褐飞虱E3泛素连接酶基因NlHECTD2的克隆和功能分析  

Cloning and functional analysis of the E3 ubiquitin ligase gene NlHECTD2 in Nilaparvata lugens (Hemiptera: Delphacidae)

作　　者：罗怡 郭莹莹 熊振泽 宋阳 刘一鹏 俞晓平 申屠旭萍 LUO Yi;GUO Ying-Ying;XIONG Zhen-Ze;SONG Yang;LIU Yi-Peng;YU Xiao-Ping;SHENTU Xu-Ping(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,College of Life Science,China Jiliang University,Hangzhou 310018,China)

机构地区：[1]中国计量大学生命科学学院,浙江省生物计量及检验检疫技术重点实验室,杭州310018

出　　处：《昆虫学报》2023年第7期992-998,共7页Acta Entomologica Sinica

基　　金：国家自然科学区域联合基金项目(U21A20223,3164001);浙江省重点研发计划项目(2022C02047,2019C02015);浙江省“三农九方”科技协作计划项目(2023SNJF034)。

摘　　要：【目的】探明褐飞虱Nilaparvata lugens E3泛素连接酶基因NlHECTD2的分子特性、表达模式及生物学功能。【方法】基于褐飞虱转录组数据,PCR克隆NlHECTD2全长cDNA序列并进行生物信息学分析;利用qRT-PCR检测NlHECTD2在卵、1-5龄若虫、初羽化24 h内雌成虫和1-5日龄雌成虫中及3日龄雌成虫头、胸、腹、肠道和卵巢中的表达量;初羽化褐飞虱雌成虫单头注射0.05μL ds NlHECTD2(5000 ng/μL),利用qRT-PCR检测RNAi后24,48和72 h时NlHECTD2的表达量,并检测褐飞虱血淋巴中类酵母共生菌(yeast-like symbiont,YLS)的数量、褐飞虱雌成虫存活率、单雌产卵量和卵孵化率。【结果】克隆获得了褐飞虱NlHECTD2基因全长cDNA序列(GenBank登录号:XM_039427060.1),其开放阅读框长2850 bp;NlHECTD2编码950个氨基酸,无信号肽,包含1个具有泛素连接酶(E3)亚类的C端催化结构域。系统发育分析表明,NlHECTD2与半翅目其他昆虫HECTD2s聚为一支,与烟粉虱Bemisia tabaci HECTD2亲缘关系最为接近。qRT-PCR结果显示,NlHECTD2具有明显的时空表达特异性,在褐飞虱3日龄雌成虫腹中的表达量最高。RNAi结果显示,与注射ds GFP对照比较,注射ds NlHECTD2能够显著抑制NlHECTD2的表达量,使褐飞虱血淋巴中的YLS数量极显著下降,导致褐飞虱雌成虫存活率、单雌产卵量及卵孵化率极显著下降。【结论】NlHECTD2与褐飞虱体内YLS释放到血淋巴这一过程紧密相关,并在褐飞虱的生长发育及繁殖过程中发挥重要作用,可作为褐飞虱防控的潜在靶标。【Aim】To explore the molecular characteristics,expression patterns and biological function of the E3 ubiquitin ligase gene NlHECTD2 of Nilaparvata lugens.【Methods】Based on the transcriptome data of N.lugens,the full-length cDNA sequence of NlHECTD2 was cloned by PCR and bioinformatically analyzed.qRT-PCR was used to detect the expression levels of NlHECTD2 in the egg,1st-5th instar nymphs,female adult newly emerged within 24 h and 1-5-day-old female adults,and the head,throax,abdomen,gut and ovary of the 3-day-old female adult of N.lugens.A single newly emerged female adult of N.lugens was injected with 0.05μL ds NlHECTD2(5000 ng/μL)and the expression level of NlHECTD2 was detected by qRT-PCR at 24,48,and 72 h after RNAi,and the number of yeast-like symbionts(YLSs)in the hemolymph,survival rate of the female adult of N.lugens,number of eggs laid per female and egg hatching rate were also detected.【Results】The full-length cDNA sequence of NlHECTD2(GenBank accession number:XM_039427060.1)was successfully cloned from N.lugens.The open reading frame(ORF)of NlHECTD2 is 2850 bp in length and encodes 950 amino acids.NlHECTD2 has no signal peptide and contains a C-terminal catalytic domain with a subclass ubiquitin ligase(E3).Phylogenetic analysis indicated that NlHECTD2 was clustered together with other hemipteran HECTD2s,and had the highest homology with HECTD2 of Bemisia tabaci.The qRT-PCR results showed that NlHECTD2 had obvious spatiotemporal expression specificity,with the highest expression level in the abdomen of the 3-day-old female adult of N.lugens.The RNAi results showed that compared with the control(injected with ds GFP),injection of ds NlHECTD2 significantly inhibited the expression level of NlHECTD2 and extremely significantly decreased the number of YLSs in the hemolymph of N.lugens,which led to an extremely significant decrease in the survival rate of female adult,number of eggs laid per female and egg hatching rate of N.lugens.【Conclusion】NlHECTD2 is closely related to the process of Y

关 键 词：褐飞虱 NlHECTD2 类酵母共生菌 RNA干扰 基因功能 

分 类 号：Q966[生物学—昆虫学]