小菜蛾保幼激素受体基因PxMet-1和PxMet-2的分子特性、表达模式与功能分析

doi:10.16380/j.kcxb.2019.07.002

昆虫学报 ›› 2019, Vol. 62 ›› Issue (7): 787-798.doi: 10.16380/j.kcxb.2019.07.002

小菜蛾保幼激素受体基因PxMet-1和PxMet-2的分子特性、表达模式与功能分析

彭露^1,2,3,4, 杨一帆^1,2,3,4, 邹明民^1,2,3,4, 王清^1,2,3,4, 尤民生^1,2,3,4,*

(1. 福建农林大学, 闽台作物有害生物生态防控国家重点实验室, 福州350002; 2. 福建农林大学应用生态研究所, 福州350002; 3. 福建农林大学, 教育部害虫生态防控国际合作联合实验室, 福州350002; 4. 福建农林大学, 福建省昆虫生态重点实验室, 福州350002)

出版日期:2019-07-20 发布日期:2019-07-09

Molecular characterization, expression profiling and functional analysis of juvenile hormone receptor genes PxMet-1 and PxMet-2 in Plutella xylostella (Lepidoptera: Plutellidae)

PENG Lu^1,2,3,4, YANG Yi-Fan^1,2,3,4, ZOU Ming-Min^1,2,3,4, WANG Qing^1,2,3,4, YOU Min-Sheng^1,2,3,4,*

(1. State Key Laboratory of Ecological Pest Control for Fujian-Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3. Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 4. Fujian Provincial Key Laboratory of Insect Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

Online:2019-07-20 Published:2019-07-09

摘要/Abstract

摘要：

【目的】本研究旨在明确小菜蛾Plutella xylostella保幼激素受体基因Met的分子特性与表达模式，分析其生殖调控作用，为筛选有效控制小菜蛾的新靶标奠定基础。【方法】根据本课题组已有的小菜蛾基因组数据库，采用PCR技术克隆小菜蛾两个Met基因的cDNA全长序列；利用qPCR测定其在小菜蛾不同发育阶段及成虫不同组织中的表达模式；基于RNAi解析其在小菜蛾雌成虫生殖发育中的作用。【结果】克隆获得小菜蛾PxMet-1(GenBank登录号: MK697672)与PxMet-2(GenBank登录号: MK697673)的cDNA序列，开放阅读框(ORF)全长分别为1 575和2 100 bp，预计分别编码524和699个氨基酸，理论分子质量分别为60.5和70.7 kD，预测等电点分别为6.73和5.50。PxMet-1和PxMet-2都具有4个保守结构域，即1个helix-loop-helix结构域(bHLH)、2个PAS保守结构域及1个PAC保守基序。系统发育树分析表明，小菜蛾PxMet-1和PxMet-2聚为不同的两支，但两者均与鳞翅目昆虫Met聚在一起。表达模式分析表明，小菜蛾PxMet1与PxMet-2在蛹期(化蛹后1-3 d)与雌成虫期(羽化后0-72 h)均有表达；PxMet-1的表达量在蛹期(化蛹后1-3 d)无明显差异，但均显著高于雌成虫期(羽化后0-48 h)，在羽化后72 h达到高峰；而PxMet-2在雌成虫期(羽化后0-48 h)的表达量呈先上升后下降的趋势，在羽化后12 h出现表达高峰，且成虫期(羽化后0-36 h)的表达量显著高于蛹期。PxMet-1与PxMet-2在成虫脂肪体中的表达量显著高于其他组织。注射PxMet-1+PxMet-2 dsRNA 24 h后，小菜蛾PxMet-1与PxMet-2的表达量均受到显著抑制；同时干扰PxMet-1和PxMet-2后，小菜蛾成熟卵子数目显著减少，羽化后3 d内单雌产卵量显著下降。【结论】抑制Met基因表达能够显著降低小菜蛾雌虫的卵子形成与产卵量。本研究为探索保幼激素的生殖调控机理奠定了基础，在实践上有助于筛选小菜蛾种群遗传调控的潜在靶标。

