Screening And Analysis Of Transcription Factors Regulating Tea Plant F3'5'H Gene

Catechins are the main phenolic compounds in Camellia sinensis,which have strong free radical scavenging and antioxidant capacity.At the same time,they play an important role in the formation of the bitter taste of tea.Previous studies have shown that the expression of Cs F3'5'H gene is positively correlated with catechin content.The end product of Cs F3'5'H gene is flavanols.The increase of flavanols will increase the bitter taste of tea and affect the quality of tea.The expression of Cs F3'5'H gene is regulated by transcription factors.However,there are few studies on the upstream cis acting elements and transcription factors of Cs F3'5'H gene.In this study,the promoters of Cs F3'5'H1,Cs F3'5'H2 and Cs F3'5'H3 genes were cloned from the leaves of "Fuding Dabai " tea plant,and the possible cis acting elements were predicted by bioinformatics methods.The expression differences of Cs F3'5'H1,Cs F3'5'H2 and Cs F3'5'H3 genes in different tissues of tea plant were analyzed,and the expression differences of Cs F3'5'H1,Cs F3'5'H2 and Cs F3'5'H3 genes in different time after different hormone treatments were analyzed.Finally,yeast one hybrid method was used to screen transcription factors interacting with the promoter region of Cs F3'5'H,determine the function of transcription factors,and reveal the regulation mode of transcription factors on Cs F3'5'H gene expression,so as to provide theoretical basis and scientific basis for studying the mechanism of action of Cs F3'5'H gene,regulating catechin biosynthesis and improving tea quality.The main findings are as follows:(1)The promoter sequences of Cs F3'5'H1(TPIA accession number: Tea 026294),Cs F3'5'H2(TPIA accession number: Tea 013315)and Cs F3'5'H3(TPIA accession number: Tea 034051)were obtained from tea genome,and the promoter sequences of Cs F3'5'H1,Cs F3'5'H2 and Cs F3'5'H3 were cloned successfully.(2)The possible cis acting elements in the promoters of Cs F3'5'H1,Cs F3'5'H2 and Cs F3'5'H3 genes were predicted by bioinformatics methods.The results showed that the sequence contained not only basic elements,such as TATA box and CAAT box,but also cis acting elements related to hormones,environment and light regulation.Among them,the promoter of Cs F3'5'H1 gene contains 6 hormone regulatory elements,4environment related elements and 3 light regulatory elements;the promoter of Cs F3'5'H2 gene contains 3 hormone regulatory elements,1 environment and 1 light regulatory element;the promoter of Cs F3'5'H3 gene contains 4 light regulatory elements,3 hormone regulatory elements and 2 elements related to environmental regulation.And the expression of GFP vector was similar to the result of fluorescence quantification.(3)The expression levels of Cs F3'5'H1 gene,Cs F3'5'H2 gene and Cs F3'5'H3 gene in different tissues of tea plant have the same characteristics,that is,the highest expression level in bud,followed by that in old leaf and stem,low expression level in fruit,and almost no expression level in root and flower.(4)Gibberellin,Methyl Jasmonate,Abscisic Acid and Ethrel could promote the relative expression of Cs F3'5'H1,Cs F3'5'H2 and Cs F3'5'H3 genes,and could induce the up regulation of the three genes in the tender leaves of tea plants.The relative expression of Cs F3'5'H2 was significantly higher than that of Cs F3'5'H1 and Cs F3'5'H3,which was mainly due to the tissue specificity of the gene.(5)Fifteen proteins interacting with Cs F3'5'H1 gene promoter and five proteins interacting with Cs F3'5'H gene promoter were screened by yeast one hybrid method.The physicochemical properties,hydrophobicity and subcellular localization of these20 proteins were predicted by bioinformatics method.In combination with previous studies,we finally identified HSF24 transcription factor and zinc finger protein transcription factor interacting with Cs F3'5'H1 gene promoter.