Cloning and expression analysis of PLR gene in Schisandra chinensis / 中草药

ABSTRACT

Objective: To obtain more information for further researches on mechanism of pinoresinol lariciresinol reductases (PLR) gene, which is the key enzyme gene involved with lignans synthesis in Schisandra chinensis, ScPLR gene and its promoter were cloned and analyzed, and the expression pattern of ScPLR gene at fruit development stages was also illustrated. Methods: On the basis of PLR gene sequence obtained by transcriptome sequencing, specific primers were designed, the open reading frame (ORF) of ScPLR gene was then cloned, and the bioinformation of ScPLR gene was analyzed through online software. Meanwhile, the promoter of ScPLR gene was amplified by TAIL-PCR method and analyzed. The expression patterns of ScPLR from fruits at different development stages were analyzed preliminarily. Results: The length of ScPLR gene ORF was 837 bp, which encoded 278 amino acids residues with molecular weight of 31419.85 and theoretical pI of 8.97; ScPLR consisted of a membrane structure, which was a hydrophobic stable protein without signal peptide, and mainly composed of α-helix and random curl; Subcellular localization prediction result showed that ScPLR protein is mainly located in cytoplasm; The results of phylogenetic analysis revealed that ScPLR is closest related to Ricinus communis PLR. The length of ScPLR gene promoter was 994 bp, which had regulatory elements including TATA-box, CAAT-box, also cis-regulatory elements related to light regulation, auxin response, anaerobic induction, defense and stress response, the presence of various cis-acting elements fully reflected the high efficiency and complexity of promoter regulation on gene expression at the transcriptional level. qRT-PCR results showed that ScPLR expression level displayed obvious up-regulation at fruit swelling stage, then down-regulation at fruit coloring period. Conclusion: The ScPLR gene and its promoter were cloned and analyzed, the trend of high expression level of ScPLR before fruit coloring period was consistent with the dynamic change of lignans accumulation in S. chinensis, which will lay foundation for the further research on function and expression regulation of ScPLR gene in lignans biosynthesis pathway.