Screening And Analysis Of Transcription Factors Regulating TCS Gene In Camellia Sinensis

As an important secondary metabolite in tea plants,caffeine can stimulate nerves to relieve fatigue for humans,and its content is closely related to the quality of tea.Therefore,revealing the regulation mechanism of caffeine synthesis in tea plants is conducive to improving the quality of tea.Existing studies have shown that the tea tree caffeine synthase TCS is the most important enzyme in the tea tree caffeine biosynthesis,but there are few studies on the expression and regulation mode of tea tree TCS gene.In order to reveal the expression mode of TCS gene regulated by transcription factors and its regulation of caffeine synthesis mechanism,this study used Fuding Dabai tea plant species as materials to clone the promoter sequence of TCS gene,construct bait strains,and screened by yeast one-hybrid technology to obtain candidate transcription factors that regulate TCS genes,and revealed the expression patterns of transcription factors that regulate TCS genes.The main results obtained from the experiment are as follows:1.Cloning and analysis of TCS gene promoter sequenceAccording to the tea plant genome data,the promoter sequences of TEA022575 and TEA024636 genes were obtained by PCR amplification experiments.The promoter sequences of the TEA022575 and TEA024636 genes obtained by Plantcare and Soft Berry were analyzed,and the cis-acting elements and transcription initiation positions were analyzed.It was found that they not only contained plant hormone response elements such as ABRE and ERE,but also contained light response element G-Box,GT1-motif and environmental response components MYB,MYC,etc.2.Expression analysis of TCS gene in different tissue of tea plants.By analyzing the results of q-PCR of TCS genes in different tissues of tea plants,it was found that the expression of TEA022575 gene in flowers was the highest,followed by stems;the expression of TEA024636 genes in roots and buds was relatively high,and there was almost no expression in other parts.Expression;and after the construction of GFP vector genetically transformed tobacco,the plant expression is similar to the results of q-PCR,indicating that different TCS genes may have tissue specificity in tea plants.3.Analysis of the response of TCS gene to hormone treatment.The experimental results showed that the expression of TCS had a responsive effect on ABA,GA3,ethephon and methyl jasmonate.Among them,TEA022575 had obvious response to gibberellin and methyl jasmonate,while the response of TEA024636 to abscisic acid and methyl jasmonate was low,indicating that different TCS genes have different response patterns to hormone changes in tea plants.4.Screen the transcription factors that regulate the expression of TCS genes.In this study,the bait expression vectors p HIS2-575 and p HIS2-636 containing the TCS gene promoter were successfully transformed into the Y187 yeast strain,and the bait strain was successfully obtained.Using the yeast one-hybrid test,9 and 12 positive clones were screened from TEA022575 and TEA024636,respectively,and auxin inhibitory protein ARP and an unknown tea tree protein was obtained from the positive clones for further study.