Screening And Fuctional Analysis Of Cotton DREB/CBF Genes That Are Involved In Plant Response And Defense To Abiotic Stress

Cotton is one of the most important economic crops in the world, but as the global ecological environment is worsening, abiotic stress such as drought, salinity, low temperatures has caused a great loss of cotton production, which are the main factors of restricting the increase of its production. To solve the issue of agriculture, by means of genetic engineering, excavating resistance related functional genes, transforming the resistance related functional genes into plant genomes and acquiring the new species of plants with resistance, is the most effective way to cope with adversity stress and to improve cotton production. Studies have shown that the over-expression of particular stress response transcription factors can regulate the expression of multiple downstream functional genes of stress resistance, which plays an important role in the process of enhancing crop tolerance.DREB/CBF (Dehydration Responsive Element Binding Protein/C-repeat Binding Factor) is a kind of important plant transcription Factors, in response to adversity stress response and plants play an important role in responding to adversity stress response and in the process of plant stress resistance. Compared to the traditional model plants such as Arabidopsis, rice, the research about identification of cotton resistance transcription factors and mechanism of stress resistance is less. Therefore, screening and identifying the stress resistance related DREB/CBF genes in cotton and analyzing the molecular mechanism of stress resistance, which provides a new scientific proof of improving the resistance of cotton by genetic engineering means.This study identified cotton DREB/CBF gene using bioinformatics methods as well as analyzed the structure characteristics of these genes, evolutionary relationships and drought induced expression pattern. We also studied cotton CBF2 and CBF3 gene in the process of plant abiotic stress response function and mechanism by excess expression method. The main results are as follows:1. Isolation, identification and expression analysis of cotton CBF gene participating in the abiotic stress response.Through Blast homology genes, a total of 21 members of cotton CBF gene family were identified and analyzed from the aspects of gene structure and evolution. The study results showed that the CBF gene family of cotton resembles arabidopsis and rice CBF gene in structure, and all of them contain a AP2/EREBP conservative domain having a composition of 58 amino acids. According to evolutionary relationships,21 cotton CBF genes were divided into four subfamilies (Ⅰ-Ⅳ). At the same time, with the use of RT-PCR and fluorescence quantitative PCR method, expression modes of the 21 cotton CBF genes were analyzed in cold, dry (mannitol treatment) and salt stress. The results showed the expression quantity of 12 genes induced by cold stress rise significantly; Under drought stress the expression quantity of 10 genes induced by drought stress have a significant increase; While under salt stress the expression quantity of 11 genes increased significantly, but after being induced by the stress each gene expression time and expression quantity are different, which showed redundancy phenomenon of gene function may exist in these gene.2. Structure and expression analysis of cotton GhCBF3 geneAccording to the induced expression pattern under adversity stress of the cotton CBF genes, a high expression gene induced in drought and salt stress was selected from the candidate genes and cloned from cotton. The total length of the gene cDNA span is 462 bp, no introns, encoding 153 amino acids, and we named it GhCBF3 (GeneBank number is KP780186). Further expression analysis results showed that after NaCl, mannitol and ABA treatment, the expression of GhCBF3 gene significantly increased, but the highest expression quantity was in leaf and root. The research results of GhCBF3 protein sub-cellular localization showed that GhCBF3:eGFP green fluorescence signal was expressed in the nucleus, and suggested that GhCBF3 gene may function in the cell nucleus. The self-activation analysis showed that GhCBF3 protein did not have the self-activation phenomenon. In the promoter sequence regions of GhCBF3 gene there were more than one AREB cis-acting element responding to ABA signal, and the histochemistry analysis results of the promoter showed that under normal conditions, in arabidopsis thaliana cotyledon, leaf veins, root tip, especially