Functional Analysis Of CsBT1 Gene,a Negative Regulator Of Tea Flavonoid Metabolism

As an important secondary metabolite in plants,flavonoids have important physiological functions in regulating growth and stress response of plants.Related studies have confirmed that apple Md BT1 regulates anthocyanins and procyanidins biosynthesis through interaction with Md MYB1 and Md MYB9 transcription factors.Flavonoids have been studied more clearly in the biosynthetic pathways in plants,but the transcriptional regulation mechanism of this metabolic pathway is not yet clear.In this experiment,the CsBT1 gene of MYB transcription factor binding protein was screened and cloned from tea plants,and its biosynthetic mechanism of anthocyanins and proanthocyanidins was analyzed.In this paper,the CsBT1 gene was first cloned from tea plants,and the role of CsBT1 gene in anthocyanin and proanthocyanidin synthesis was verified by in vitro protein interaction,Arabidopsis and tobacco genetic transformation system.The main results are as follows:1.The CsBT1 gene expression level of tea plants has tissue expression specificity and variety expression specificity.The expression of this gene was highest in the second and third leaves,and lowest in the first leaf.In addition,CsBT1 gene expression was significantly different in different tea color varieties.The expression of CsBT1 was highest in the conventional variety Shuchazao,the anthocyanin-rich purple bud variety Zijuan was the middle expression,and the yellowed variety was the lowest.2.Using the genetic transformation of Arabidopsis thaliana,the functional study of CsBT1 gene was carried out.In Arabidopsis thaliana,the leaves of this gene mutant are purple due to the deletion of the BT1 gene,and the complementary Arabidopsis leaves are green,and the traits are restored.The Arabidopsis pap1 trait is that the leaves are purple,and the leaves change from purple to green after the gene is transformed.The above results can be speculated that the CsBT1 gene of tea plants is involved in the biosynthesis of flavonoids,and its regulatory site may be upstream of the MYB transcription factor.3.The tobacco genetic transformation system was used to further verify the function of the CsBT1 gene.Compared with the control,the tobacco flower overexpressing CsBT1 was found to have a lighter flower color and a lower anthocyanin content.The total anthocyanins in the flower were also reduced by DMACA staining.The above conclusions speculate that tea tree CsBT1 participates in the negative regulation of procyanidins and anthocyanins biosynthesis at the same time.Regulatory sites need further verification.4.Protoplast localization analysis found that the CsBT1 protein was localized in the cytoplasm;yeast two-hybrid experiments and Pull down results showed that CsBT1 had an interaction with CsMYB5 b and CsMYB6 a proteins,but had no interaction with WD40 and b HLH.Based on the above test results,we speculate that CsBT1 is upstream of the MYB transcription factor and is involved in the flavonoid metabolism of tea plants.