昆虫学报 ›› 2023, Vol. 66 ›› Issue (2): 138-149.doi: 10.16380/j.kcxb.2023.02.003

• 研究论文 • 上一篇    下一篇


李凯龙*, 王鑫, 马明勇, 彭兆普, 陈昂, 陈武瑛, 罗香文, 刘勇, 张德咏   

  1. (湖南省农业科学院植物保护研究所, 长沙 410125)
  • 出版日期:2023-02-20 发布日期:2023-04-07

Molecular characteristics and functional analysis of nuclear receptor gene NlE75 in Nilaparvata lugens (Hemiptera: Delphacidae)

LI Kai-Long*, WANG Xin, MA Ming-Yong, PENG Zhao-Pu, CHEN Ang, CHEN Wu-Ying, LUO Xiang-Wen, LIU Yong, ZHANG De-Yong   

  1.  (Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, China)
  • Online:2023-02-20 Published:2023-04-07

摘要: 【目的】蜕皮激素通过信号级联反应调节昆虫的蜕皮和变态过程,而核受体基因E75是信号通路的早期响应基因。本研究旨在分析褐飞虱Nilaparvata lugens NlE75的分子特性及生物学功能,为褐飞虱的防治和新农药的开发提供新分子靶标。【方法】本研究基于序列比对和同源检索以及褐飞虱转录组数据获得NlE75的cDNA序列,并利用RT-PCR和RACE技术对其进行cDNA克隆; 利用生物信息学软件预测分析NlE75编码蛋白的结构特性; 利用MEGA5.0邻接法(neighbor-joining, NJ)构建系统进化树。利用RT-qPCR检测NlE75及其转录本在褐飞虱不同发育阶段(卵、1-5龄若虫和成虫)、5龄若虫不同组织(头、胸、腹、表皮、翅芽、脂肪体、中肠和足)中和3龄若虫被注射0.2 μg/头20-羟基蜕皮激素(20-hydroxyecdysone, 20E)后的表达谱。通过注射法进行RNAi敲降褐飞虱3龄若虫20E合成关键基因NlCYP314A1后,利用RT-qPCR检测3龄若虫中NlE75的表达量;通过注射法进行RNAi下调3和5龄若虫NlE75及3龄若虫中其转录本的表达后,利用RT-qPCR检测3龄若虫中NlE75及其转录本的表达量,观察敲降NlE75对褐飞虱3和5龄若虫蜕皮和变态的影响,分析NlE75的5个转录本的相互调控关系。【结果】克隆获得褐飞虱NlE75的5个转录本,分别命名为NlE75A, NlE75B, NlE75C, NlE75DNlE75E(GenBank登录号分别为ON086330, ON086331, ON086332, ON086333和ON086334),其编码蛋白均具有典型的核受体保守结构域;系统进化分析发现其与人体虱Pediculus humanus corporis和德国小蠊Blattella germanica的E75蛋白聚在一起。RT-qPCR结果显示, NlE75基因5个转录本具有不同的发育和组织表达特性; NlE75A和NlE75D的表达量在褐飞虱各发育阶段均较低,NlE75A, NlE75BNlE75C的表达动态与NlE75一致,NlE75D在4龄和5龄蜕皮前表达量增至最高,而NlE75E在成虫中的表达量最高; NlE75 5个转录本在5龄若虫各个组织中均有表达,但NlE75B, NlE75C, NlE75DNlE75E在足中高表达,而NlE75A在脂肪体中高表达。20E诱导后4和8 h时,处理组NlE75的表达量较对应的对照组分别上调了4.7和5.0倍,而RNAi干扰褐飞虱NlCYP314A1后48 h时处理组NlE75的表达量较对照组(注射dsGFP)极显著下调了47.8%; RNAi结果表明在3龄若虫中注射dsNlE75后导致褐飞虱蜕皮困难,出现畸形虫体;在5龄若虫中注射dsNlE75导致褐飞虱变态终止,出现畸形虫体;3和5龄若虫注射dsNlE75均无法完成蜕皮过程并最终100%死亡。只有对5个转录本均干扰后才出现畸形表型,而对单一转录本干扰后则不出现畸形表型,说明NlE75基因5个转录本之间存在着复杂的调控关系。【结论】褐飞虱中NlE75基因具有5个转录本,具有不同的发育和组织表达特性;RNAi结果表明,NlE75基因5个转录本存在明显的功能冗余现象,NlE75基因在褐飞虱蜕皮过程发挥重要作用。

