防治花生蛴螬高效特异启动子的功能分析及调控元件作用机制初探

Peanut roots and pods were damaged by subterranean pests white grubs which caused severe reduction. If the insecticidal genes from Bacillus thuringiensis could be highly expressed in peanut roots and control newly hatched larvae of white grubs effectively during growing season, it will provide an new way to control subterranean pests white grubs without chemical pesticide. Insecticidal genes were not able to be expressed highly in peanut roots driven by constitutive promoters, and no studies about root-specific promoter and regulatory elements were reported. Three promoters, AhRam, AhGAox and AhBsp have been cloned through the method of mining tissue-specific promoters based on gene digital expression profile. The project is planned to determine the core promoter and key regulatory elements; explore the mechanism of recruitment transcription factors by regulatory elements through analyzing the effects of the arrangement of key regulatory elements on the promoter activity; and assay the expression level of cry8-like gene cloned by our lab and its activity against white grubs. The aim is to choose efficient promoters suitable for control of white grubs, guide the design of root-specific promoters, lay the molecular foundation for development of peanut varieties resistant to subterranean insect pests. This will be important for academic significance and applictions.

地下害虫蛴螬等啃食花生嫩根、果荚，造成花生严重减产，如能将来自苏云金芽胞杆菌的杀虫基因在花生根部高效表达，在整个生长期都能有效抵御蛴螬危害，将为此类地下害虫的绿色防控开辟新的途径。针对组成型启动子无法指导杀虫基因在花生根部的高效表达，国内外又缺乏花生根特异启动子及调控元件研究的现状，申请人通过基因数字表达谱挖掘组织特异启动子的方法，目前已克隆三个启动子AhRam、AhGAox和AhBsp，本项目拟确定三个启动子的核心区及关键调控元件；通过分析关键调控元件排列方式对启动子表达特异性的影响，探索根特异元件招募转录调控因子机制；以课题组克隆的对蛴螬高毒力新基因资源为材料，评价候选根特异启动子指导cry8-like基因的表达水平及对蛴螬的防治效果。筛选可用于防治蛴螬的根部特异启动子，指导根部特异启动子的定向设计，为抗地下害虫转基因花生品种的创制奠定分子基础，具有重要的理论意义和潜在的应用价值。

地下害虫蛴螬等啃食花生嫩根、果荚，造成花生严重减产。针对组成型启动子无法指导杀虫基因在花生根部的高效表达，国内外又缺乏花生根特异启动子及调控元件研究的现状，根据花生基因数字表达谱结果，筛选了4个候选根特异表达的contig，并且克隆了4个候选基因的启动子序列。根据启动子区表达元件预测结果，对4个候选启动子分别设计了截短片段，构建了相应启动子片段驱动GUS基因的表达载体，转化烟草进行功能验证。GUS染色结果表明，AhMtan、AhNDrp、AhGAox和AhNramp启动子都为根特异表达启动子，四个启动子的核心区分别为起始密码子上游235 bp、1360 bp、518 bp和920 bp片段。通过设计启动子截短实验及人工合成启动子，发现ROOTMOTIFTAPOX1元件数量影响启动子根特异表达特性。构建了根特异启动子AhNramp、AhMtan和SPSR驱动的cry-8like基因表达载体，启动子AhNramp可驱动的cry-8like基因在花生根中的高效表达。共发表文章3篇，其中SCI源文章2篇，获得国家发明专利2项。