-12 hours, pick a single clone for PCR positive detection, and use 35S-F (5'-CCACTATCCTTCGCAAGACCCT-3') and OE-GhJAZ1 as primers -R(5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAACTCGGCTGGGGACTACTAC-3'), the PCR reaction conditions were: 94°C pre-denaturation for 5 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 28 cycles; 72°C for 5 min. The positive single clone is activated to extract the plasmid, which is to obtain the overexpression plasmid pK2GW7.0-GhJAZ1 for transformation.

B. Construction of Interfering Expression Vector Plasmids

Primers were designed according to the obtained SEQ ID NO:1 sequence to construct the non-conserved region interference expression vector, and the linker bases of BP-LR reaction were added to both ends of the primers, and the primer sequences were RNAi-GhJAZ1-F(5'- GGGGACAAGTTTGTACAAAAAAAGCAGGCTTGGGTTCTTCAGTGGAGCCT-3') and RNAi-GhJAZ1-R (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGATGCTGTGGAGTTACTTATCTGGT-3'), using T-GhJAZ1 plasmid as a template for PCR amplification, the PCR reaction conditions were: 94°C pre-denaturation for 5min; 94°C for 30sec, 58°C 30sec, 72°C 30sec, 28 cycles; 72°C extension for 5min, amplified by PCR to obtain a PCR product with attB linkers at both ends. The GhJAZ1 interference fragment of the PCR product was connected to the plant expression vector pHELLSGATE 4 through BP reaction (among them: BP enzyme was purchased from Invitrogen, the United States; reacted at room temperature for 4 hours, and the vector map is shown in Figure 3D; the intermediate vector pHELLSGATE 4 was donated by CSIRO Plant Industry, Australia ), transform Escherichia coli competent cell TOP10 with reaction product. 35S-F (5'-CCACTATCCTTCGCAAGACCCT-3') and RNAi-GhJAZ1-R (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGATGCTGTGGAGTTACTTATCTGGT-3') were used to perform PCR detection on the single clones picked, and the positive single clones were activated to extract plasmids. The PCR reaction conditions were: 94°C pre-denaturation for 5 minutes; 94°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, 28 cycles; 72°C for 5 min. The positive plasmids were detected by single enzyme digestion with xbaI and xhoI respectively (both xbaI and xhoI were purchased from New England Biolabs, the United States, digested for 5 hours at 37°C), and the digested products were run on gel for detection, and the plasmid with the correct band pattern was determined to be constructed correctly The interference expression vector plasmid pHELLSGATE4-GhJAZ1 (that is, the pHellsgate4-JAZ1 vector plasmid shown in FIG. 3D ).

C. Vector transformation of Agrobacterium

Transform the constructed pK2GW7.0-GhJAZ1 vector and the interference expression vector pHELLSGATE4-GhJAZ1 into Agrobacterium strain EHA105, pick a single clone colony and inoculate it in LB liquid medium containing 100mg/L spectinomycin at 150rpm, shake at 28°C for 24h, Specific primers were used for positive detection of bacterial liquid respectively (pK2GW7.0-GhJAZ1 carrier detection primers are OE-GhJAZ1-F and OE-GhJAZ1-R (see Example 2 for the primer sequence), pHELLSGATE4-GhJAZ1 carrier detection primers are RNAi-GhJAZ1-F and RNAi-GhJAZ1-R, the primer sequences described in Example 2), positive bacterial liquid according to the bacterial liquid: fresh LB: 40% glycerol volume ratio of 3:3:2 was added to a 2mL centrifuge tube and mixed Mix well and store at -70°C. Cotton hypocotyls were then transformed by Agrobacterium-mediated transformation.

The formula of LB medium described above and below is: peptone 10g/L, yeast extract 5g/L, NaCl 5g/L; use 5mM NaOH to adjust the pH of the medium to 7.2; use distilled water to make up to 1L; Sterilize under high pressure steam at 125°C for 15-20min. LB solid medium needs to add 8g of agar per liter.

