麦长管蚜肠道RNAi靶标基因的筛选鉴定与抗蚜转基因小麦新种质创制

Aphids are major agricultural pests which cause significant yield losses of wheat each year in China. Increased outbreak of aphids are reported regularly in Chinese wheat production area. Present attempts to improve the aphid resistance of wheat through conventional breeding in China are being confronted by slow progress due to lack of aphid resistant wheat germplasm, the complexity of plant-aphid interactions and the rapid development of resistant pest biotypes. Therefore, exploring new type of aphid resistant genes and generation of transgenic wheat engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. In this project, Illumina's RNA sequencing (RNA-seq) is used to screen potential RNA interference (RNAi) targets from the guts of grain aphid upon feeding on wheat plants; In vitro dsRNA synthesis, dsRNA added to artificial diet and qRT-PCR analysis are employed to select optimal RNAi targets in grain aphid. Vectors harboring the dsRNAs of the RNAi targets will be constructed by using gateway system and will be transferred into wheat through Agrobacterium-mediated transformation. Southern blot and Northern blot analyses will be performed to characterize the integration, expression of the target SiRNAs. Aphid bioassay will be used to determine the aphid resistance of transgenic wheat plants and their effects on the expression of the aphid corresponding target genes upon feeding on these plants. The aims of this project are to identify the optimal RNAi targets in the gut of grain aphid and their potential functions, and generate a novel type of aphid resistant transgenic wheat through plant mediated RNAi technology.

蚜虫是危害我国小麦生产的主要害虫之一。由于小麦抗蚜种质资源匮乏，常规育种难以奏效，致使蚜虫为害面积逐年增加，给小麦生产造成巨大经济损失。挖掘和利用新型抗蚜基因，培育转基因抗蚜小麦新种质，具有重要科学意义和应用前景。本项目拟在对取食前、后麦长管蚜肠道转录组测序和基因表达谱分析基础上，利用dsRNA体外蚜虫饲喂试验和qRT-PCR分析，筛选和鉴定麦长管蚜肠道中与消化吸收相关的RNAi靶标基因；采用Gateway技术构建靶标基因dsRNA植物表达载体；农杆菌介导转化小麦，获得转基因小麦；Southern 和Northern blot分析，检测靶标基因dsRNA在转基因小麦中整合和SiRNA表达特性；通过蚜虫抗性鉴定，解析转基因小麦抗蚜特性及其对蚜虫靶标基因表达影响。目的旨在获得和明确麦长管蚜肠道中与消化吸收相关的关键RNAi靶标基因及其功能，并利用植物介导的RNAi技术创制抗蚜转基因小麦新种质。

蚜虫是危害小麦生产的主要害虫之一，目前我国小麦品种中抗蚜种质资源匮乏，蚜虫为害面积日趋增加，给我国小麦生产造成巨大经济损失。挖掘和利用新型、安全抗蚜基因对通过基因工程培育小麦抗蚜新种质具有重要意义。植物介导的RNAi技术已成为农作物抗虫基因工程的热点之一, 但在小麦抗蚜中的应用研究尚无报道。本项目率先完成了麦长管蚜转录组测序和取食前、后的肠道转录组测序及基因差异表达分析；通过体外双链RNA饲喂，筛选出10个蚜虫致死效果较好的RNAi靶标基因；将三个RNAi靶标基因分别通过基因枪和农杆菌介导的遗传转化，转入小麦和烟草，蚜虫生测结果表明，RNAi转基因小麦和烟草表现出明显的抗蚜特性。本研究结果为通过RNAi系统性传导而沉默其相应的基因，进而抑制蚜虫危害，培育抗蚜转基因小麦新种质奠定了基础。相关研究获得授权专利6项，PCT专利1项；发表论文6篇，其中SCI论文5篇（BMC Genomics;Theor Appl Genet; Funct Integr Genomics; Pest management science; International Journal of Molecular Science）。培养毕业博士研究生1名，硕士研究生2名。