利用植物病毒表达载体来筛选玉米黏虫有效干扰RNA的研究

RNA interference is a technology used for silencing of target genes via sequence-specific manner. There are already many examples and even practical implementation of RNAi-based technologies for development of anti-pathogenic crops. In 2007, researchers from Monsanto and CAS reported the development of transgenic corn and cotton resistant to insect herbivore using RNAi technology, providing a species-specific and environmentally sound anti-pest strategy. Recently, outbreaks of Mythimna separata severely threatens corn production in Northern China, calling for new control approaches. We have successfully carried out dsRNA-mediated RNAi experiment in some insect species including mythimna separata. We propose here to develop Mythimna separate-resistant plants using RNAi technology. Our primary hypothesis is that if we can identify and deliver to Mythimna separata interfering RNAs that down-regulate expression of Mythimna separata genes essential for their development and insecticide resistance, they will result in a debilitating phenotype. We will take transcriptome sequencing technique to find unigenes in mythimna separata and analyze RNAi machinery genes.The interfering sequences against the candidate genes will be cloned into plant viral vectors like TMV and TRV. Plant hosts such as N. Benthamiana will be infected with the recombinant virus carrying insect sequences, and produce the dsRNA and siRNA for the insert sequences. The infected plants will be fed to Mythimna separata, and the RNAi effects toward endogenous complementary genes would then be evaluated by these high-throughput screening method. The effective sequences, especially those which caused phenotypic effects, will be cloned into plant binary vector. The recombinant binary vectors will be transformed into plant host to express hairpin dsRNA for the insert sequences, and the transgenic plants will be evaluated for the RNAi efficacy toward the feeding herbivore insects.

RNAi是序列特异的基因沉默技术。RNAi在植物抗病领域得到了广泛的应用，2007年美国Monsanto公司及中国科学院研究人员利用RNAi 技术分别获得了抗虫转基因玉米和棉花，为农业害虫的防控提供了新的思路和途径。近几年，玉米黏虫在我国华北地区爆发成灾，迫切需要探索有效的控制途径。申请者在前期研究中已对黏虫在内的几种昆虫成功进行了dsRNA介导的RNAi实验。本研究拟对玉米黏虫进行转录组测序，通过生物信息学方法组装一些新的unigene，分析RNAi 过程相关基因及黏虫RNAi机制。同时挑选对黏虫生长发育及抗药性产生至关重要的基因做为候选基因，利用不同植物病毒载体如TMV、TRV等高通量表达针对上述候选基因的干扰序列，筛选出抑制这些关键基因的干扰RNA序列。随后将上述干扰序列以hairpin dsRNA形式转基因表达，进一步探讨通过RNAi技术培育抗玉米黏虫转基因植物的技术路线和方法。

RNAi是序列特异的基因沉默技术，在植物抗病领域得到了广泛的应用，并被用于抗虫作物的培育。玉米黏虫在我国华北地区爆发成灾，迫切需要探索有效的控制途径。本项目中我们首先对玉米黏虫进行了转录组测序，找到了12类RNAi机制相关的基因，为利用RNA干扰技术来控制玉米黏虫奠定了基础。我们从注释到NR数据库中的Unigenes中找到了98条序列与RNAi机制相关，这些序列分别被注释到12类基因。从中选择35条Unigenes进行了RT-PCR验证，27条可以获得预期长度的基因片段，它们分属于8类基因，分别编码Sid、 Dicer、Argonaute、 PIWI、Loquacious、SR-C、Juvenile hormone esterase和juvenile hormone epoxide hydrolase。我们挑选了对玉米黏虫生长发育至关重要的Chitinase等基因做为候选基因，设计合成和检测了抑制这些基因的干扰RNA 序列。为了达到RNAi效果，我们设计了四个实验。第一，体外合成针对靶基因的dsRNA，并与人工饲料混匀，饲喂玉米黏虫。第二，细菌体内合成了dsRNA, 并与人工饲料混合，饲喂玉米黏虫。第三，为了检测植物体内表达的dsRNA所介导的RNAi效应，本研究将干扰片段克隆到植物病毒载体TMV（PJL36），将克隆载体侵染了本氏烟草并饲喂了玉米黏虫。第四，利用gateway技术构建了针对两个Chitinase基因（MsChi1和MsChi2）的RNAi干扰载体，将表达MsChi1和MsChi2基因hairpin dsRNA的pCB2004B重组载体转化水稻，成功获得了转基因T0代，T1代及T2代苗，进行了玉米黏虫的喂饲实验。随后，我们通过测定以下指标来检测了上述四种方法介导的RNAi效应。荧光定量PCR结果表明，相应靶标基因的表达量显著降低，同时我们也利用序列特异的探针检测到了对应的siRNA,说明通过体外合成的dsRNA和植物体病毒复制所产生的干扰RNA被玉米黏虫吸收后均引起了RNAi效应。另外，MsChi1、MsChi2基因的干扰导致了受试玉米黏虫的体重减少及死亡率增加。本研究为利用RNAi来防治玉米黏虫提供了实验依据及其理论基础。