目的 从五味子中克隆木脂素生物合成途径松脂醇-落叶松脂素还原酶（Pinoresinol-lariciresinol reductases，PLR）基因及其启动子序列，并进行生物信息学与表达分析。方法 根据转录组PLR测序序列设计特异性引物，克隆ScPLR并进行生物信息学分析；采用TAIL-PCR对ScPLR启动子序列扩增，并进行序列分析；采用实时荧光定量PCR（qRT-PCR）分析果实不同发育时期ScPLR的表达。结果 ScPLR基因开放阅读框（ORF）全长837 bp，编码278个氨基酸；ScPLR蛋白相对分子质量31 419.85，理论等电点8.97；具有跨膜结构，无信号肽，为稳定性疏水蛋白，主要由α-螺旋和无规卷曲构成；亚细胞定位预测ScPLR主要定位于细胞质；系统进化显示ScPLR与蓖麻PLR亲缘关系较近。克隆到ScPLR基因启动子长994 bp，具有TATA-box、CAAT-box基本元件、光调控、生长素响应、厌氧诱导、防御和应激反应的多种调控元件；qRT-PCR结果显示ScPLR表达量呈现果实膨大期快速增加而进入着色期迅速降低的变化趋势。结论 获得了ScPLR基因及其启动子序列，ScPLR果实着色期之前高水平表达的趋势和木脂素的动态变化相一致，为进一步研究该基因功能及表达调控奠定了理论基础。

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.

R282.12

2018年全国中药资源普查项目（1102-01042918001）