关键词: 小菜蛾, 保幼激素受体; Met, 分子特性, 表达模式, 生殖调控, 害虫控制

Abstract: 【Aim】 This study aims to clarify the molecular characteristics and expression patterns of juvenile hormone receptor genes (Met) and to analyze their functions in reproductive regulation in the diamondback moth, Plutella xylostella, so as to provide basis for screening new targets for effectively controlling the diamondback moth. 【Methods】 Based on the genome database of P. xylostella we obtained previously, the fulllength cDNAs of two Met genes were cloned using PCR, and their expression patterns in different developmental stages and adult tissues of P. xylostella were detected via qPCR. Moreover, the role of the two Met genes in the reproductive development of female adults of P. xylostella was analyzed by RNAi. 【Results】 The cDNA sequences of PxMet-1 (GenBank accession no. MK697672) and PxMet-2 (GenBank accession no. MK697673) were cloned from P. xylostella, and they contain ORFs of 1 575 bp encoding 524 aa and 2 100 bp encoding 699 aa, respectively. The predicted molecular mass of PxMet-1 and PxMet-2 are 60.5 and 70.7 kD, and the theoretical isoelectric points are 6.73 and 5.50, respectively. PxMet-1 and PxMet-2 both contain a basic helix-loop-helix (bHLH) domain, two PAS motifs and one PAC motif. The results of phylogenetic tree analysis showed that PxMet-1 and PxMet-2 were clustered into different branches, but clustered into the same branch with lepidopteran Mets. The expression profiles showed that PxMet-1 and PxMet-2 were both expressed in the pupal stage (1-3 d after pupation) and female adult (0-72 h after eclosion). The expression level of PxMet-1 in the pupal stage (1-3 d after pupation) showed no significant difference, but was significantly higher than that in female adult (0-48 h after eclosion), and its expression peaked at 72 h after eclosion. In the female adult (0-48 h after eclosion), the mRNA level of PxMet-2 firstly increased, reached a peak at 12 h after eclosion, and then decreased. Moreover, the expression level of PxMet-2 at 0-36 h after eclosion was significantly higher than that in the pupal stage. The expression levels of PxMet-1 and PxMet-2 in the fat body of adult were significantly higher than those in other tissues. Injection of dsPxMet-1 and dsPxMet-2 significantly inhibited the expression of PxMet-1 and PxMet-2 within 24 h. In the female adults of P. xylostella with both PxMet-1 and PxMet-2 silenced, oogenesis was significantly inhibited, and the number of eggs laid per female within 3 d after eclosion significantly reduced. 【Conclusion】 Inhibition of the expression of PxMet-1 and PxMet-2 can significantly reduce egg maturation and oviposition of female P. xylostella. This study establishes a theoretical foundation for reproductive regulation mechanisms by JH in P. xylostella and facilitates screening potential targets used for genetic manipulation of P. xylostella populations.

Key words: Plutella xylostella, juvenile hormone receptor, Met; molecular characteristic, expression profile, reproductive regulation, pest management

彭露, 杨一帆, 邹明民, 王清, 尤民生. 小菜蛾保幼激素受体基因PxMet-1和PxMet-2的分子特性、表达模式与功能分析[J]. 昆虫学报, 2019, 62(7): 787-798.

PENG Lu, YANG Yi-Fan, ZOU Ming-Min, WANG Qing, YOU Min-Sheng. Molecular characterization, expression profiling and functional analysis of juvenile hormone receptor genes PxMet-1 and PxMet-2 in Plutella xylostella (Lepidoptera: Plutellidae)[J]. Acta Entomologica Sinica, 2019, 62(7): 787-798.

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小菜蛾保幼激素受体基因PxMet-1和PxMet-2的分子特性、表达模式与功能分析

Molecular characterization, expression profiling and functional analysis of juvenile hormone receptor genes PxMet-1 and PxMet-2 in Plutella xylostella (Lepidoptera: Plutellidae)

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