Cloning And Expression Analysis Of SCPL1and SCPL2Gene Related To The Acyl Transfer Function In Camellia Sinensis

Galloylated catechins, as the main components of tea polyphenols and the most important secondary metabolites in tea plant, are also the main chemical components to determine the quality of tea. Looking for key genes involved in galloylated catechins biosynthesis is of great significance for the study of the biosynthetic pathway of galloylated catechins, laying the foundation for tea breeding and the improvement of tea quality.Previous research has basically proved the biosynthetic pathway of galloylated catechins. with which1-O-galloyl-β-D-glucose O-galloyltransferase （ECGT） is directly correlated and might belong to the SCPL acyltransferase family.Using RACE, RT-PCR, prokaryotic expression, eukaryotic expression systems and western blot,HPLC technology, the two sequences SCPL1and SCPL2selected from the Tea Transcriptome Data were cloned, and the recombinant proteins of it were expressed and functionally verified, which provided technical support for screening and functional verification of ECGT gene in tea plant.The main research achievements were as follows.1. The sequences of CsSCPL1and CsSCPL2were cloned by RACE clone technology, the open reading frame （ORF） of the two sequences were analyzed by DNAMAN software. The results showed the complete ORF of SCPL1gene is1455bp, coding484amino acids, predicting molecular weight55.2KD, isoelectric point4.65; the complete ORF of SCPL2gene is1458bp, coding485amino acids, predicting molecular weight54.5KD, isoelectric point5.31.Via SignalP3.0software, it is predicted that41amino acids of CsSCPL1’s N-terminal was signal peptide sequence,43amino acids of CsSCPL2’s N-terminal was signal peptide sequence. Evolutionary tree indicated that the identities of amino acids of CsSCPLl and XP₀04289771[Fragaria vesca subsp.vesca] respectively was55.05%and the identities of amino acids of CsSCPL2and BAH89272.1[Diospyros kaki] respectively is53.16%, together belonged to SCPL Clade I A subgroup, predicting that the two genes were likely to have acyltransferase function.2. Expression levels of CsSCPL1and CsSCPL2in tea leaves at different development stages and different organs were studied by qRT-PCR. The results indicated that the expressions of CsSCPL1and CsSCPL2existed in bud, first leaf, second leaf, third leaf, forth leaf, stem and root, the relative expression level of CsSCPL1in bud was highest, the relative expression of CsSCPL2in root and bud were the higher, the relative expression levels of the latter in second leaf, third leaf, forth leaf, stem were higher than the former.3. Expression host Rosetta （DE3） was transformed by the recombinant plasmids of pET-SCPL1and pET-SCPL2constructed and the expression of target protein was induced with IPTG. The results showed that the two recombinant proteins were both accumulated in inclusion body. The recombinant protein of CsSCPL2could be used for the preparation of antibodies, which will be convenient for Western-Blot detection. The results showed that antibodies could react with the recombinant protein in prokaryotic expression, crude and purified ECGT enzyme solutions from tea plant.4. Saccharomyces cerevisiae host WT11was transformed by the recombinant plasmids of pYES-DEST52-SCPL1and pYES-DEST52-SCPL2constructed and the expression of target protein was induced with galactose; the results of SDS-PAGE indicated that the protein bands of CsSCPL2protein expression were consistent with the subunits of predicted protein; however, the enzyme reaction activity with ECGT.5. Agrobactrium tumefaciens host EHA105was transformed by the recombinant plasmids of pCB2004-SCPL1constructed. Tobacco leaf was infected by Leaf-disc method, getting transgenic plant after screening, which laid the foundation for the follow-up functional verification of SCPL1.