Studied On The Bioinformatics Of SCPL And Function Of CsSCPL3in Tea Plant [Camellia Sinensis]

Whether there is galloyl group at the C-3postion of catechins makes a difference ingallated catechins and non-galloylated catechins in tea plant. Our previous works haveshown that the enzyme catalyzed the gallate acylation of catechins was an acyltransferasewhich may belong to SCPL family.In order to investigate genes involved in gallic acyltransferase, we first used thebioinformatics to select SCPL in tea transcriptome and genome database, then performedmotifs and phylogenetic analysis and function prediction. The full-length cDNA ofCsSCPL3is cloned in tea plant by RACE, and the tissue-specific expression and functionof the gene was predicted by quantitative RT-PCR,prokaryotic expression,eukaryoticexpression and heterologous model plant expression.1.49CsSCPL proteins were divided into three groups by Arabidopsis classificationmethod.9、8、17and15ESTs belonged to Clade IA, Clade IB, CladeⅡ and CladeⅢ,respectively. And acyltransferase-related proteins belonged to Clade IA class.2. The CsSCPL3from Clade IA was cloned by RT-PCR, the cDNA full length ofCsSCPL3was1686bp, containing a1413bp complete open reading frame, CsSCPL3codes471amino acid. The size of expected encoded protein was54.7KD, and itsisoelectric point was5.81.3. CsSCPL3gene was predicted and analyzed by bioinformatics. The results showedthat CsSCPL3protein had S10conserved domain and belonged to α/β hydrolase foldsuperfamily. The CsSCPL3protein contained SCPL family’s characteristic structures, suchas one substrate binding and three catalysis conserved regions, a number ofN-glycosylation sites and a conserved catalytic triad Ser-Asp-His amino acid activecatalytic site. It is predicted that CsSCPL3protein might be secreted proteins. CsSCPL3protein had four protein modified ways, including N-glycosylation site, protein kinase Cphosphorylation sites, casein kinase Ⅱp hosphorylation sitesand N-myristoylation sites.4. Quantitative RT-PCR analysis showed that the CsSCPL3gene is expressed in bud,leaf, stem and root. The relative expression of CsSCPL3in leaves was significantly higherthan that in stems and roots, while CsSCPL3was highly expressed in the mature leavesthan that in young bud.5. The CsSCPL3gene was constructed into expression vector pET-32a(+) for overexpression in prokaryotic cells, and optimal inducing conditions of inducing time and temperature were studied. The optimum expression condition was4hours at37℃.TheSDS-PAGE showed that recombinant proteins with formula weight70KD were inducedsuccessfully by IPTG, which coincided with the prediction. Unfortunately, the fusionproteins were existed with inclusion body protein.6. Recombinant plasmids pYES-dest52-CsSCPL3was successfully constructed, whichcould be successfully expressed in S.cerevisiae. The HPLC results showed thatrecombinant protein had no activity yet.7. Recombinant plasmid pCB2004-CsSCPL3was constructed then transformed intoAgrobacterium tumefaciens EHA105by electroporation. Transgenetic tobacco withpCB2004-CsSCPL3was obtained and the function of CsSCPL3in it remained to beverified.