淡色库蚊保幼激素受体基因CpMet的表达特性和功能分析

doi:10.11853/j.issn.1003.8280.2021.06.005

摘要/Abstract

摘要： 目的了解淡色库蚊保幼激素受体基因CpMet的表达特性以及生殖调控作用。方法采用荧光定量PCR法检测CpMet基因在淡色库蚊不同发育阶段、成虫不同组织及激素处理后的表达特性；利用RNA干扰技术解析CpMet基因在淡色库蚊雌成虫生殖调控中的作用。所有数据用平均值±标准误表示，采用SPSS 13.0软件进行统计学分析。结果 CpMet在淡色库蚊各个发育阶段均有表达，在卵期表达量显著高于其他阶段，其次是蛹期和成虫期，幼虫期表达量很低。CpMet在雌成蚊卵巢中表达水平较高，其次是脂肪体。进一步研究发现在保幼激素类似物烯虫酯处理6、12和24 h后，雌性成蚊的CpMet表达量分别上调1.27、4.15和1.26倍，表明CpMet受保幼激素的正调控。用蜕皮激素20E处理蚊蛹24 h后，CpMet基因的表达量急剧增加，表明CpMet基因表达受20E的诱导。注射dsCpMet 48 h后CpMet mRNA表达水平显著下调了86.29%；注射dsCpMet的雌蚊所产的卵块平均含卵数下降了49.89%，卵的孵化率降至73.36%。结论干扰CpMet基因表达能够显著降低雌蚊的产卵量，表明CpMet基因参与淡色库蚊生殖调控，可作为蚊虫控制的潜在靶标。

关键词: 淡色库蚊, Met基因, 表达模式, 功能分析

Abstract: Objective To investigate the expression characteristics of the juvenile hormone receptor gene CpMet of Culex pipiens pallens and its regulatory effects on reproduction. Methods Real-time quantitative PCR was used to determine the expression characteristics of the CpMet gene in different developmental stages of Cx. pipiens pallens, in different tissues of adult mosquitoes, and after hormone treatment. RNA interference technology was used to analyze the role of the CpMet gene in regulating the reproduction of female adult Cx. pipiens pallens. All data were expressed as the mean ±standard error of the mean and analyzed by SPSS 13.0 softwere. Results CpMet was expressed in all developmental stages of Cx. pipiens pallens. The expression level of CpMet was the highest in the egg stage, followed by the pupal and adult stages, and was very low in the larval stage. CpMet had a high expression level in the ovary of female adult mosquitoes, followed by the fat body. After treatment with the juvenile hormone analogue methoprene for 6 h, 12 h, and 24 h, the expression of CpMet was up-regulated by 1.27 times, 4.15 times, and 1.26 times in female adult mosquitoes, respectively, indicating that CpMet was positively regulated by juvenile hormone. After treating mosquito pupae with ecdysone 20E for 24 h, the expression of CpMet was increased sharply, indicating that 20E could induce CpMet gene expression. At 48 h after dsCpMet injection, the mRNA expression of CpMet was significantly decreased by 86.29%. In the egg masses produced by female mosquitoes with dsCpMet injection, the mean number of eggs dropped by 49.89%, and the hatching rate of eggs dropped to 73.36%. Conclusion Interfering with CpMet gene expression can significantly reduce the egg production of female Cx. pipiens pallens mosquitoes, indicating that CpMet can regulate the reproduction of Cx. pipiens pallens and can be used as a potential target for mosquito control.

Key words: Culex pipiens pallens, Met gene, Expression pattern, Functional analysis

中图分类号:

R384.1
Q753

周长印, 关晴晴, 戴雨琪, 刘洪霞, 钱坤. 淡色库蚊保幼激素受体基因CpMet的表达特性和功能分析[J]. 中国媒介生物学及控制杂志, 2021, 32(6): 680-685.

ZHOU Chang-yin, GUAN Qing-qing, DAI Yu-qi, LIU Hong-xia, QIAN Kun. Expression characterization and functional analysis of juvenile hormone receptor gene CpMet in Culex pipiens pallens[J]. Chinese Journal of Vector Biology and Control, 2021, 32(6): 680-685.

