Cloning and Prokaryotic Expression of Glycosyltransferase Genes from Paris polyphylla var. yunnanensis / 中国实验方剂学杂志

ABSTRACT

Objective:To clone the full-length glycosyltransferase genes （<italic>PpUGT</italic>1，<italic>PpUGT</italic>7） related to saponins biosynthesis in <italic>Paris polyphylla</italic> var. <italic>yunnanensis</italic>，and perform bioinformatics analysis，relative expression analysis and prokaryotic expression analysis. Method:Total RNA was isolated from <italic>P. polyphylla </italic>var. <italic>yunnanensis </italic>with use of the Eastep^® Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and the prokaryotic expression vectors were built to heterologously express recombinant protein in <italic>Escherichia coli.</italic> Result:The open reading frame （ORF） of <italic>PpUGT</italic>1 was 1 827 bp，encoding 608 amino acids，and was predicted as a steroid glycosyltransferase；the ORF of <italic>PpUGT</italic>7 was 1 380 bp，encoding 459 amino acids，and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively，and both of them were hydrophilic proteins，no transmembrane domain，no signal peptides，both showing high similarity and conservativeness with homologous sequences. The results of Real-time PCR showed that the expression level of <italic>PpUGT</italic>1 was root>leaf>flower>stem；the expression level of <italic>PpUGT</italic>7 was stem>leaf>flower>root. In addition，PpUGTs proteins were expressed in <italic>E. coli</italic>. in a soluble form. Conclusion:The genes of <italic>PpUGT</italic>1 and <italic>PpUGT</italic>7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs， and their recombinant proteins were successfully expressed in <italic>Escherichia coli</italic>. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in <italic>Paris polyphylla </italic>var. <italic>yunnanensis</italic>.