橡胶树HbMVK启动子的克隆与缺失表达分析

摘要

为了研究橡胶MVK基因启动子精细结构及功能,笔者利用PCR技术对橡胶树HbMVK基因启动子进行了克隆,并对其进行了生物信息学分析.结果表明,该启动子序列全长1 696 bp,包含CAAT-box,TATA-box,CAT-box,LTR,GARE-motif,TCA-element等元件.此外,根据元件分布,构建橡胶树HbMVK基因启动子系列缺失和全长序列表达载体并转化拟南芥.T2代转基因拟南芥中,报告基因GUS组织化学染色结果表明:HbMVK启动子全长序列和5'端-1 386 bp缺失,HbMVK启动子驱动的GUS基因只在转基因拟南芥实生苗胚轴中有极微弱表达.5'端-1 221 bp和-725 bp缺失,HbMVK启动子驱动GUS基因表达的活性较强,而5'端-325bp缺失,HbMVK启动子则完全失去活性,这表明启动子核心调控元件位于-725 bp到-325 bp之间.%Mevalonate kinase (MVK) is an important enzyme in natural rubber biosynthesis pathway of rubber tree (Hevea brasiliensis).The promoter of HbMVK gene from rubber tree was cloned by using PCR and analyzed with bioinformatics tools to observe the fine structure and function of rubber tree MVK promoter.The HbMVK promoter sequence is 1 696 bp in length,containing some elements or mortifs such as CAAT-box,TATA-box,CAT-box,LTR,GARE-motif,TCA-element,etc.Based on the element distribution,plant expression vectors were constructed with HbMVK promoter and its serious deletion fragments were fused to reportergene GUS (encoding β-glucuronidase),which were transformed into Arabidopsis thaliana.GUS histochemical staining of T2 transgenic seedlings showed that the full-length HbMVK promoter and its-1386 bp deletion fragment weakly drove GUS expression in transgenic seedling hypocotyls.The 5'-1 221 bp and-725 bp deletion fragments of HbMVK promoter were observed to strongly drive GUS expression,and the-325 bp deletion fragment of HbMVK promoter completely lost promoter activity,suggesting that the core promoter regulatory elements were located between-325 bp and-725 bp.This study might provide an important basis for further research on the regulation of HbMVK expression.