Improving RNA interference in the southern green stink bug, Nezara viridula
Improving RNA interference in the southern green stink bug, Nezara viridula
复制标题
DOI：
10.1007/s10340-021-01358-3
复制
发表时间：
2021-03-09
影响因子：
4.8
通讯作者：
Palli, Subba Reddy
中科院分区：
文献类型：
--
作者：
Gurusamy, Dhandapani;Howell, Jeffrey L.;Palli, Subba Reddy
南方绿臭虫，Nezara viridula，是许多重要作物的世界性害虫。对病毒乳杆菌若虫和成虫的RNA进行测序并组装成转录组。通过搜索转录组，确定了参与RNA干扰（RNAi）途径的蛋白质编码基因的同源基因和RNAi介导的害虫控制的潜在靶基因。与RNAi难处理的秋粘虫相比，病毒乳虫的水样唾液、管腔和血淋巴的dsRNase活性较低。通过对7个管家基因60S RP L12、40S RP S17、18S rRNA、ef1 - α、Actin、Ubiquitin和β - tubulin在不同发育阶段和dsRNA处理下的评估，确定60S RP L12和40S RP S17为逆转录酶定量PCR （RT-qPCR）的最佳内参基因。在病毒北蝽中检测了1微克针对7个已知RNAi靶基因（SNF7、IAP1、IAP2、IAP5、PP1、GPCR和ATPase1）同源物的dsRNA。测试的7个dsrna中有3个导致67-100%的死亡率。将以SNF7为靶点的dsRNA注射到病毒乳螨成虫体内，死亡率为100%，而喂食dsSNF7的成虫和若虫死亡率均为60%。通过喂食或注射传递dsRNA可以有效地降低靶基因的表达。这些数据表明，病毒北蝽中存在RNAi途径成分，通过注射或喂食传递dsRNA可诱导靶基因表达沉默和死亡，表明可以开发基于RNAi的方法来管理这种害虫。
The southern green stink bug, Nezara viridula, is a worldwide pest of many important crops. RNA from N. viridula nymphs and adults was sequenced and assembled into a transcriptome. Orthologs of genes coding for proteins involved in RNA interference (RNAi) pathways and potential target genes for RNAi-mediated control of this pest were identified by searching the transcriptome. The watery saliva, lumen and hemolymph collected from N. viridula showed lower dsRNase activity than that in the lumen and hemolymph collected from the fall armyworm, Spodoptera frugiperda, which is refractory to RNAi. Seven housekeeping genes, 60S RP L12, 40S RP S17, 18S rRNA, EF1-alpha, Actin, Ubiquitin and beta-Tubulin, were evaluated across developmental stages and dsRNA treatments and identified 60S RP L12 and 40S RP S17 as the best reference genes for use in reverse transcriptase quantitative PCR (RT-qPCR). One microgram of dsRNA targeting the orthologs of seven known RNAi target genes (SNF7, IAP1, IAP2, IAP5, PP1, GPCR and ATPase1) was tested in N. viridula. Three out of seven dsRNAs tested induced 67-100% mortality. Injection of dsRNA targeting SNF7 into N. viridula adults caused 100% mortality, while feeding dsSNF7 induced 60% mortality in both adults and nymphs. Delivery of dsRNA by either feeding or injection efficiently knocked down target gene expression. These data demonstrate that RNAi pathway components are present in N. viridula and delivering dsRNA by either injection or feeding induces silencing of target gene expression and mortality, suggesting that RNAi-based methods could be developed to manage this pest.