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

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.

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。

本项目通过genome-walking从棉花基因组中克隆了D-7基因上游1876bp 的D-7 基因启动子序列，利用生物信息学分析其含有 CAAT-box、TATA-box、LTR、ABRE 和 HSE 等顺式作用元件，通过遗传转化实验确定对干旱、低温、盐和 ABA等不同的非生物胁迫有响应。构建了系列 D-7 基因启动子5'端缺失片段与eGFP连接的表达载体，利用棉花子叶瞬时表达系统和农杆菌介导拟南芥的遗传转化实验检测上述启动子活性，确定了D-7 启动子核心区，同时构建了具有不同干旱元件的系列棉花诱导型启动子和添加了28 bpSynJ（人工合成的1个增强子序列）的启动子，以及棉花干旱胁迫响应蛋白基因（DREB1）启动子，对拟南芥进行转化，筛选出T2代转基因植株；对转基因植株进行各种胁迫处理及相关分析；利用棉花叶片瞬时表达系统对筛选出的具有显著诱导效果的启动子进行分析；构建了D-7:ASGNA（ASGNA：抗虫基因）及干旱胁迫诱导型启动子G5:ASGNA，对棉花进行了遗传转化。上述研究成果对棉花 D-7基因5’上游的调控序列与各种胁迫因子的关系有了较好的解析；确定重要调控元件的准确位置；构建出适应于干旱胁迫诱导的启动子及新的组成性启动子，授权专利两项，有望应用于实践，培育新的干旱胁迫诱导型的转基因抗虫棉。已发表相关文章4篇，另有两篇待发表；参加棉花年会3次，共计10人，参加生物化学与分子生物学年会2次，共计6人，参加植物生物学大会2次，共4人；培养研究生5名（已毕业）。