in stomatal guard cells, GUS signal can be observed. And GUS activity was enhanced after arabidopsis thaliana seedlings were treated with abiotic stress, suggesting that proGhCBF3 can respond to external environment, such as high salt, drought and ABA signal.3. Analysis of the role of GhCBF3 gene in plant response and defense to abiotic stressFor the research of GhCBF3 gene function, the GHCBF3 excessive expression vector was constructed and transformed into arabidopsis thaliana, by screening to obtain transgenic plants. We selected the arabidopsis thaliana strains whose exogenous gene expression quantity were high, medium and low for the analysis of the phenotype and the research of function. Results showed that the transgenic plants with excessive GhCBF3 gene expression had no significant difference in growth phenotype compared with wild type plants. When treated by drought and salt stress, GhCBF3 transgenic arabidopsis thaliana tolerance to drought and salt stress was much more significantly enhanced than contrast. In addition, the ABA sensitivity experiments showed transgenic plants with GhCBF3 excessive expression had significantly increased sensitivity of exogenous ABA. Under high salt, drought and ABA treatment, the expression quantity of transgenic arabidopsis RD29A Marker genes,associated with drought stress and ABA, COR15A, RD22 and P5CS increased remarkably.GhCBF3 gene not only increased the sensitivity of transgenic arabidopsis seeds to ABA, but also promoted closure of transgenic arabidopsis leaf stomatal. In addition, after exogenous ABA treatment, AREB /ABF2 and AREB2 /ABF4 gene expression quantity in transgenic arabidopsis is significantly higher than that in wild-type arabidopsis, suggesting that GhCBF3 gene could regulate the expression of AREB1/ABF2 and AREB2/ABF4 gene in transgenic arabidopsis, and then affect the sensitivity of transgenic arabidopsis stoma to ABA, accelerate the closure of stoma. The overall results showed that GhCBF3 was involved in the ABA response.4. Analysis of the role of GhCBF2 gene in plant response and defense to abiotic stressWe identified another CBF gene from 21 candidates genes induced by abiotic stress, named it GhCBF2 gene, encoding a length of 226 amino acid sequences of proteins.GhCBF2 contains a typical AP2/EREBP conserved domain composed by 58 amino acids, and conservative AP2/EREBP domain structure can form three beta folding and 1 alpha helix structure.GhCBF2 gene expression analysis results showed that after NaCl and mannitol treatment, the GhCBF2 gene expression quantity in the cotyledon, root and leaf were significantly increased; When treated with ABA, amount of GhCBF2 gene expression in each tissue was increased significantly, showing that GhCBF2 gene may play an important regulatory role in cotton response to drought and salt stress. Tissue expression result showed GhCBF2 gene expression quantity had the highest amount in root and leaf tissue. The research results of GhCBF2 protein subcellular localization showed that the GhCBF2:eGFP green fluorescence signal was in the nucleus, and illustrating that GhCBF2 protein was located in the nucleus. For the analysis of GhCBF2 gene resistance function, We selected L6, Lll and L21 respectively from transgenic arabidopsis strains to represent the high, medium and low three kinds of transgenic strains with different target gene expression quantity, used for gene function analysis. Results confirmed that the transgenic strains with GhCBF2 gene high expression had strongest ability of resistance to salt and drought, illustrating that GhCBF2 gene played an important role in the resistance to salt and drought in the seeds and seedling period of the transgenic plants. In this experiment, we determined the proline, relative water content, chloroplast pigment content and a series of physiological indexes in transgenic arabidopsis and wild-type arabidopsis, and the results showed that when the transgenic arabidopsis and wild-type arabidopsis were treated with NaCl solution processing and dry processing, the leaf relative water content and the chloroplast pigment content in wild-type arabidopsis decreased more significantly than that in transgenic arabidopsis, but the free proline content was less than that in transgenic arabidopsis thaliana, indicating that GhCBF2 transgenic Arabidopsis had greater tolerance to salt and drought stress. Under high salt, drought treatment, the expression quantity of transgenic arabidopsis RD29A Marker genes,associated with abiotic stress, COR15A, and ERD6 increased remarkably.