Screening And Analysis Of Transcription Factors Regulating The HCT Gene Of Tea Plant

Chlorogenic acid as an important of phenolic substances in tea,has many beneficial effects to human body health,which also plays an important role in the formation of tea flavor and helping tea plants to resist a variety of stresses.Therefore,revealing the regulatory network of chlorogenic acid in tea plants is beneficial to improve the quality of tea and the ability of tea plants to resist stress.Existing reports have found that HCT plays an important role in the biosynthesis of chlorogenic acid in tea plants,which works in two biosynthetic pathways of chlorogenic acid.However,the regulatory mechanism involved has not been fully understood.In order to reveal the mode of transcription factor regulation of HCT gene expression and then regulate the biosynthesis of chlorogenic acid in tea plants,Fuding Dabai tea was chosen as research material to clone the promoter sequence of HCT gene by PCR in our study.A bait strain was constructed based on the promoter sequence of HCT gene,then the yeast onehybrid technology was used to screen the transcription factors that interact with the HCT gene,so as to reveal the mode of transcription factor regulation of HCT gene expression.Therefore,the research was carried out and the following results were obtained:(1)The corresponding HCT gene promoter sequence in Fuding Dabai tea was cloned by PCR technology,and bioinformatics software was employed to the HCT gene promoter sequence.The results showed that it contained the hormone-related elements like ABRE,ERE and P-Box,and also contained the light-responsive components,such as G-Box,Box 4,TCT-motif and GT1-motif,etc.,in addition,it also included many environmental response-related components,such as MYB,MYB-like,MYC and LTR.The above result indicated that the expression of HCT gene may be under the coregulation of hormones,light and environment.(2)The results of the analysis of HCT gene expression in different parts of tea plants showed that compared with fruits and roots,the expression levels of HCT gene in buds,leaves and stems were relatively high,but it was almost not expressed in flowers;(3)Hormone treatment experiments showed that the expression of HCT had a response to the three hormones of ABA,GA3 and ethephon.The response of tea tree to GA3 was relatively slow,while the effect of ethephon was rapid and long-lasting;(4)In this study,we successfully constructed a cDNA library of Fuding Dabai Tea and a yeast strain of p HIS2-HCT bait containing the HCT gene promoter.Six positive clones were successfully screened,but after the analysis of their sequencing results,we did not get transcription factors.This study showed that the expression of HCT gene was tissue-specific and responded to the three hormones of ABA,GA3 and ethephon.In addition,the construction of the Fuding Dabai Tea cDNA library and the summary of the problems in the experimental process laid the foundation for further optimizing the experiment and screening out the transcription factors that regulate the HCT gene.