Genetic transformation and screening identification of embodiment 3GhJAZ1 gene

A. Preparation of Cotton Hypocotyls

The test material was Yuzao No. 1 upland cotton (YZ1). The plump and consistent YZ1 seeds were selected, the seed coat was peeled off, and sterilized with 0.1% mercury chloride solution for 10-12 minutes. During this period, the seeds were rinsed with sterile water three times. Seeds were placed on the surface of MS medium. After 1 day of dark culture at 30°C, seedlings were supported, and dark culture was continued for 4-5 days.

B. Activation of Agrobacterium

Take out the glycerol tube of the EHA105 strain containing the target gene (i.e. the cloned GhJAZ1 gene of the present invention) stored in the ultra-low temperature refrigerator and melt it on ice, add 10 μl to 2 ml of LB liquid containing 100 mg/L spectinomycin, and culture with shaking at 28°C 1 day, add 20ul of the activated bacteria solution and 15-20ml of fresh LB liquid containing 100mg/L spectinomycin, culture overnight at 28°C, absorb 1ml of the turbid bacteria solution and centrifuge at 8000-10000rpm for 30s in a 2ml sterile centrifuge tube Bacteria, resuspend the bacteria in 20ml of MGL medium containing 50mg/L acetosyringone (AS) (see the description below for specific components), shake and culture at 28°C for 30-40min, and use to infect hypocotyls.

Genetic Transformation of YZ1 Hypocotyl and Regeneration of Transgenic Plants

The method and procedure of Agrobacterium-mediated transformation of cotton hypocotyls refer to Jin Shuangxia, State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (Jin Shuangxia 2006, http://cdmd.cnki.com.cn/Article/

CDMD-10504-2006190162.htm) reported method. Specific steps are as follows:

(1) Take 15-20 hypocotyls of sterile seedlings each time and cut them into 0.5-0.8cm segments into 50ml sterile Erlenmeyer flasks, add activated EHA105 containing target vectors pK2GW7.0-GhJAZ1 and pHELLSGATE4-GhJAZ1 The Agrobacterium bacteria liquid was infected for 10 minutes, and shaken several times during the period;

(2) Pour off the bacteria solution, place the hypocotyls on sterile filter paper to blot the surface bacteria solution, put it on a clean bench and blow it for 10-15 minutes, and then insert it into the 2,4-D induction medium without antibiotics (specifically The ingredients are described later), co-cultivated at 19°C for 48-60h in the dark;

(3) After the end of the co-cultivation, the hypocotyl sections were inserted into the 2,4D induction medium containing kanamycin (100mg/L) and cephalosporin (100mg/L) (see the description below for the specific components), at 28 Cultivate under low light;

(4) After 3 weeks, transfer to induction medium containing antibiotic indolebutyric acid (IBA) (see the description below for specific components) and continue to subculture until embryogenic callus appears;

(5) The embryogenic callus is successively inserted into the embryonic differentiation medium (see the description below for the specific components) to be subcultured until the somatic embryo matures, and the mature cotyledon embryos are inserted into the rooting medium (see the description below for the specific components) to germinate, until a complete plant is obtained.

MGL medium used in this example: tryptone 5g/L, NaCl 5g/L, MgSO ₄ ·7H ₂ O 0.1g/L, KH ₂ PO ₄ 0.25g/L, mannitol 5g/L, glycine 1g/L L, make up to 1L with distilled water. 2,4-D induction medium: based on MS, add 2,4-D 0.1mg/L, cytokinin (KT) 0.1mg/L, glucose 30g/L, Phytagel 2.5g/L, use Make up to 1L with distilled water. Adjust the pH to 5.9.

IBA induction medium: MS-based medium, supplemented with IBA 0.5mg/L, KT 0.1mg/L, glucose 30g/L, Phytagel 2.5g/L, supplemented with distilled water to 1L. Adjust the pH to 5.9.

