Functional Analysis Of Heat Stress Transcription Factor BrHSFA7a In Pakchoi

Pakchoi[Brassica rapa ssp.Chinensis]is a variety of Brassica rapa in the genus Brassica of the family Cruciferae,which is commonly cultivated in the Yangtze-Huaihe River Basin of China.It is one of the leafy common vegetables in people’s life because of its fast growth,high yield and rich nutrition.Therefore,it is important to study the heat resistance mechanism of Pakchoi,to clarify its regulatory network,and to lay the theoretical foundation for the selection and breeding of new heat-resistant varieties of Pakchoi.In this study,the BrHSFA7a gene was successfully cloned from Pakchoi and transferred into Arabidopsis thaliana for functional verification,and then screened and verified its upstream reciprocal genes.The main findings are as follows.1.A total of 51 HSF family genes were identified in Pakchoi using the Brassica rapa genome,and extensive bioinformatics analysis of the chromosomal localization,amino acid physicochemical properties,phylogenetic relationships,gene structure and promoter cis-acting elements of the family genes were performed.The chromosomal localization results showed that 51 Br HSF family genes were randomly distributed on 10 chromosomes.Physicochemical analysis showed that the Br HSF family genes encode 124-480 amino acids,with protein molecular weights ranging from 14.65-53.68 k Da and isoelectric points of 4.66-10.12,respectively.Gene structure analysis showed that different genes contained different structural domains and introns numbers.The results of promoter cis-acting element analysis showed that the promoter regions of Brassica rapa HSF family genes contain several hormone,stress and growth-related response elements.2.Analysis of the promoter sequence of the BrHSFA7a gene predicted that the Br NAC002gene may interact with it through special bonding site.The interaction between BrHSFA7a and Br NAC002 proteins were verified by yeast two-hybrid,and bimolecular fluorescence complementation assay in vivo.3.The BrHSFA7a and Br NAC002 genes of Pakchoi were cloned,overexpression vectors were transferred to Arabidopsis thaliana,and the gene function was verified for the transgenic Arabidopsis T2 generation.Under high temperature stress,to overexpressed BrHSFA7a transgenic plants had a better phenotype with lower relative conductivity（REL）,hydrogen peroxide（H₂O₂）,malondialdehyde（MDA）,and superoxide anion(O₂^·-)content than wild type,indicating that the BrHSFA7a gene could enhance heat tolerance in Arabidopsis.Similarly,under high temperature stress,overexpression of Br NAC002 plants wilted severely and had higher relative conductivity（REL）,malondialdehyde（MDA）and hydrogen peroxide（H₂O₂）contents than the wild type,while RT-q PCR results showed that Br NAC002 repressed the transcription level of BrHSFA7a gene,indicating that Br NAC002may act as a blocker of BrHSFA7a and negatively participate in the heat stress response of Pakchoi regulation.