三叶青ThMYBPAR基因的克隆及组织差异表达分析  

Cloning and analysis of tissue-specific expression patterns of ThMYBPAR gene in Tetrastigma hemsleyanum

作　　者：王子玥 杨锋 王红燕 邢巧月 白岩[1,2] WANG Ziyue;YANG Feng;WANG Hongyan;XING Qiaoyue;BAI Yan(Zhejiang Provincial Key Laboratory of Resources Protection and Innovation of Traditional Chinese Medicine,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;College of Food and Health,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China)

机构地区：[1]浙江农林大学浙江省中药资源保护与创新重点实验室,浙江杭州311300 [2]浙江农林大学食品与健康学院,浙江杭州311300

出　　处：《浙江农林大学学报》2025年第2期273-280,共8页Journal of Zhejiang A&F University

基　　金：浙江农林大学人才启动项目(2024LFR011);浙江省“尖兵”“领雁”研发攻关计划项目(2023C02054)。

摘　　要：【目的】黄酮类成分是三叶青Tetrastigma hemsleyanum药材品质的物质基础,探究ThMYBPAR基因对三叶青块根和叶片中黄酮类成分的合成调控作用,可为三叶青药材品质形成的分子机制提供理论依据。【方法】以近3年生的三叶青块根及叶片为材料,测定总黄酮和原花青素的质量浓度以及抗氧化能力,设计引物克隆ThMYBPAR基因,利用生物信息学分析ThMYBPAR蛋白的序列特征和系统进化关系,通过激光共聚焦显微镜观察其亚细胞定位,采用实时荧光定量PCR(RT-qPCR)分析ThMYBPAR基因在三叶青块根和叶片中的表达模式,并分析ThMYBPAR基因表达水平与黄酮类成分的相关性。【结果】三叶青块根中总黄酮及原花青素质量浓度分别是0.55和1.77 g·L^(-1),显著高于叶片(P<0.05),而且块根提取物的ABTS自由基清除率高达90%,说明三叶青块根和叶片间的药材品质存在差异。根据前期转录组数据克隆得到1个长度为888 bp的ThMYBPAR基因,可编码产生310个氨基酸组成的蛋白,与葡萄Vitis vinifera基因中的VvMYBPAR基因高度相似,具有典型的R2和R3 DNA结合功能结构域。系统进化分析结果表明:三叶青中的ThMYBPAR属于R2R3-MYB第5亚家族转录因子,且主要定位在细胞核中。RT-qPCR分析结果显示:ThMYBPAR基因主要在三叶青块根中表达,且表达量与总黄酮和原花青素质量浓度呈极显著正相关(P<0.01)。【结论】ThMYBPAR基因参与调控三叶青中原花青素的合成机制,推测不同表达水平可能是影响三叶青块根和叶片品质的主要原因。[Objective]Flavonoids are essential components that determine the quality of Tetrastigma hemsleyanum.This study aims to investigate the regulatory role of the ThMYBPAR gene in flavonoid biosynthesis in both the tuberous root and leaves of T.hemsleyanum,thereby providing a theoretical foundation for understanding the molecular mechanisms underlying quality formation in this plant species.[Method]The tuberous root and leaves of 3-year-old T.hemsleyanum plants were analyzed by determining their content of proanthocyanidins,total flavonoids,and antioxidant capacity.Primers were designed for the cloning of the ThMYBPAR gene.The sequence characteristics and evolutionary relationships of the ThMYBPAR protein were assessed through bioinformatics analysis.Additionally,the expression patterns of the ThMYBPAR gene in both tuberous root and leaves were examined using real time fluorescence quantitative PCR(RT-qPCR),alongside an investigation into the correlation between ThMYBPAR gene expression levels and flavonoid content.[Result]The content of total flavonoids and proanthocyanidins in the tuberous root were 0.55 and 1.77 g·L^(-1) respectively,which were significantly higher than those in the leaves(P<0.05).Moreover,the ABTS free radical scavenging rate of the tuberous root extract reached 90%,indicating notable differences in the quality of herbal material between the tuberous root and leaf samples.Previous transcriptomic analyses had demonstrated that the ThMYBPAR gene,which consisted of 888 bp and encoded a protein comprising 310 amino acids,showed a significant degree of similarity to the VvMYBPAR gene found in grapevines(Vitis vinifera).The protein contained distinctive DNA-binding functional domains,R2 and R3,and was classified within the R2R3-MYB transcription factor subfamily 5,with a preferential localization in the nucleus.RT-qPCR analysis revealed that the expression of the ThMYBPAR gene was primarily detected in the tubers of T.hemsleyanum,exhibiting a positive correlation with the content of total flavonoids a

关 键 词：三叶青 基因克隆 ThMYBPAR基因 原花青素 

分 类 号：S718.3[农业科学—林学]