棉花诱导型启动子D7-P的克隆、鉴定及其重要调控元件的功能解析

利用转基因技术培育棉花新品种是当前的热点研究问题，但目前转基因棉花培育中常用的组成型启动子存在明显不足。本研究采用与植物抗性密切相关的第三组Lea基因D-7的启动子为研究对象，通过外源接头介导的TAIL-PCR技术从棉花中克隆D-7基因的启动子序列，利用生物信息学进行顺式作用元件的分析预测；并构建全长和 5′端系列缺失及定点突变片段与报告基因连接的表达载体，转化拟南芥；通过细胞生物学和分子生物学技术分析不同胁迫条件下转基因植株中报告基因的表达，探讨温度、ABA、干旱等因素对启动子的调控机制及主要顺式作用元件的精确位置；在此基础上，利用Over-lap PCR将核心启动子元件与各类胁迫诱导元件进行拼接、组合，构建出可响应混合或单一胁迫处理的多种启动子；以eGFP为报告基因，利用转基因拟南芥，棉花叶片瞬时表达系统对上述启动子进行诱导特性及诱导效率分析，以获得多个具有实用价值的新型诱导型启动子。

Using transgenic technology to cultivate new cotton varieties is popular topic in cotton transgenic research. However,constitutive promoter CaMV 35S used in transgenic cotton still exist some obvious defects. In this project, we focus on the promoter of D-7, a group three of Lea gene, which has a close relationship with stress tolerance of plant. The upstream region of the D-7 gene was cloned using adaptors mediated TAIL-PCR for directional genome walking. Based on the bioinformatic analysis of the sequence, a series of expression vectors carrring eGFP driven by the promoters with the deletion or point mutation at 5' end was constructed, then transformed into Arabidopsis thaliana, respectively. The expression of eGFP in the transgenic plants was evaluated by molecular biology and cell biology techniques.The regulating mechanism of different stresses treatment on promoter activity will be explored, the exact position of cis-elements present in promoter will be also identified. On the basis of the studies above-mentioned , a multiple of promoters with response to mixed or single stress treament will developed by the combination of core element with kinds of stress responsive elements using over-lap PCR. The inducing features and efficiency of the promoters will be investigated by the expression of reporter gene eGFP in transgenic plants and transient expression system of cotton. A multiple of novel promoters will be obtained for cotton transgenic engineering based on the results from the project.