Prediction And Verification Of Transcription Regulators For CsTSI Based On Co-expression Network

L-Theanine is the most abundant free amino acid in tea plant(Camellia sinensis).It is one of the main secondary metabolites of tea plant and plays an important role in tea quality and health.It is of great significance for the molecular regulatory network of theanine synthesis to identify the transcriptional factors that regulate theanine synthase CsTSI and analyze their functions.The research purpose of this subject is to mine the transcriptional factors of CsTSI.The main content and results of the paper are as follows:1.Using the tea plant transcriptome sequencing samples in the NCBI database to achieve a two-way co-expression network of 16 functional genes and 221 transcription factors related to theanine biosynthesis with a cutoff 0.7 of Pearson correlation coefficient.2.Twenty-two transcription factors related to CsTSI were screened through the co-expression network,which were analyzed for tissue specificity and the expression levels of tea seedling roots treated with ethylamine hydrochloride at different time points.Using the online website Wo LF PSORT to predict the subcellular localization of transcription factors and found that they are all located in the nucleus.Using the Plant CARE website to predict and analyze the cis-acting elements on the CsTSI promoter,it was found that the CsTSI promoter has many elements that were respond to light and hormones.At the same time,the presence of MYB and W-BOX elements on the promoter is predicted,indicating that it may be regulated by members of the MYB and WRKY family,and the transcription factors screened by the co-expression network happen to have many members of these two families.3.The 1927 bp CsTSI promoter sequence and the full-length open reading frames(ORF)sequences of 16 transcription factors were cloned.The cloned transcription factors and CsTSI promoter were subjected to a yeast one-hybrid experiment to verify the interaction.We found that 16 transcription factors failed to interact with the CsTSI promoter.But some of them can interact with other functional genes of theanine metabolic pathway,indicating that they are indirectly related to CsTSI,and finally achieve the purpose of regulating theanine synthesis in the root.4.The key sequences of the CsTSI promoter,S56(-1～-563bp)and S2(-1231～-1520bp),were successfully predicted by experiments with transient infection of tobacco.The two sequences were used as the bait sequences for yeast single hybrid library screening.The bait vectors have been constructed,transferred into Y1 H Gold yeast cell to become bait strains,and the minimum Ab A inhibitory concentration of the bait strains has been determined.The above research provides some information for transcription factors that may be related to theanine metabolism.Most of these transcription factors are the highest in the roots of tea plant,and some are only expressed in the roots.We cloned the CsTSI promoter sequence and predicted the key sequences on the promoter by transient infection experiment.