Study On Molecular Mechanism Of Tea Leaves Color Change And Functional Identification Of CsMYB Gene

Tea（Camellia sinensis L.）is a kind of perennial evergreen commercial crop,containing high levels of secondary metabolic products with both commercial and medicinal value.Young shoot purple-related tea plant is a kind of characteristic germplasm resources.Young leaves of tea plant turn out to purple is considered associated with the accumulation of anthocyanins in leaves.This research was carried out to screen the differencs between tender purple leaves（TPL）and mature green leaves（MGL）of tea plant from physiology and biochemistry level,transcriptional level and protein level.Finally,Gateway techonogy system was applied to construct the overexpression vector of CsMYB75,which was successfully transferred to Agrobacterium tumefaciens GV3101.A preliminary study on the function of CsMYB75 gene was carried out by transient expression.This study identifies several genes and proteins that are differentially expressed in the development of young shoot purple-related tea plant,and provides a theoretical basis for the molecular mechanism behind the leaf color change and screening high anthocyanin tea germplasm resources.（1）During leaf growth and maturation,the content of secondary metabolic compounds decreases,resulting in higher content in TPL,and lower content in MGL samples.Compared to MGL samples,TPL samples had higher content of anthocyanin,total polyphenols and total catechins,a higher carotenoid-to-chlorophyll ratio and lower content of soluble sugars（glucose,fructose and sucrose）.TPL samples showed a lower photosynthetic ability,demonstrated by a lower CO2 assimilation and carbohydrate accumulation rate.（2）The aim of this study was to analyze the differential expression of genes in these two tissues,with a cDNA-AFLP（amplified fragment length polymorphism）approach.Using cDNA-AFLP with 256 primer combinations,differential transcript profiling generated 148 matched transcript-derived fragments（TDFs）.Among these TDFs,77 genes were upregulated and 71 were downregulated.These were grouped into11 functional categories which are important for final tea quality parameters.（3）The changes of protein profile in the development of young shoot purple-related tea plant（Camellia sinensis）were investigated to study the mechanism of leaf color change using two-dimensional electrophoresis（2-DE）.The results showed that a total of about 900 protein spots were detected on the gels in which 48 spots showed a significant change in protein abundance based on the two-fold difference.46 spots were identified using MALDI-TOF/TOF.We classified these 46 protein spots into seven functional categories.These results indicated that the function of protein mainly concentrated on carbohydrate and energy metabolism（50%）.26 spots were up-regulated while 20 spots were down-regulated compared to the control among 46 differentially expressed proteins spots.Quantitative real-time PCR analysis showed that the expression patterns at the mRNA and protein levels were correlated with each other.（4）Coloning three full-length genes related to anthocyanin biosynthesis using SMART-RACE technology:Flavonoid 3’-monooxygenase（TDF#3341₁f）,UDP-glycosyltransferase（TDF#2421₁d）and UDP glucose:flavonoid 3-O-glucosyltransferase（TDF#4411₁f_b）.（5）Screening a MYB gene from transcriptome data,which is similar to R2R3MYB75 of Arabdopsis,named CsMYB75 and coloing the ORF。（6）Constructing overexpression vector of CsMYB75 gene with Gateway system and successfully transferred to Agrobacterium tumefaciens GV3101.（7）By Transient Expression of tobacco,a preliminary study on the function of CsMYB75 gene which can regulate the anthocyanin biosynthetic pathway was carried out.This can provide a basis for cultivation tea germplasm resource with high anthocyanin content.