Genome-Wide Analysis, Cloning And Expression Of UGTS Family In Camellia Sinensis (L.) O. Kuntze

There are many glycosides in Camellia sinensis which decide the quality of tea.UDPG–dependent uridine diphosphate glycosyltransferases (UGTs) are the enzymescatalyze aglycones formed glucosides. Many glycosyltransferase genes involve inflavonoid biosynthesis which promote the glycosylation of flavonol, anthocyanidin,catechin and phenolic acid. Research on the function of these genes is of great significancefor the selective breeding of Camellia sinensis and quality control of tea. The UGT genesin Camellia sinensis were classified, named, cloned and expressed combiningbioinformatics, RACE, prokaryotic expression system, qRT-PCR technology. The keyresearch results are as follows:1. The UGT genes in Camellia sinensis was screened by bioinformatics.68UGTgenes in Camellia sinensis were classified. The UGTs were divided into twelve groups byArabidopsis classification method and named by nomenclature system.2. Three UGT genes involve in flavonoid biosynthesis (UFGT) CsUGT73E1,CsUGT78E1, CsUGT72F1were cloned by homology cloning method. The amino acidnumber of three UFGT genes was475,459and465respectively. The isoelectric pointpredicted was53.72kDa,49.48kDa and50.57kDa respectively. Analysis of signal peptideshowed three genes have no signal peptide.3. CsUGT78E1shares59%identity and75%similarity with VvGT1in Vitis vinifera(accession number: P51094.2, PDB id:2c9z), which meets homologous modelingconditions. The homology modeling of CsUGT78E1was set up via SWISS-MODELWorkspace software according the structure of VvGT1and the three-dimensional structureof CsUGT78E1enzyme was predicted, which provided positive information for substratespecificity of enzymes. It suggested the protein from CsUGT78E1could catalyzeanthcyandin, quercetin, and kaempferol to form their glucosides by taking UDP-glucose asactive donor.4. The recombinant plasmids of pET32a-CsUGT73E1, pET32a-CsUGT78E1,pET32a-CsUGT72F1, pGEX-4T-2-CsUGT73E1, pET28a-CsUGT78E1andpET28a-CsUGT72F1were constructed and transformed into host rosetta (DE3). The geneswere induced by0.1mmol/L IPTG with the temperature of37℃,25℃,16℃. But,unfortunately, the fusion proteins were existed consistently with inclusion body protein. 5. The CsUGT78E1inclusion body protein was renatured and was purified by affinityresin. The results showed that inclusion body protein renatured had no activity yet.6. The qRT-PCR expression of CsUGT73E1, CsUGT78E1and CsUGT72F1wereanalyzed in tea leaves at different development stages, and leaves with different hormoneand hurt processing. The results showed that the expression level of CsUGT73E1is thehighest in the bud. However, the expression level of CsUGT78E1and CsUGT72F1is thehighest in the mature leaf. The expression level of three genes appeared the differencewhen leaves subjected to GA, damage, ABA processing.