饲喂法RNAi介导的HvCHS1基因沉默抑制茄二十八星瓢虫的存活和发育  

Silencing of Chitin Synthase 1(HvCHS1) by Dietary RNAi Inhibits the Survival and Development of the 28-Spotted Potato Ladybird,Henosepilachna vigintioctopunctata

作　　者：骆旭铭 陈诗敏 潘广 杨春晓 潘慧鹏 LUO Xuming;CHEN Shimin;PAN Guang;YANG Chunxiao;PAN Huipeng(College of Plant Protection,South China Agricultural University/Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province,Guangzhou 510642,China;National Modern Agricultural Industry Science and Technology Innovation Center of Guangzhou,Guangzhou 510520,China;South China Agricultural University,State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangzhou 510642,China)

机构地区：[1]华南农业大学植物保护学院/广东省生物农药创制与应用重点实验室,广州510642 [2]广州国家现代农业产业科技创新中心,广州510520 [3]华南农业大学亚热带农业生物资源保护与利用国家重点实验室,广州510642

出　　处：《中国生物防治学报》2022年第5期1083-1093,共11页Chinese Journal of Biological Control

基　　金：国家自然科学基金(31972269,32172500)。

摘　　要：几丁质的合成与降解之间的平衡关系影响着昆虫的生长发育,而几丁质合成酶1(chitin synthase 1,CHS1)是昆虫几丁质合成过程中的关键酶之一。本研究旨在使用饲喂法RNAi(RNAinterference)探究CHS1对茄二十八星瓢虫Henosepilachnavigintioctopunctata存活和发育的影响。基于从茄二十八星瓢虫基因组数据中获得的Hv CHS1序列,通过生物信息学方法分析其理化性质及与其他昆虫之间的同源性;运用RT-qPCR技术分析该基因的时空表达特性;通过饲喂体外合成和菌液表达的dsRNAs对茄二十八星瓢虫进行RNAi,统计死亡率、化蛹率,并通过苏木精-伊红(H&E)染色观察Hv CHS1基因干扰后的表皮变化。Hv CHS1的开放阅读框为4680 bp,编码1559个氨基酸残基,相对分子质量为177.5 kDa。Hv CHS1在表皮中的表达水平高于其他组织。此外,通过摄食体外合成的ds Hv CHS1导致茄二十八星瓢虫存活率和化蛹率显著下降,存在明显的剂量依赖效应,摄食50 ng/μL、250 ng/μL和500 ng/μL ds HvCHS1的1龄幼虫死亡率分别为42.00%、82.00%和94.00%,摄食50 ng/μL、100 ng/μL和200 ng/μL ds Hv CHS1的4龄幼虫化蛹率分别为17.04%、34.07%和68.52%。而摄食菌液表达的ds Hv CHS1对1龄和3龄幼虫的致死率分别为64.00%和60.00%,但对成虫的存活率无影响。最后,H&E染色显示,摄取ds Hv CHS1可抑制幼虫腹部新的角质层形成。本研究表明,HvCHS1参与幼虫蜕皮过程中新角质层的形成,可作为一个潜在的RNAi靶标基因,用于茄二十八星瓢虫的生物防治。The growth and development of insects are affected by the balance between the synthesis and degradation of chitin,and chitin synthase 1(CHS1) is one of the key enzymes involved in insect chitin synthesis.This study aims to investigate the effects of CHS1 on survival and development of Henosepilachna vigintioctopunctata using dietary RNAi.HvCHS1 sequence obtained from the genome data of H.vigintioctopunctata was analyzed for physicochemical properties and homology with other insects through bioinformatics methods.The temporal and spatial expression patterns of HvCHS1 were analyzed by RT-qPCR.The RNAi(RNA interference) of HvCHS1 was carried out by ingestion of in vitro synthesized and bacterially expressed dsRNAs.The mortality and pupation rate of H.vigintioctopunctata were recorded,and the epidermal changes were observed by the hematoxylin and eosin(H&E) staining.The open reading frame of HvCHS1 is 4680 bp,encoding 1559 amino acid with a relative molecular mass of 177.5 kDa.The expression levels of HvCHS1 were greater in cuticle than in other tissues.In addition,ingestion of ds HvCHS1 led to a significant decrease in the survival rate and pupation rate of H.vigintioctopunctata with a significant dose-dependent effect.The mortality rates of 1st instar larvae were 42.00%,82.00% and 94.00% when the larvae were fed with 50 ng/μL,250 ng/μL and 500 ng/μL ds HvCHS1,respectively.The pupation rates of 4th instar larvae fed with 50 ng/μL,100ng/μL and 200 ng/μL ds HvCHS1 were 17.04%,34.07% and 68.52%,respectively.Moreover,ingestion of bacterially expressed ds HvCHS1 resulted in high larval mortality of 64% and 60% in the 1st and 3rd instar larvae,respectively,whereas it had no impact on the survival rate of adults.Finally,H&E staining assays revealed the ingestion of ds HvCHS1 prevented new abdominal cuticle formation.It is obvious that HvCHS1 is responsible for the new cuticle formation during larvae ecdysis,and it is a potential RNAi target gene for the bio-control of H.vigintioctopunctata.

关 键 词：茄二十八星瓢虫 几丁质合成酶1 饲喂法RNAi 死亡率 化蛹率 

分 类 号：S188[农业科学—农业基础科学]