Embryo differentiation medium: based on MS, add 1.9g/L KNO ₃ , KT 0.1mg/L, glucose 30g/L, Gln 1.0g/L, Asn0.5g/L, Phytagel 2.5g/L, with Make up to 1L with distilled water. Adjust the pH to 5.9.

Rooting medium: take 1/2MS as the base medium, add glucose 15g/L, Phytagel 2.5g/L, supplement to 1L with distilled water. Adjust the pH to 5.9.

The basic MS medium formula described in the above medium formula is: macroelements (KNO ₃ 1.9g/L, NH ₄ NO ₃ 1.65g/L, KH ₂ PO ₄ 0.17g/L, MgSO ₄ 7H ₂ O 0.37 g/L, CaCl ₂ 2H ₂ O 0.44g/L), trace elements (KI 0.83mg/L, H ₃ BO ₃ 6.2mg/LMnSO ₄ 4H2O 22.3mg/L, ZnSO ₄ 7H ₂ O 8.6mg/L L, Na ₂ MoO ₄ 2H ₂ O 0.25mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl ₂ 0.025mg/L), iron salt (Na ₂ EDTA 37.3mg/L, FeSO ₄ 7H ₂ 0 27.8mg/L), organic components (inositol 100mg/L, Gly 2mg/L, VB ₁ 0.1mg/L, VB ₆ 0.5mg/L, VB ₅ 0.5mg/L).

C. Identification of transgenic plants

(1) Positive detection of transgenic plants and pure line detection

Genomic DNA was extracted from the young leaves of transgenic plants. The DNA was extracted using the Plant Genomic DNA Extraction Kit of Tiangen Biochemical (Beijing) Technology Co., Ltd. (see the instructions of the kit for specific operation steps), and the 35s promoter forward primer 35s-S (5'-CCACTATCCTTCGCAAGACCCT-3') and target gene GhJAZ1 reverse primer RNAi-GhJAZ1-R (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGATGCTGTGGAGTTACTTATCTGGT-3') were used to detect whether there was a corresponding T-DNA insertion by PCR. The PCR reaction conditions were: 94°C pre-denaturation for 5 minutes; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 28 cycles; 72°C for 5 min.

Peel off the seed coats of the harvested T ₁ generation seeds, sterilize with 0.1% mercuric chloride solution for 10-12min, shake constantly during this period, wash 3 times with sterile water, and place the seeds in cotton sterile seedling medium (containing 100mg/ L Kanamycin) surface. After 1 day of dark culture at 30°C, seedlings were supported, transferred to a light room (3000Lux, 15h light/9h dark) for culture, and observed for resistance segregation after 5-6 days (plants with long lateral roots were identified as positive transgenic plants). Afterwards, each generation of individual plants was reserved for inbreds for screening until no resistance segregation occurred, which was the transgenic pure line, which was used for the next step of phenotypic analysis and functional identification.

(2) Transgenic RNA level detection

The inverted leaf of the main stem of the T ₃ generation transgenic cotton plants was used to extract RNA, and the RNA was extracted by the modified guanidine isothiocyanate method in our laboratory (Zhu Longfu et al. 2005).

