目的 克隆多花黄精Polygonatum cyrtonema的环阿屯醇合酶（cycloartenol synthase，CAS）基因PcCAS，对其进行生物信息学、重组蛋白表达以及亚细胞定位等分析，为进一步研究多花黄精皂苷生物合成机制提供基础资源。方法 基于多花黄精转录组数据筛选并克隆PcCAS基因，利用生物信息学方法分析其编码蛋白的理化性质、二级结构、三级结构等。利用qRT-PCR对PcCAS进行组织特异性表达分析。构建酵母表达载体转入毕赤酵母进行酵母表达分析。构建亚细胞定位表达载体并侵染烟草叶片，对PcCAS蛋白进行烟草异源表达分析。结果 PcCAS的开放阅读框（open reading frame，ORF）长度为2 286 bp，编码759个氨基酸，属于亲水蛋白，无信号肽。根茎中总皂苷含量与PcCAS表达量显著高于茎、叶、花组织，PcCAS表达量与总皂苷含量呈正相关。酵母表达得到的重组蛋白大小符合86 400的预期。烟草亚细胞定位显示PcCAS蛋白主要定位于内质网中。结论 PcCAS蛋白属于氧化角鲨烯环化酶（oxidosqualene cyclase，OSC）超家族，定位于内质网中，正向调控总皂苷物质的合成。

Objective To clone the cycloartenol synthase (CAS) gene PcCAS from Polygonatum cyrtonema, perform bioinformatic and recombinant protein expression analyses, and determine its subcellular localization etc, thereby providing essential resources for further elucidating the pivotal role of PcCAS in saponin biosynthesis. Methods Based on the transcriptome data of P. cyrtonema, the PcCAS gene was screened and cloned, and bioinformatics approaches were employed to analyze the physicochemical properties, secondary structure, and tertiary structure of its encoded protein. qRT-PCR was used to analyze the tissue-specific expression of PcCAS. A prokaryotic expression vector was constructed and transferred into Escherichia coli for prokaryotic expression analysis. Yeast expression vectors were constructed and transformed into Pichia pastoris for subsequent yeast expression analysis. A subcellular-localization vector was built and infiltrated into Nicotiana benthamiana leaves for the analysis of heterologous expression in tobacco. Results The open reading frame (ORF) of PcCAS is 2 286 bp in length and encodes 759 amino acids. The protein is hydrophilic and lacks a signal peptide. The total saponin content and PcCAS expression level in the rhizome are significantly higher than those in the stem, leaf, and flower, indicating the expression level of PcCAS is positively related with the total saponin content. Yeast-expressed recombinant protein matched the expected molecular weight of 86 400. Subcellular-localization assays in tobacco leaves demonstrated that the PcCAS protein is predominantly localized to the endoplasmic reticulum. Conclusion PcCAS protein belongs to the OSC superfamily of enzymes and is localized in the cytoplasm, positively regulating the total saponin biosynthesis.

R282.12

湖北省自然科学基金资助项目（2025AFD159）；国家现代农业产业技术体系建设专项（CARS-21）；中央财政林业科技推广示范项目（鄂[2024]TG25号）；恩施州科技计划项目（启航专项）(33)