Study On The Regulation Mechanism Of Leaves Turning Purple In Zikui Tea Plant

Anthocyanins are water-soluble pigments that widely exist in plants.It has many health protection functions,including anti-inflammatory,anti-oxidation,etc.Zikui(C.sinensis cv.Zikui),a cultivar developed from a purple-bud mutation line of N61(a line from C.sinensis cv.Meitan-Taicha in Guizhou),is a new purple-leaf tea line cultivar with purple all year round,and the anthocyanin content in the leaves could be as high as 4.97 mg/g FW,which had important promotion and application value.However,the molecular mechanism underlying purple-leaf formation in Zikui remains unclear.In this study,the leaves Zikui were used as the main research object,and the leaves(green)of N61 cultivar were combined for metabolome and transcriptome analysis,and it was found that bovine petunidin 3-O-glucoside was the key metabolite determining the purple-leaf characteristics in Zikui.Furthermore,the key regulatory gene Cs MYB90 of Zikui leaves was obtained by joint analysis of transcriptome and metabolomics,and its gene function was verified.The main results are as follows:(1)The total anthocyanins in the leaves of Zikui and N61 were measured by the p H differential method.The results showed that the anthocyanin content in the leaves of Zikui increased first and then decreased.The anthocyanin content in the leaves of Zikui was the highest at 15 days at 4.97 mg/g FW,which was 3.19 times that of N61during the same period.It then decreased to 3.59 mg/g FW at 30 days and 3.36 mg/g FW at 45 days.In contrast,we did not observe any differences in the anthocyanin content of N61 in the previous five periods,and it was lower than that of Zikui in all periods.(2)The metabolomics of leaves of Zikui and N61 at 15 and 45 days were analyzed by LC-MS technology.The results showed that petunidin 3-O-glucoside was the most abundant among all of the metabolites in the leaves of Zikui at 15 days,and its relative content was 3.96×10~7,followed by cyanidin 3-O-galactoside(1.58×10~7)and cyanidin 3-O-glucoside(1.44×10~7),which were117.76、10.68 and 9.14 for N61in the same period,respectively.It indicated the key substances related to the purple-leaf phenotype in Zikui.(3)Eleven transcription factors were identified by combined analysis of metabolomics and transcriptomics,among which the Cs MYB90 gene was strongly correlated with petunidin 3-O-glucoside,cyanidin 3-O-galactoside,and cyanidin 3-O-glucoside(PCC>0.8).In addition,two F3’H genes and two ANS genes were positively correlated with anthocyanin accumulation,and three PPOs were negatively correlated with anthocyanin accumulation.(4)The Cs MYB90 gene was transformed into tobacco by Agrobacterium-mediated method.The leaves of transgenic tobacco were purple,and the anthocyanin content in leaves was significantly higher than that of the wild type,which was 13.04times that of the wild type.These results provide a theoretical basis for the molecular mechanism of anthocyanin biosynthesis in purple-leaf tea plants,and provide a series of candidate genes for the breeding of anthocyanin-rich cultivars.