The cDNA was synthesized using 2 μg total RNA as a template, mixed with 1 μl 500 μg/ml oligo-dT(15) primer (purchased from Promega), and DEPC-water, with a total volume of 14 μl; then denatured at 70°C for 5 minutes and quenched on ice; Then add 10μl containing 5μl RT buffer, 1.25μl 10mM dNTP, 1.75μl DEPC-water, 1μl Mixed solution of Ribonuclease Inhibitor (purchased from Promega, USA) and 1 μl Superscript III reverse transcriptase (purchased from Invitrogen, USA); warm bath at 42°C for 1h to synthesize the first strand; after the reaction, treat at 70°C for 15min to reverse transcribe Superscript III Enzyme inactivation. Each cDNA was diluted to 200 μl and stored at -20°C until use. Using the above cDNA synthesized by reverse transcription as a template, specific PCR amplification (amplification The product is 203bp long). GhUbiquitin7 (GenBank accession number: DQ116441) gene was specifically amplified (amplified product 198bp in length), used as an internal reference for relative quantitative analysis. The total volume of the PCR reaction system is 20μl, cDNA template 1ul, 10×Taq enzyme reaction buffer 2μl, 25mM MgCL ₂ 1.2ul, 2mM dNTP 1.5ul, 10uM primer 0.2ul, 0.3 unit Taq enzyme 0.2μl, add ddH ₂ O to 20 μl. The reaction program was: denaturation at 94°C for 5 min, 34 cycles (GhJAZ1) or 28 cycles (GhUbiquitin7) at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and extension at 72°C for 5 min. 10 μl of the obtained PCR product was detected by 0.8% agarose gel electrophoresis.

The results show that the expression levels of the GhJAZ1 gene cloned in the present invention are different in different transgenic lines. In the follow-up functional verification study, the overexpression lines J39 and J92 and the interference lines JR3 and JR13 with appropriate expression levels were selected for analysis (see Figure 4).

Example 4: Functional verification of the GhJAZ1 gene using transgenic cotton

Specific steps are as follows:

A. Low temperature treatment experimental design

The identification of cotton low temperature resistance is carried out by using artificial climate chamber to simulate the low temperature environment, and the low temperature environment is set to 8°C. Select the transgenic material with stable expression and meet the requirements and the control, and select two stable expression lines (interference: JR3, JR13; overexpression: J39, J92) for the interference and overexpression materials, and select 8 lines for each experiment. The seedlings with the same plant growth potential at the stage of two leaves and one heart were used for the experiment, and three biological replicates were set up. During the treatment, the plant phenotype (such as the wilting degree of the leaves) was recorded in time, and after one day of treatment, the materials were recovered at room temperature, and the wilting and dead leaves were photographed.

In addition, the sampling experiment was designed. When the cotton seedlings grew to two leaves and one heart, the seedlings with the same growth were selected for low temperature treatment, and six sampling time points were set at 0h, 1h, 3h, 6h, 12h, and 24h. For one true leaf, two plants were regarded as one sample, and three biological replicates were set. The samples are used for the measurement of relevant physiological indicators, such as malondialdehyde, proline, leaf soluble sugar and free proline, leaf cell membrane ion leakage rate, superoxide dismutase activity and catalase activity, etc. Resistance of materials to low temperature stress.

B. Determination of Physiological Indexes of GhJAZ1 Transgenic Plants and YZ1 Plants After Low Temperature Treatment

1) Determination of relative conductivity (leaf cell membrane ion leakage)

Plant cell membrane plays an important role in maintaining the microenvironment and normal metabolism of cells. Under normal circumstances, cell membranes are selectively permeable to substances. When plants are affected by adversity, the cell membrane is destroyed, and the membrane permeability increases, so that the electrolyte in the cell leaks out, so that the conductivity of the plant cell extract increases. The degree of membrane permeability increase is related to the intensity of adversity stress, and also related to the strength of plant stress resistance. Therefore, the conductivity method has become an accurate and practical method for identifying the stress resistance of plants in crop resistance cultivation and breeding.

For the different transgenic lines of GhJAZ1, take a leaf after low-temperature treatment, rinse it with double distilled water for 3 times, gently blot the water on the surface of the leaf with absorbent paper, put the leaf in 15ml of double distilled water, rinse it and dry it, then centrifuge Add 10ml of double-distilled water into the tube, pump air at 0.05MPa for 20min in a vacuum desiccator, shake slowly at 25°C for 2h, then measure the conductivity S1, and measure the blank conductivity C1 of water at the same time, then place the sample in a boiling water bath Treat in medium for 20 minutes, measure the conductivity S2 after cooling to room temperature, treat the water in the same way, and measure the blank conductivity C2. Ion leakage = (S1-C1)/(S2-C2)*100%. From the ion leakage of leaf cell membranes of different lines after treatment, the overexpression lines had lower ion leakage than the control and interference lines (see Figure 5C), indicating that overexpression of the GhJAZ1 gene can improve the resistance of cotton plants to low temperature stress. tolerance.

