褐飞虱五个miRNA的克隆鉴定与表达谱分析  

Cloning,identification and expression profiling of five miRNAs of the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)

作　　者：王妮 张超 尤元政 周文武 祝增荣[1,2] WANG Ni;ZHANG Chao;YOU Yuan-Zheng;ZHOU Wen-Wu;ZHU Zeng-Rong(State Key Laboratory of Rice Biology and Breeding,Key Laboratory of Molecular Biology of Crop Pathogens and Insects,Ministry of Agriculture and Rural Affairs,Institute of Insect Sciences,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China;Zhejiang Yuhang Rice Science and Technology Institute,Yuhang 311121,China)

机构地区：[1]浙江大学农业与生物技术学院昆虫科学研究所,水稻生物育种全国重点实验室,农业农村部作物病虫分子生物学重点实验室,杭州310058 [2]浙江余杭稻米科技小院,余杭311121

出　　处：《昆虫学报》2024年第9期1289-1298,共10页Acta Entomologica Sinica

基　　金：国家重点研发计划项目(2021YFD1401105);浙江省“三农六方”科技协作项目(CTZB-F190625LWZ-SNY1);国家自然科学基金面上项目(31871962)。

摘　　要：【目的】本研究对前期鉴定所获得的5个微小RNA(microRNA, miRNA)进行表达和序列验证,检测其在褐飞虱Nilaparvata lugens不同发育阶段和成虫组织中的表达模式,旨在为探究miRNA调控褐飞虱生长发育和繁殖的作用机制提供理论和实验依据。【方法】采用Stem-loop RT-PCR对褐飞虱5个miRNA(miR-1a-3p, miR-71-3p, miR-184-3p, miR-190-5p和miR-281-5p)进行表达验证。结合Sanger测序验证5个miRNA。利用qRT-PCR检测这5个miRNA在褐飞虱不同发育阶段(卵、 1-5龄若虫和雌雄成虫)、 1-5日龄雌成虫和雌成虫组织(头、胸、腹、足、翅、中肠、马氏管和卵巢)中的表达量。通过注射miRNA类似物agomir和抑制剂antagomir对褐飞虱雌成虫这5个miRNA分别进行过表达和抑制分析。【结果】Stem-loop RT-PCR和Sanger测序结果显示miR-1a-3p, miR-71-3p, miR-184-3p, miR-190-5p和miR-281-5p在褐飞虱体内真实存在和表达。发育表达谱分析发现,miR-1a-3p和miR-71-3p在褐飞虱整个发育阶段均有不同程度的表达,且分别在雄成虫和2龄若虫中高表达;miR-184-3p和miR-281-5p在褐飞虱雌雄成虫中表达量最高,在卵和若虫中表达量较低,且在卵和若虫间的表达量无显著差异;miR-190-5p在卵中表达量显著高于其他发育阶段的。miR-1a-3p的表达量在雌成虫4日龄时显著降低;miR-71-3p和miR-184-3p的表达量在雌成虫3日龄时达到最高;miR-190-5p和miR-281-5p在雌成虫1日龄时的表达量较高,在2-4日龄时的表达量显著下降。组织表达谱分析发现,miR-1a-3p在足部和卵巢中显著高表达;miR-71-3p主要在雌成虫卵巢特异性地高表达;miR-184-3p在中肠中表达量最高,其次是在卵巢中的表达量;miR-190-5p和miR-281-5p均在翅中表达量最高,其次是在头和胸中(miR-190-5p)以及中肠和卵巢中(miR-281-5p)。注射miRNA类似物后,5个miRNA的表达量均比阴性对照显著上调;注射miRNA抑制剂后,除miR-281-5p表达量比阴性对照显著上调外,其余4�【Aim】This study aims to provide theoretical and experimental bases for exploring the mechanism of microRNA(miRNA)regulation on the growth,development and reproduction of Nilaparvata lugens by clarifying the expression and sequences of five miRNAs obtained in the previous identification and determining the expression profiles of the five miRNAs in different developmental stages and adult tissues of N.lugens.【Methods】The expression and sequence authenticity of five miRNAs(miR-1a-3p,miR-71-3p,miR-184-3p,miR-190-5p and miR-281-5p)of N.lugens were proved by Stem-loop RT-PCR and Sanger sequencing,respectively.The expression levels of the five miRNAs in different developmental stages(eggs,1st-5th instar nymphs,female and male adults),1-5-day-old female adults,and tissues(head,thorax,abdomen,leg,wing,midgut,Malpighian tubules and ovary)of female adults of N.lugens were detected by qRT-PCR.Overexpression and inhibition of the five miRNAs were conducted by injecting miRNA agomir and antagomir into the female adults of N.lugens,respectively.【Results】Stem-loop RT-PCR and Sanger sequencing results showed that miR-1a-3p,miR-71-3p,miR-184-3p,miR-190-5p and miR-281-5p truly exist and are expressed in N.lugens.Developmental expression profile analysis showed that miR-1a-3p and miR-71-3p were expressed in the whole developmental stages of N.lugens,and were highly expressed in male adults and 2nd instar nymphs,respectively.The relative expression levels of miR-184-3p and miR-281-5p were the highest in female adults and male adults of N.lugens,but were lower in eggs and nymphs and the relative expression levels of miR-184-3p and miR-281-5p between eggs and nymphs had no significant difference.The expression level of miR-190-5p in eggs was significantly higher than those in the other developmental stages.The expression level of miR-1a-3p in the 4-day-old female adult decreased significantly,those of miR-71-3p and miR-184-3p in the 3-day-old female adult were the highest,and those of miR-190-5p and miR-281-5p in the 1-day-

关 键 词：褐飞虱 MIRNA 表达谱 生长 发育 繁殖 

分 类 号：Q966[生物学—昆虫学]