Functional Analysis Of A Transcription Factor SlbZIP6 Under High Temperature Stress

In the process of crop cultivation,crop are often affected by drought,high temperature,low temperature and salt and other environmental stresses,which are the main reasons for the decrease of the yield and quality of crops.Understanding the mechanism of abiotic stress and improve the stress resistance of plants have become one of the hotspots in botany research.In recent years,many studies have been made on the molecular mechanisms of plant stress resistance,such as low temperature,drought and salt stress,while the research on the related aspects of high temperature stress is relatively few,that mainly focusing on the functional protein genes and the regulatory protein genes related to high temperature.Therefore,to deepening study of molecular mechanism about plant stress resistance,with systematically study on high temperature related gene expression and regulation under high temperature stress conditions,which can lay a foundation for the cultivation of new varieties of tomato high temperature resistance.It provides a new approach for high temperature resistance improvement and enriches theory of plant stress tolerance as well as creating new germplasm with improved stress tolerance.Through microarray transcriptional analysis,a tomato bZIP transcription factor SlbZIP6 was found to be involved in the stress response.In this study,SlbZIP6 was cloned in AC⁺⁺tomato and transformed into AC⁺⁺tomato.By phenotypic identification and physiological and biochemical analysis of transgenic tomato and AC⁺⁺tomato,it was found that overexpression of SlbZIP6 reduced the ability of tomato to resist high temperature stress.The expression analysis of transgenic tomato plants showed that the over expression of SlbZIP6 in tomato could induce a series of expression related to high temperature related genes,indicating that SlbZIP6 participated in regulating the gene expression of plant defense against high temperature.The main results are as follows:1 Cloning of SlbZIP6 and the construction of vectorWith tomato leaf cDNA as a template,the target fragment of about 1365bp was obtained by PCR,and SlbZIP6 was successfully cloned.Bioinformatics shows that SlbZIP6 transcription factor encodes 454 amino acids,and the predicted molecular formula is C₂₀₅₇H₃₃₃₅N₆₂₅O₇₀₅S₁₆ and the molecular weight is 48.61kD.SlbZIP6 protein has no signal peptide sequence,and has no transmembrane domain,and is located in nucleus.The results of cluster analysis show that SlbZIP6 protein is highly homologous with pepper CaCPRF2,tobacco NtCPRF2 and soybean GmHBF-1.2 Analysis of inducible expression factorsBy using exogenous hormones and abiotic stresses to AC⁺⁺,qRT-PCR was used to analyze the expression of SlbZIP6 gene in different tissues and locations under exogenous hormones and abiotic stresses.Identification of factors that can effectively induce the differential expression of SlbZIP6 gene.The results showed that the expression of SlbZIP6 gene in AC⁺⁺was obviously up-regulated under high temperature stress.Under the drought and salt treatment,the SlbZIP6 gene also has a more obvious upward trend of expression.Under ABA treatment,the SlbZIP6 gene was rapidly expressed in AC⁺⁺,indicating that SlbZIP6 might be involved in the regulation of ABA.Under the treatment of SA,the expression of SlbZIP6 gene was obviously up-regulated in the later period,suggesting that SlbZIP6 may play a role in the regulation of SA.Under the treatment of GA,JA and ethylene,the expression of SlbZIP6 gene was not affected by the treatment.3 Obtained the transgenic tomato plants over-expression or RNAi of SlbZIP6 geneUsing agrobacterium mediated genetic transformation method,the SlbZIP6 gene in the over-expression vector was successfully introduced into tomato genome,and obtained transgenic plants.At the same time,the RNAi expression vector was transferred into tomato,and obtained the transgenic tomato plants successfully.4 Over-expression SlbZIP6 reduced the ability of tomato to resist high temperature The transgenic tomato plants and wild type AC⁺⁺tomatoes were also treated at high temperature.The results showed that the growth of tomato plants transferred to SlbZIP6 gene was not as good as that of wild type AC⁺⁺tomato.The over expression of SlbZIP6 reduced the heat tolerance of tomato.The physiological and biochemical results of high temperature treatment have shown that over-expression SlbZIP6 plants accumulated less proline than AC⁺⁺tomato after stress treatment,while MDA accumulation increased significantly compared with AC⁺⁺tomato.In addition,the relative conductivity of overexpressing plants increased significantly.5 Over-expression SlbZIP6 may reduce the high temperature resistance of tomatoes by participating in the regulation of high temperature related genes.Through the analysis of high temperature related genes with SlbZIP6 over-expression and wild type AC⁺⁺in tomato,it was found the expression of HsfA2,HsfB1,Hsp90,Hsp100 was down,the expression of SOD and APX increased,and the expression of AREB1,PP2C2,NCED and TAS14 decreased in over-expression SlbZIP6plants,compared with wild type.It is indicated that SlbZIP6 can regulate the molecular mechanism of plant by heat expression of these genes.