Punjab Agricultural University, Regional Station, Faridkot, Punjab, 151203, India.
Forest Research Institute, Dehradun, Uttaranchal, India.
BMC Mol Biol. 2019 Feb 18;20(1):6. doi: 10.1186/s12867-019-0123-1.
Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time.
From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of CDS identified showed positive Blast hits. The homologs of most of the core RNAi machinery genes were identified in this transcriptome. To select reference genes for reverse-transcriptase real-time quantitative PCR (RT-qPCR) experiments, 14 housekeeping genes were identified in the transcriptome and their expression was analyzed by (RT-qPCR). UbiCE in adult, 28s in nymphs and SOD under starvation stress were identified as the most stable reference genes for RT-qPCR. Feeding dsSNF7 and dsAQP caused 16.4- and 14.47-fold reduction in SNF7 and AQP mRNA levels respectively, when compared to their levels in dsGFP fed control insects. Feeding dsSNF7 or dsAQP also caused 62 and 72% mortality in T. tabaci. Interestingly, simultaneous feeding of dsRNAs targeting SNF7 or AQP and one of the RNAi pathway genes (Dicer-2/Aubergine/Staufen) resulted in a significant reduction in RNAi of target genes. These data suggest the existence of robust RNAi machinery in T. tabaci.
The current research is the first report of the assembled, analyzed and annotated RNAseq resource for T. tabaci, which may be used for future molecular studies in this insect. Reference genes validated across stages and starvation stress provides first-hand information on stable genes in T. tabaci. The information on RNAi machinery genes and significant knockdown of the target gene through dsRNA feeding in synthetic diet confirms the presence of efficient RNAi in this insect. These data provide a solid foundation for further research on developing RNAi as a method to manage this pest.
烟粉虱是洋葱和棉花的严重害虫。由于缺乏对其基因组或转录组的信息，因此在分子水平上对这种昆虫知之甚少。为了在这种昆虫中启动分子研究，对其进行了 RNA 测序；进行了从头转录组组装和分析。使用 RNAseq 数据鉴定了这种昆虫的参考和 RNAi 途径基因。此外，首次在烟粉虱中证明了取食 RNAi。
从组装的转录组中，鉴定出 27836 个编码序列（CDS），每个 CDS 的平均大小为 1236bp。大约 85.4%的 CDS 鉴定出有阳性 Blast 命中。该转录组中鉴定出大多数核心 RNAi 机制基因的同源物。为了选择用于逆转录实时定量 PCR（RT-qPCR）实验的参考基因，在转录组中鉴定出 14 个管家基因，并通过（RT-qPCR）分析其表达。成虫中的 UbiCE、若虫中的 28s 和饥饿胁迫下的 SOD 被鉴定为 RT-qPCR 最稳定的参考基因。与喂食 dsGFP 的对照昆虫相比，喂食 dsSNF7 和 dsAQP 分别导致 SNF7 和 AQP mRNA 水平降低 16.4-和 14.47 倍。喂食 dsSNF7 或 dsAQP 也导致烟粉虱 62%和 72%的死亡率。有趣的是，同时喂食靶向 SNF7 或 AQP 的 dsRNA 和 RNAi 途径基因（Dicer-2/Aubergine/Staufen）之一，导致靶基因的 RNAi 显著降低。这些数据表明烟粉虱中存在强大的 RNAi 机制。
本研究首次报道了组装、分析和注释的烟粉虱 RNAseq 资源，可用于该昆虫的未来分子研究。在不同阶段和饥饿胁迫下验证的参考基因提供了烟粉虱中稳定基因的第一手信息。RNAi 机制基因的信息以及通过合成饮食中的 dsRNA 喂食显著敲低靶基因，证实了这种昆虫中存在有效的 RNAi。这些数据为进一步研究将 RNAi 作为一种管理这种害虫的方法提供了坚实的基础。