关键词: 褐飞虱, 核受体; NlE75, 转录本, RNAi, 蜕皮

Abstract: 【Aim】 Ecdysone regulates the molting and metamorphosis processes of insects through signaling cascade, and the nuclear receptor gene E75 is an early response gene of the signaling pathway. The objective of this study is to analyze the molecular characteristics and biological function of NlE75 in the brown planthopper, Nilaparvata lugens, so as to provide new molecular target for the management of N. lugens and development of new pesticides. 【Methods】 The cDNA sequence of NlE75 was obtained based on sequence alignment, homology search and N. lugens transcriptome data, and cloned by RT-PCR and RACE technologies. The structure characteristics of the protein encoded by NlE75 were predicted using bioinformatics software, and the phylogenetic tree was constructed using the neighbor-joining method of MEGA 5.0. The expression profiles of NlE75 and its transcripts in different developmental stages (egg, 1st-5th instar nymphs and adult), different tissues (head, thorax, abdomen, cuticle, wing bud, fat body, midgut and leg) of the 5th instar nymph, and the 3rd instar nymph of N. lugens after injection of 0.2 μg/individual of 20-hydroxyecdysone (20E) were detected by RT-qPCR. After knockdown of NlCYP314A1 in the 3rd instar nymph by RNAi via dsRNA injection, the expression level of NlE75 in the 3rd instar nymphs was detected by RT-qPCR. After downregulating the expression of NlE75 in the 3rd and 5th instar nymphs and its transcripts in the 3rd instar nymphs by RNAi via dsRNA injection, the expression levels of NlE75 in the 3rd instar nymphs were detected by RT-qPCR, the effects of knockdown of NlE75 on the molting and metamorphosis of the 3rd and 5th instar nymphs of N. lugens were observed, and the regulatory relationships of five transcripts of NlE75 were analyzed. 【Results】 Five NlE75 transcripts NlE75A, NlE75B, NlE75C, NlE75D and NlE75E (GenBank accession numbers are ON086330, ON086331, ON086332, ON086333 and ON086334, respectively) of N. lugens were cloned, and their encoded proteins all have typical nuclear receptor conserved domains. Phylogenetic analysis revealed that NlE75A-E clustered with the E75 proteins of Pediculus humanus corporis and Blattella germanica. RT-qPCR results showed that five NlE75 transcripts had different developmental and tissue expression characteristics. The expression levels of NlE75A and NlE75D in various developmental stages of N. lugens were low, the expression dynamics of NlE75A, NlE75B and NlE75C were consistent with that of NlE75 and the expression level of NlE75D increased to the highest level before the molting of both the 4th and 5th instar nymphs, while the expression levels of NlE75E in adults were the highest. The five NlE75 transcripts were expressed in various tissues of the 5th instar nymphs, with NlE75B, NlE75C, NlE75D and NlE75E highly expressed in the leg, and NlE75A highly expressed in the fat body. The expression levels of NlE75 in treatment groups significantly increased by 4.7 and 5.0 times, respectively, at 4 and 8 h after 20E induction, as compared to those in the corresponding control groups. The expression level of NlE75 in treatment group was extremely significantly down-regulated by 47.8% at 48 h after RNAi of NlCYP314A1, as compared with that in the control group (injected with dsGFP). RNAi results showed that injection of dsNlE75 into the 3rd instar nymphs resulted in the failure of molting and phenotypic defects, while injection of dsNlE75 into the 5th instar nymphs resulted in the failure of metamorphosis and phenotypic defects, and the 3rd and 5th instar nymphs injected with dsNlE75 could not complete the molting process and eventually died 100%. The phenotypic defects appeared only when all the five transcripts of NlE75 were knocked down, but did not appear when a single transcript was knocked down, suggesting that there is a complex regulatory relationship between the five transcripts. 【Conclusion】 There are five NlE75 transcripts in N. lugens, with different developmental and tissue expression characteristics. RNAi results suggest that five NlE75 transcripts have obvious functional redundancy and NlE75 plays an important role in the molting of N. lugens.

Key words: Nilaparvata lugens, nuclear receptor, NlE75, transcript, RNAi, molting