参考文献

[1] Roy S,Saha TT,Zou Z,et al. Regulatory pathways controlling female insect reproduction[J]. Annu Rev Entomol,2018,63:489-511. DOI:10.1146/annurev-ento-020117-043258.
[2] Gwadz RW,Spielman A. Corpus allatum control of ovarian development in Aedes aegypti[J]. J Insect Physiol,1973,19(7):1441-1448. DOI:10.1016/0022-1910(73)90174-1.
[3] Clifton ME,Noriega FG. Nutrient limitation results in juvenile hormone-mediated resorption of previtellogenic ovarian follicles in mosquitoes[J]. J Insect Physiol,2011,57(9):1274-1281. DOI:10.1016/j.jinsphys.2011.06.002.
[4] Clifton ME,Noriega FG. The fate of follicles after a blood meal is dependent on previtellogenic nutrition and juvenile hormone in Aedes aegypti[J]. J Insect Physiol,2012,58(7):1007-1019. DOI:10.1016/j.jinsphys.2012.05.005.
[5] Attardo GM,Hansen IA,Raikhel AS. Nutritional regulation of vitellogenesis in mosquitoes:implications for anautogeny[J]. Insect Biochem Mol Biol,2005,35(7):661-675. DOI:10.1016/j.ibmb.2005.02.013.
[6] Li M,Mead EA,Zhu JS. Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression[J]. Proc Natl Acad Sci USA,2011,108(2):638-643. DOI:10.1073/pnas.1013914108.
[7] Zou Z,Saha TT,Roy S,et al. Juvenile hormone and its receptor,methoprene-tolerant,control the dynamics of mosquito gene expression[J]. Proc Natl Acad Sci USA,2013,110(24):E2173-E2181. DOI:10.1073/pnas.1305293110.
[8] Li M,Liu PC,Wiley JD,et al. A steroid receptor coactivator acts as the DNA-binding partner of the methoprene-tolerant protein in regulating juvenile hormone response genes[J]. Mol Cell Endocrinol,2014,394(1/2):47-58. DOI:10.1016/j.mce.2014. 06.021.
[9] Saha TT,Roy S,Pei GF,et al. Synergistic action of the transcription factors Krüppel homolog 1 and Hairy in juvenile hormone/Methoprene-tolerant-mediated gene-repression in the mosquito Aedes aegypti[J]. PLoS Genet,2019,15(10):e1008443. DOI:10.1371/journal.pgen.1008443.
[10] Hansen IA,Attardo GM,Park JH,et al. Target of rapamycin-mediated amino acid signaling in mosquito anautogeny[J]. Proc Natl Acad Sci USA,2004,101(29):10626-10631. DOI:10. 1073/pnas.0403460101.
[11] Livak KJ,Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-△△Ctmethod[J]. Methods,2001,25(4):402-408. DOI:10.1006/meth.2001.1262.
[12] 李志清. 家蚕BmMet基因的鉴定、克隆及功能分析[D]. 重庆:西南大学,2009. Li ZQ. Identification,cloning and functional analysis of BmMet,a Methoprene-tolerant gene in the Silkworm,Bombyx mori[D]. Chongqing:Southwest University,2009.
[13] 姚云. 褐飞虱Met基因的克隆及其功能研究[D]. 杭州:中国计量学院,2015. Yao Y. Molecular cloning and functional study of Methoprene-tolerant(Met) from Nilaparvata lugens (Stål)[D]. Hangzhou:China Jiliang University,2015.
[14] 李亚丽. 桔小实蝇保幼激素信号通路Met和Kr-h1基因功能初探[D]. 重庆:西南大学,2016. Li YL. Functional analysis of juvenile hormone signaling pathway genes Met and Kr-h1 in Bactrocera dorsalis (Hendel)(Diptera:Tephritidae)[D]. Chongqing:Southwest University,2016.
[15] Guo EE,He QY,Liu SM,et al. MET is required for the maximal action of 20-hydroxyecdysone during Bombyx metamorphosis[J]. PLoS One,2012,7(12):e53256. DOI:10.1371/journal.pone.0053256.
[16] Konopova B,Jindra M. Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum[J]. Proc Natl Acad Sci USA,2007,104(25):10488-10493. DOI:10.1073/pnas.0703719104.
[17] Lozano J,Belles X. Role of Methoprene-tolerant (Met) in adult morphogenesis and in adult ecdysis of Blattella germanica[J]. PLoS One,2014,9(7):e103614. DOI:10.1371/journal.pone. 0103614.
[18] Smykal V,Bajgar A,Provaznik J,et al. Juvenile hormone signaling during reproduction and development of the linden bug,Pyrrhocoris apterus[J]. Insect Biochem Mol Biol,2014,45:69-76. DOI:10.1016/j.ibmb.2013.12.003.
[19] Song JS,Wu ZX,Wang ZM,et al. Krüppel-homolog 1 mediates juvenile hormone action to promote vitellogenesis and oocyte maturation in the migratory locust[J]. Insect Biochem Mol Biol,2014,52:94-101. DOI:10.1016/j.ibmb.2014.07.001.
[20] Parthasarathy R,Tan A,Sun Z,et al. Juvenile hormone regulation of male accessory gland activity in the red flour beetle,Tribolium castaneum[J]. Mech Dev,2009,126(7):563-579. DOI:10.1016/j.mod.2009.03.005.
[21] Parthasarathy R,Tan AJ,Palli SR. bHLH-PAS family transcription factor methoprene-tolerant plays a key role in JH action in preventing the premature development of adult structures during larval-pupal metamorphosis[J]. Mech Dev,2008,125(7):601-616. DOI:10.1016/j.mod.2008.03.004.
[22] Guo W,Wu ZX,Song JS,et al. Juvenile hormone-receptor complex acts on Mcm4 and Mcm7 to promote polyploidy and vitellogenesis in the migratory locust[J]. PLoS Genet,2014,10(10):e1004702. DOI:10.1371/journal.pgen.1004702.
[23] Marchal E,Hult EF,Huang J,et al. Methoprene-tolerant (Met) knockdown in the adult female cockroach,Diploptera punctata completely inhibits ovarian development[J]. PLoS One,2014,9(9):e106737. DOI:10.1371/journal.pone.0106737.
[24] Holtof M,Van Lommel J,Gijbels M,et al. Crucial role of juvenile hormone receptor components methoprene-tolerant and Taiman in sexual maturation of adult male desert locusts[J]. Biomolecules,2021,11(2):244. DOI:10.3390/biom11020244.