Functional Analysis Of UGT84A22 And C4H Genes In Camellia Sinensis By Eukaryotic Expression System

Phenolic compounds are an important secondary metabolite in tea plant,which play a crucial role in dominating tea flavor.It is also the primary functional ingredients which impact on human health.Phenolic compounds and its derivatives are complicated and various.The biosynthetic pathways involve shikimic acid pathway,phenylpropanoid metabolic pathway and flavonoid synthetic pathway,meanwhile also share some derivative pathways,such as methylation,acylation,glycosylation,hydroxylation and so on.Thus,researching on the genes as well as their functions help to further understand metabolic regulation in tea plant and have vital significance in breeding and mechanism of quality formation.In this paper,the UGT multigene family in Camellia sinensis transcriptome database were identified,classified and analyzed by bioinformatics technology,then the CsUGT84A22 gene was functionally identified by eukaryotic expression system?The full-length cDNA of C4 H was cloned from tea plant by rapid amplification of cDNA ends,then use eukaryotic expression system to verify the gene function.Its promoter sequence was isolated by genome walking method.The CsC4 H gene's tissue-specific expression profiles was performed by Quantitative RT-PCR.Fusion proteins were then transiently expressed in tobacco leaf and analyzed by confocal laser scanning microscopy for fluorescence of CsC4H-GFP.The main research results are as follows:1.Based on the highly conserved PSPG motif,homology search or the keyword search annotated as “UDP-glycosyltransferase”,178 CsUGTs were identified in NCBI database and the transcriptome sequencing database obtained from our lab.Among them,129 CsUGTs have a C-terminal consensus sequence.2.132 CsUGTs encoding peptides ranged from 250 to 522 amino acids were selected to constructed a phylogenetic tree by MEGA6.0 software.Phylogenetic analysis showed that CsUGTs can be clustered into 16 groups,without group N and Q which have been identified in other plants,but an novel separated group was identified and designated as group R.Sequence logos for each phylogenetic group were generated using the Weblogo application to get a graphical representation of the PSPG motif sequence conservation and the relative frequency of each amino acid in each position.3.Based on the results of phylogenetic analysis,CsUGT84A22 was selected in this experiment as a target gene for eukaryotic expression.The recombinant plasmids of pYES-Cs UGT84A22 was constructed and transformed into expression of host Saccharomyces cerevisiae WT11.The yeast cells harboring expression plasmids were induced by galactose,and then were fully broken by acid-washed glass beads,next to centrifuge to collect the supernatant.Then,the supernatant was used for activity measurements.Enzymatic products were analyzed by HPLC techniques.The results show that rCsUGT84A22 could catalyze the glucose conjugation of a wide range of phenolic acids to form glc-ester.4.The full-length cDNA of C4 H was cloned from tea plant based on EST sequence by RACE technology with a 1 518 bp open reading frame encoding a protein of 505 amino acids.The deduced protein molecular weight was 58.15 kD and its theoretical isoelectric point was 9.29.A 1 840 bp promoter sequence was isolated by genome walking method.The promoter region not only has the basic transcriptional elements of TATA-box and CAAT-box,but also has many potential inducible and tissue-specific cis-acting elements.5.Quantitative RT-PCR analysis showed that the CsC4 H gene expressed in bud,leaf,stem and root.Subcellular localization of CsC4 H indicated that the fusion proteins located in the membrane of chloroplasts,endoplasmic reticulum and plasma membrane.6.The recombinant plasmids of pYES-CsC4 H was constructed and transformed into expression of host Saccharomyces cerevisiae WT11.The substrate was added into yeast culture for activity measurements.Then the yeast cells were centrifuged and the supernatant loaded onto HPLC for assay.The results show that rCsC4 H could catalyze the cinnamic acid para-hydroxylated to generate p-coumaric acid.