2) Determination of chlorophyll content

Low temperature can inhibit the activity of enzymes related to chlorophyll synthesis and affect the absorption of mineral elements such as N and Mg by roots, thereby affecting the biosynthesis of chlorophyll; low temperature may change the ultrastructure of chloroplasts and break the spatial position of chlorophyllase and its substrate chlorophyll. The isolation thus promotes the decomposition of chlorophyll; it may also affect the synthesis and degradation of chlorophyll by affecting the activity of certain isoenzymes. Low temperature may also induce aging accelerators, which increase the activity of chlorophyllase and promote the decomposition of chlorophyll. Different plants and leaves of different parts of plants have different effects of low temperature on chlorophyll content.

For the different transgenic lines of GhJAZ1, take the second leaf after low temperature treatment, and sample the whole leaf, grind it with liquid nitrogen and add 1.3ml 80% acetone to extract overnight, and use a microplate reader to measure the absorbance of the samples at wavelengths of 663nm and 645nm respectively. Chlorophyll Ca=12.72 A663–2.59 A645, chlorophyll Cb=22.88A645–4.67A663, total chlorophyll CT=Ca+Cb. From the total chlorophyll content of leaves of different lines after treatment, the overexpression lines had higher total chlorophyll content than the control and interference lines (see Figure 5D), indicating that the chlorophyll content was more affected by low temperature than the control and interference lines. small.

3) Determination of malondialdehyde (MDA) content

Membrane lipid peroxidation occurs in plant organ aging or under abiotic stress conditions, and malondialdehyde is one of the products. It is usually used as an indicator of membrane lipid peroxidation, indicating the degree of cell membrane lipid peroxidation and plant response to adversity conditions strength.

The malondialdehyde content was measured on leaves of 8℃ low temperature treatment for 3h and untreated control plants. Weigh about 0.1 g of leaves, cut them into pieces, add 1 mL of 10% trichloroacetic acid (TCA), grind them on ice with a pre-cooled mortar, and pour them into a 2 mL centrifuge tube. Centrifuge at 3000rpm/min for 10min, take 0.4mL of supernatant to a new 2mL centrifuge tube, add 0.67% thiobarbituric acid (TBA) 0.4mL to it, mix and boil in 100°C boiling water for 30min, After cooling, centrifuge again. The absorbance values of the supernatant at 450nm, 532nm and 600nm were measured with a microplate reader. And calculate the MDA concentration according to the formula, and then calculate the MDA content (μmol/g) in the unit fresh weight tissue. The result is calculated according to the formula: C/μmol/L=6.45(A532-A600)*(V1*V)/(V2*W). Among them, V1 is the total amount of the reaction solution, V2 is the amount of the extract solution in the reaction, V is the total amount of the extract solution, and W is the sample mass. Finally, it was found that the samples treated at low temperature led to membrane lipid peroxidation, and the degree of membrane lipid peroxidation in the overexpression line was significantly lower than that in the interference line and the control under normal conditions and low temperature stress (see Figure 5E).

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Claims (5)

1. A GhJAZ1 gene isolated from cotton, the nucleotide sequence of which is the sequence shown in base 1-792 in SEQ ID NO: 1 in the sequence table. 2. A GhJAZ1 gene isolated from cotton, the protein sequence of which is shown in SEQ ID NO:2. 3. An overexpression vector pK2GW7.0-GhJAZ1, characterized in that it contains the gene as claimed in claim 1. 4. the application of the gene overexpression described in claim 1 in improving the tolerance of cotton to low temperature stress. 5. the application of the overexpression vector pK2GW7.0-GhJAZ1 described in claim 4 in improving the tolerance of cotton to low temperature stress.