Cloning,Bioinformatics and Prokaryotic Express Analysis of PeCS Gene Encoding Chorismate Synthase in Perilla frutescens
The aim of this study was to explore the key genes in the biosynthetic pathway of phenolic acid,an active medicinal ingredient of Perilla frutescens L.Based on the transcriptome sequencing data of Perilla fru-tescens,chorismate synthase(CS)genes of differentially expressed genes in phenolic acid synthesis pathway were screened out,and the open reading frame(ORF)of PeCS gene was obtained.Bioinformatics technology was used to preliminarily predict the function of PeCS gene.The prokaryotic expression vector pET30a-PeCS was con-structed and transformed into Escherichia coli BL21(DE3).The expression was induced by IPTG and detected by SDS-PAGE.The expression level of PeCS in different tissues(roots,stems and leaves)of Perilla frutescens was detected by real-time quantitative fluorescence PCR(RT-qPCR).The length of open reading frame(ORF)of PeCS gene was 877 bp,encoding 291 amino acids,which was predicted by bioinformatics to be a hydrophilic protein,located in chloroplasts.The relative molecular weight of the protein was 31 659.16.The PeCS gene encoded protein contained no signal peptide and no transmembrane region,and its subcellular structure was located in chloroplasts.The constructed prokaryotic expression vector pET30a-PeCS was expressed in Escherichia coli BL21(DE3)under the optimal conditions of 15 ℃ and 0.5 mmol/L IPTG culture for 16 h.The PeCS recombinant protein mainly existed in the form of inclusion body,and the molecular weight of the protein was consistent with the prediction.RT-qPCR analysis showed that PeCS gene was expressed in different organs and tissues of Perilla frutescens,with the highest expression level in leaves and the lowest expression level in stems.The successful cloning of PeCS gene of Perilla frutescens branching acid synthase is helpful to further elucidate the synthesis pathway and regulatory mechanism of Perilla frutescens phenolic acid.
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