The Functional Analiysis Of Flavonoid Metabolism Related CsbHLHs In The Tea Plant (Camellia Sinensis)

The metabolic pathway of flavonoids is one of the important secondary metabolism pathway in plants,which plays an important role in the growth and development of plants and defensing adverse environment.bHLH transcription factor,the secondary family of transcription factors in plants next to MYB family,can interact with MYB and WD proteins to form a triple complex to participate in the regulation of flavonoid metabolic pathway.This paper focuses on the study of tea bHLH transcription factors and their regulatory roles in the flavonoid metabolism pathway.The main results are as follows:1.In this experiment,206 tea plant CsbHLH genes were screened from the Shuchazao transcriptome database firstly,and they were put together with Arabidopsis AtbHLH to construct a phylogenetic tree.Their tissue expression specificity and inducible expression specificity were analyzed.2.Two CsbHLH transcription factors CsTT8 and CsMYC were screened.The homologous cloning method was used to amplify the open reading frames of CsTT8 and CsMYC genes.The open reading frame of CsTT8 was 2058 bp long and encoded 686 amino acids.The open reading frame of CsMYC was 1998 bp long and encoded 666 amino acids.3.Bioinformatics analysis showed that CsTT8 and CsMYC in tea plant are homologous to AtTT8 and AtMYC1 in Arabidopsis.The yeast two-hybrid result showed that both CsTT8 and CsMYC could interact with CsMYB5 s and CsWD40 proteins;CsMYC could complement the symptoms of mutant gl3.These results suggest that they are likely to participate in the regulation of flavonoid metabolic pathway.4.Using fluorescence quantitative PCR technique,the tissue expression and induction specificity of two genes were detected.The results showed that the expression of both genes were high in the aboveground parts and low in the roots;CsTT8 and CsMYC were expressed differently under the induction of various hormones and abiotic stresses treatment.5.Using tobacco genetic transformation system to verify the function of CsTT8 and CsMYC,the results showed that,compared with the control,the content of anthocyanin and PA in CsTT8 overexpressed tobacco increased slightly,while the content of CsMYC overexpressed tobacco anthocyanin and PA decreased and the color of their flowers is white,and the whole flower is smaller than the wild type.The real-time fluorescence quantitative PCR results showed that most of the structural genes involving in the flavonoid biosynthesis pathway in transgenic tobacco flowers were down-regulated.