Perkin L C, Elpidina E N, Oppert B
USDA, Agricultural Research Service, Center for Grain and Animal Health Research, 1515 College Avenue, Manhattan, KS, USA.
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Insect Mol Biol. 2017 Feb;26(1):35-45. doi: 10.1111/imb.12269. Epub 2016 Oct 22.
Tribolium castaneum is a major agriculture pest damaging stored grains and cereal products. The T. castaneum genome contains 26 cysteine peptidase genes, mostly cathepsins L and B, and seven have a major role in digestion. We targeted the expression of the most highly expressed cathepsin L gene on chromosome 10, TC011001, by RNA interference (RNAi), using double-stranded RNA (dsRNA) constructs of different regions of the gene (3', middle, 5' and entire coding regions). RNA sequencing and quantitation (RNA-seq) was used to evaluate knockdown and specificity amongst the treatments. Overall, target gene expression decreased in all treatment groups, but was more severe and specific in dsRNA targeting the 3' and entire coding regions, encoding the proteolytic active site in the enzyme. Additional cysteine cathepsin genes also were down-regulated (off-target effects), but some were up-regulated in response to RNAi treatment. Notably, some serine peptidase genes were increased in expression, especially in dsRNA targeting 5' and middle regions, and the response was similar to the effects of dietary cysteine protease inhibitors. We manually annotated these serine peptidase genes to gain insight into function and relevance to the RNAi study. The data indicate that T. castaneum larvae compensate for the loss of digestive peptidase activity in the larval gut, regardless of the mechanism of disruption.
赤拟谷盗是一种主要的农业害虫，会损害储存的谷物和谷类产品。赤拟谷盗基因组包含26个半胱氨酸蛋白酶基因，主要是组织蛋白酶L和B，其中7个在消化过程中起主要作用。我们通过RNA干扰（RNAi），使用该基因不同区域（3'端、中间、5'端和整个编码区）的双链RNA（dsRNA）构建体，靶向位于10号染色体上表达量最高的组织蛋白酶L基因TC011001的表达。RNA测序和定量分析（RNA-seq）用于评估各处理之间的基因敲低效果和特异性。总体而言，所有处理组的靶基因表达均下降，但在靶向3'端和整个编码区的dsRNA处理中更为严重且具有特异性，这些区域编码该酶的蛋白水解活性位点。其他半胱氨酸组织蛋白酶基因也被下调（脱靶效应），但有些基因在RNAi处理后会上调。值得注意的是，一些丝氨酸蛋白酶基因的表达增加，尤其是在靶向5'端和中间区域的dsRNA处理中，且这种反应与膳食半胱氨酸蛋白酶抑制剂的作用相似。我们对这些丝氨酸蛋白酶基因进行了手动注释，以深入了解其功能以及与RNAi研究的相关性。数据表明，无论破坏机制如何，赤拟谷盗幼虫都会补偿幼虫肠道中消化蛋白酶活性的损失。
