野生大豆鲨烯单加氧酶基因GsSQM1在抗棉铃虫反应中的分子调控机制及功能的解析

Wild soybeans have excellent insect-resistant traits, and are relatives of cultivated soybean. Plant brassinosteroids (BRs) are potential natural materials of plant insect-resistant, squalene monooxygenase is a key enzyme in the early stage of BR synthesis pathway. It is important to study the molecular regulation mechanism of squalene monooxygenase gene in wild soybean after wild soybean damaged by insects and how the gene affects the BR constituents in transgenic plant. The present study use yeast one-hybrid library system and yeast two-hybrid library system to screening regulatory factors of squalene monooxygenase gene 1(GsSQM1). Meanwhile, the present study also use promoter site mutation、EMSA、BIFC and pull down technologies to further validation the results of upstream regulatory factors interactions and finally clarify the molecular regulator mechanisms in wild soybean after damaging by insect. On the other hand, the present study also try to clarify how the GsSQM1 gene affect plant resistance to insect pest in heterologous plant Arabidopsis thaliana and homologous plant wild soybean by transgenic technology (over expression/RNAi). These research results will provide new theoretical basis and technological approaches for using germplasm resources of wild soybean for insect resistance genetic improvement of soybean varieties.

野生大豆具有优良的抗虫性状，与栽培大豆是近缘种。植物油菜素甾醇类物质是潜在的天然植物抗虫物质，鲨烯环氧酶是植物油菜素甾醇类物质前期合成的关键限速酶。研究该酶基因在野生大豆虫害后的分子调控机制及在转基因植物中的抗虫功能具有积极的意义。本项目拟通过酵母单杂交文库和酵母双杂交文库系统筛选棉铃虫害后野生大豆鲨烯单加氧酶基因（GsSQM1）的调控因子。同时，本研究辅以启动子位点突变实验、凝胶阻滞实验（EMSA）、双分子荧光互补（BIFC）实验和pull down实验验证结果，明确GsSQM1基因的虫害响应分子调控机制。另一方面，通过转基因手段在模式植物拟南芥（GsSQM1过表达）和野生大豆（GsSQM1过表达\ GsSQM1沉默干扰）中影响GsSQM1基因表达，探明其在同源和异源植物中的对植物抗虫能力的影响。研究结果将为利用野生大豆种质资源进行大豆抗虫品种遗传改良提供新的理论依据和技术途径。

棉铃虫是一种杂食性的害虫，在大豆种植发育过程中可以啃食叶片造成大豆严重减产。野生大豆具有优良的抗虫特性，是珍贵的抗虫种质资源来源。野生大豆ED059被鉴定了具有显著的抗虫性，可以作为抗虫优异基因来源。本项目利用前期的工作用野生大豆ED059中的油菜素内酯合成限速酶基因GsSQM1基因作为出发点，构建了超表达转基因大豆材料，基因编辑敲除大豆材料。抗虫性实验表明，超表达GsSQM1基因可以提高大豆的抗虫性。同时，利用超表达转基因大豆材料进行了转录组学测序和代谢组学检测。综合转录组学和代谢组学数据发现超表达大豆材料相对于未转基因大豆显著影响了大豆此生代谢合成途径和植物激素信号转导途径。基于以上分析，本项目还鉴定了一个具有潜在抗虫功能的MYB基因GsMYB15。最后本项目成功创制了野生大豆ED059的酵母筛选文库，后续的GsSQM1调控基因筛选工作正在进行中。