---

作 者 ：周兴文;李纪元*;殷恒福;范正琪;李辛雷

期 刊 ：植物研究 2013年 33卷 1期 页码：58-65

关键词：金花茶；黄酮醇合成酶；RNA干扰；表达载体构建；

Keywords:Camellia nitidissima Chi, flavonol synthase, RNA interference, construction of expression vector,

摘 要 ：利用RT-PCR和RACE技术，从我国珍稀濒危植物金花茶花瓣中获得了黄酮醇合成酶（Flavonol synthase，FLS）基因的cDNA全长，命名为CnFLS，GenBank登录号JF343560.1。根据该基因序列设计全长扩增引物P3/P4进行PCR扩增，将扩增产物插入PMD18-T载体并转化克隆菌株DH5α，提取质粒DNA酶切鉴定，通过酶切连接的方法将该基因与改造后的pCAMBIA1300表达载体连接，成功构建了该基因的正义表达载体pCAM-CnFLS。根据该基因的保守序列设计了含有酶切位点的特异引物G1/G2，扩增出250 bp的干扰片段并将其克隆到PMD18-T载体，在中间载体pUCCRNAi及表达载体pCAMBIA1300的基础上，通过多次酶切连接，成功构建了金花茶FLS基因的干扰表达载体pCAMRNAi-CnFLS。金花茶正义及干扰植物表达载体的成功构建，为进一步研究该基因的功能及其对花色的调控效应奠定了基础。

Abstract:A full-length cDNA sequence of flavonol synthase (FLS) gene was obtained from petals of Camellia nitidissima using the methods of Reverse Transcription PCR and RACE, and named CnFLS(GenBank accession No.JF343560.1). The sense expression vector pCAM-CnFLS was constructed successfully while the gene FLS amplified by the primers P3/P4 and inserted into PMD18-T vector was connected to expression vector pCAMBIA1300. The RNA interference (RNAi) expression vector was established by many times enzyme digestion and connection on the base of intermediate vector pUCCRNAi, expression vector pCAMBIA1300 and the combined vector PMD18-T with a conserved segment of 250 bp which was amplified by primers G1 and G2. The successful construction of sense and RNAi expression vectors provides a foundation for further studying on CnFLS gene functions and its effects to flower color.

---