目的 探究灰毡毛忍冬Lonicera macranthoides中查耳酮合酶LmCHS1基因对黄酮类成分合成的作用。方法 以灰毡毛忍冬叶的cDNA为模板，利用RT-PCR技术克隆得到了LmCHS1基因开放阅读框（open reading frame,ORF）序列，并对其基因序列进行生物信息学分析。通过农杆菌侵染的方式，构建本氏烟草瞬时转化体系，对LmCHS1编码蛋白进行亚细胞定位；紫外分光光度法检测烟草叶片及灰毡毛忍冬样品中总黄酮含量；构建原核表达载体，诱导大肠杆菌得到重组蛋白；利用qRT-PCR检测LmCHS1基因在灰毡毛忍冬花、茎和叶中的表达情况。结果 克隆得到的基因长度为1 182 bp，共编码393个氨基酸，分子式为C₁₉₁₄H₃₀₇₀N₅₁₄O₅₆₃S₁₇，理论等电点为6.34，属于亲水蛋白。亚细胞定位结果表明LmCHS1蛋白主要定位在细胞质，与大部分物种中具催化黄酮前体合成功能的CHS蛋白的亚细胞定位一致。原核表达得到的重组蛋白，大小为43 000。总黄酮的含量测定结果显示过表达LmCHS1烟草叶片总黄酮含量高于空载组，并具统计学差异。联合表达量和总黄酮含量结果分析发现，LmCHS1表达量与总黄酮含量呈正相关。结论 LmCHS1蛋白定位在细胞质中，正向调控黄酮类物质的合成。

Objective To investigate the function of chalcone synthase of Lonicera macranthoides (LmCHS1) gene in the flavonoid biosynthesis. Methods The open reading frame (ORF) sequence of LmCHS1 was cloned from leaf cDNA using RT-PCR technology, followed by comprehensive bioinformatic analysis. The subcellular localization of the encoded protein was determined by constructing a transient transformation system in Nicotiana benthamiana via Agrobacterium infection. Total flavonoid content was quantified by ultraviolet spectrophotometry in both tobacco leaves and L. macranthoides samples. Prokaryotic expression vectors were constructed to obtain recombinant proteins through Escherichia coli induction. qRT-PCR was employed to detect the expression levels of LmCHS1 gene in flowers, stems, and leaves of L. macranthoides. Results The cloned gene was 1 182 bp in length, encoding 393 amino acids, with a molecular formula of C₁₉₁₄H₃₀₇₀N₅₁₄O₅₆₃S₁₇ and a theoretical isoelectric point of 6.34, which belongs to a hydrophilic protein.. Subcellular localization demonstrated cytoplasmic localization of LmCHS1 protein, consistent with that of most CHS proteins which catalyze the synthesis of flavonoid precursors in other species.. The recombinant protein obtained from prokaryotic expression was 43 000 in size. Flavonoid quantification showed significantly higher content in tobacco leaves overexpressing LmCHS1 compared to empty vector controls, with statistical significance. The integrated analysis of qRT-PCR data and total flavonoid content measurements revealed a positive correlation between the expression level of LmCHS1 and the total flavonoid content. Conclusion LmCHS1 protein s localized in the cytoplasm and positively regulates flavonoid biosynthesis.

R286.12

国家自然科学基金资助项目（82373992）；国家现代农业产业技术体系（CARS-21）；湖南省中药材产业技术体系专项（HARS-11）；2020年湖南省一流专业建设点：中药资源与开发；湖南中医药大学重点学科中药学科（校行发规字[2023]2号）