Identification And Analysis Of Anthocyanin Galactosyltransferase From Tea Plant[Camellia Sinensis(L.)O.Kuntze]

Anthocyanin is a kind of natural water-soluble pigment unique to plants.It can not only endow plant tissues and organs with brilliant colors,but also has a variety of biological activities.Because anthocyanins have various biological activities in human health,there is great research interest in the development of anthocyanin-rich foods and beverages.Tea[Camellia sinensis（L.）O.Kuntze]is one of the world’s most widely consumed natural beverage consumption.The new shoots of some tea varieties appear purple due to the accumulation of high content of anthocyanin in bud and leaf tissues.Cyanidin 3-O-galactoside,delphinidin 3-O-galactoside and and their acylated anthocyanins have been identified as the main anthocyanin components in purple shoot tea,but the enzyme responsible for their biosynthesis remains unclear.UDP-galactose anthocyanidin3-O-galactosyltransferase（UA3GalT）is presumed to catalyze the galactosylation of anthocyanidin.In this study,the tea varieties’Shaanchayihao’,’Zijuan’,’Ziyan’and the green strain’51-1’and the purple mutant’51-2’of’Meitantaicha’were used as research materials.Firstly,the content of anthocyanin in them was analyzed.Then the crude enzyme was extracted from the above 5 materials,and the enzymatic activity of UA3GalT was detected.The correlation between its catalytic activity and total anthocyanin content was analyzed.Subsequently,a suspected gene CsUGT78A15 encoding anthocyanin3-O-galactosyltransferase was screened from the tea plant genome by homology comparison method,and then cloned from above five materials.The prokaryotic expression vector and plant overexpression vector were constructed to verify its function in vitro and in vivo.The main research results are as follows:1.The total anthocyanin content and component analysis of the new shoots of tea plants with one bud and two leaves showed that the total anthocyanin contents of the purple-leaved tea materials’51-2’,’Zijuan’and’Ziyan’were significantly higher than that of the green-leaved materials’Shaanchayihao’and’51-1’.The descending order of the total anthocyanin contents was:’Ziyan’,’Zijuan’,’51-2’,’51-1’and’Shaanchayihao’.There are four main kinds of anthocyanins were detected by high performance liquid chromatography in purple-leaved tea plants,and two kinds were identified as cyanidin 3-O-galactose and delphinidin 3-O-galactose,respectively.The content variation trend of these two anthocyanins in different tea shoots was consistent with that of total anthocyanins.2.Crude enzymes was extracted from 5 tea plant materials,and the activity of UA3GalT was detected with UDP-galactose as sugar donor and cyanidin as sugar ac ceptor,respectively.Except for’51-1’,the crude enzymes extracted from other samples all showed UA3GalT activity.There was a significant positive correlation between the activity of UA3GalT and the content of total anthocyanin（r=0.929,p<0.05）,indicating that the high accumulation of anthocyanin was closely related to the activity of UA3GalT in tea plants.3.The candidate gene CsUGT78A15 encoding anthocyanin 3-O-galactosyltransferase was screened from the tea plant genome by the method of homologous sequence alignment.And then cloned from above 5 tea samples.Phylogenetic analysis and multiple sequence alignment analysis showed that CsUGT78A15 may encode UA3GalT enzyme involved in the biosynthesis of galactosylated anthocyanins.The q RT-PCR analysis showed that the relative expression of CsUGT78A15 was consistent with the accumulation of anthocyanins in the purple-leaved tea samples.4.Enzymatic assays showed that r CsUGT78A15 could catalyze the synthesis of cyanidin 3-O-galactose and delphinidin 3-O galactose use UDP-galactose as sugar donor.The K_m values of the two substrates were 32.64 and 45.59μM,the V_max values were 3.29and 6.63μM?min^-1,the k_cat values were 18.61 and 37.45 min^-1,and the k_cat/K_m values were0.57 and 0.82 min^-1?μM^-1,respectively.The catalytic efficiency of delphinidin was higher than that of cyanidin.The above results indicate that CsUGT78A15 hasUA3GalT activity in vitro.5.Subcellular localization analysis showed that CsUGT78A15 was localized in both the endoplasmic reticulum and the nucleus,which was consistent with the synthesis of anthocyanins.The transient overexpression indicated that the up-regulation expression of CsUGT78A15 promoted the accumulation of cyanidin 3-O-galactoside in’Granny Smith’apples and resulted in a clear pink color in the pericarp.These indicate that CsUGT78A15also hasUA3GalT activity in plants.