Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua.
Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua.
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转录组分析和筛选 RNAi 介导的甜菜夜蛾害虫防治的潜在靶基因
DOI：
10.1371/journal.pone.0065931
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Li F
中科院分区：
文献类型：
--
作者：
Li H;Jiang W;Zhang Z;Xing Y;Li F
甜菜夜蛾（Spodoptera exigua（Hübner））是一种世界性的严重害虫，对作物造成重大损失。不幸的是，甜菜夜蛾的遗传资源极其稀缺。为了改善这些资源，我们对S.使用Illumina Solexa平台，对代表所有阶段的甜菜夜蛾进行鉴定，包括卵、第1至第5龄幼虫、蛹、雄性和雌性成虫。我们用Trinity组装了转录组，产生了31,414个重叠群。在这些重叠群中，通过针对NCBI nr数据库的Blast搜索，18，592个被注释为蛋白质编码基因。已经显示，通过dsRNA或siRNA敲低重要昆虫基因是控制昆虫害虫的可行机制。开发有效的RNAi介导的害虫控制技术的第一个关键步骤是找到合适的靶基因。为了筛选甜菜夜蛾的有效靶基因，我们选择了9个候选基因。使用RACE策略扩增这些基因的序列。然后，设计并化学合成siRNA。我们将2 μl siRNA（2 μg/μl）注射到4龄幼虫中以敲除相应的靶基因。靶基因的mRNA丰度在注射siRNA后下降到不同水平（约20-94.3%）。敲低几丁质酶7、PGCP、几丁质酶1、ATP酶、tubulin 1、arf 2、tubulin 2和arf 1等8个基因后，与阴性对照组相比，死亡率显著升高（P<0.05）。在5个基因（PGCP、几丁质酶1、微管蛋白1、微管蛋白2和解旋酶）的siRNA处理组中，约80%的存活昆虫表现出发育迟缓。在几丁质酶1-siRNA和几丁质酶7-siRNA给药组中，12.5%的存活者出现“半蜕皮”。arf 1-siRNA组和arf 2-siRNA组注射后48 h有15%的小鼠体色变黑。因此，该转录组可作为一种有价值的遗传资源，用于S.本研究为RNAi防治甜菜夜蛾提供了可能的靶点。
The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1st to 5th instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20–94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited “half-ecdysis”. In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.
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