三叶青查尔酮异构酶基因的克隆及序列分析

Cloning and sequence analysis of chalcone-flavanone isomerase gene from Tetrastigma hemsleyanum Diels et Gilg

目的 阐明三叶青中查尔酮异构酶(CFI)的结构基因信息及其在黄酮生物合成途径中表达调控机制。方法取三叶青新鲜叶片提取总RNA,以所提的总RNA为模板,随机引物(N8)作为引物进行逆转录反应;以逆转录产物为模板,使用正向引物CFI-F与反向引物CFI-R进行PCR;PCR产物经回收纯化后与克隆载体pMD18-T连接并转化至大肠杆菌DH5a,筛选阳性克隆进行测序。结果 测序结果分析显示,该基因片段长为760 bp,含一个长为714 bp的开放阅读框,编码237个氨基酸(NCBI登录号:OP358477)。NCBI数据库BLAST搜索分析显示,该基因编码产物与CFI高度同源;进化树分析表明三叶青的CFI基因与大齿牛果藤的亲缘关系最近。结论 本研究为进一步探究三叶青黄酮类成分的生物合成机制奠定基础。

Objective To elucidate the structural gene information of chalcone-flavanone isomerase(CFI)in Tetrastigma hemsleyanum Diels et Gilg(known as Sanyeqing in Chinese)and its expression and regulation mechanism in the flavonoid biosynthesis pathway.Methods Total RNA from fresh leaves of Tetrastigma hemsleyanum Diels et Gilg were extracted and used as the template,and random primers(N8)were used as primers for reverse transcription reaction;With reverse transcription products as templates,PCR was performed using forward primer CFI-F and reverse primer CFI-R;After recovery and purification,the PCR products were linked to the cloning vector pMD18-T and then transformed into E.coli DH5a.The positive clones were screened for sequencing.Results The sequencing results showed that the gene fragment was 760bp long and contained an open reading frame of 714 bp,encoding 237 amino acids(NCBI login number:OP358477).The BLAST search analysis of the NCBI database showed that the coding product of the gene was highly homologous with the CFI;Phylogenetic tree analysis shows that the CFI gene of Tetrastigma hemsleyanum Diels et Gilg has the closest genetic relationship with Nekemias grossedentata.Conclusion This study lays the foundation for further exploration of the biosynthetic mechanism of flavonoids in Tetrastigma hemsleyanum Diels et Gilg.