麻疯树异戊烯基焦磷酸异构酶基因(IPI)启动子的克隆及瞬时表达分析

Cloning and transient expression analysis of isopentenyl diphosphate isomerase gene (IPI) promoter from Jatropha curcas

摘要:

异戊烯基焦磷酸异构酶（IPI）是麻疯树（Jatropha curcas）萜类合成途径中的关键酶．IPI基因的表达和活性的高低调控了麻疯树中萜类化合物的含量．本文以麻疯树基因组DNA为模板，通过PCR扩增得到麻疯树IPI基因（JcIPI）起始密码子上游1 536 bp的启动子序列．利用在线软件PLACE和PlantCARE分析表明该序列除具备TATA-Box、CAAT-Box等启动子基本元件外，还含有AuxRR-core、TATC-box、MBS、HSE等特异性元件．为确定启动子核心启动区域，构建JcIPI启动子283、550、970、1 276和1 536 bp的5′端缺失片段，并将其分别驱动pBI121载体的葡萄糖苷酸酶（GUS）基因，构建植物表达载体．采用农杆菌介导法转化烟草，在烟草叶片中进行瞬时表达分析．GUS酶活测定结果显示，五个启动子缺失片段都具有启动活性，并随长度增加而增强．

Abstract:

The isopentenyl diphosphate isomerase (IPI) is considered to be a key enzyme involved in terpenoid biosynthesis pathway of Jatropha curcas. In order to study the expression and regulation of isopentenyl diphosphate isomerase (IPI) gene in the terpenoid biosynthesis pathway of Jatropha curcas, a 1536bp promoter fragment of IPI gene was cloned by PCR method. The bioinformatics analysis showed that the fragment contained the conserved promoter sequence, such as TATA-box, CAAT-box. Furthermore, it contained several elements related to auxin response, gibberellin response, disease-related and high temperature. To determine the optimal promoter sequence for gene expression, IPI gene promoter was deleted from its 5′ end to form promoter fragments with 283, 550, 970, 1276 and 1536bp. Such fragments were fused to a β-glucuronidase (GUS) gene. The fused genes were transformed into Nicotiana benthamiana using Agrobacterium tumefaciens-mediated method for transient expression. GUS quantitative fluorometric assays demonstrated that the five different length fragments of the promoter had promoter activities and the activities were increased with the deleted length of promoters.