蜕皮激素和保幼激素对淡色库蚊精氨酸激酶基因CpAK1和CpAK2表达的影响

doi:10.11853/j.issn.1003.8280.2024.03.004

摘要/Abstract

摘要： 目的了解淡色库蚊精氨酸激酶基因CpAK1和CpAK2的表达是否受蜕皮激素和保幼激素及其信号通路的影响。方法采用反转录实时荧光定量PCR（RT-qPCR）和蛋白免疫印迹法,分别检测蜕皮激素20-羟基蜕皮酮（20E）和保幼激素类似物（烯虫酯）处理不同时间后淡色库蚊CpAK1和CpAK2的mRNA和蛋白表达水平。RNA干扰蜕皮激素信号通路相关基因CpBR-C、CpECR以及蜕皮激素信号通路相关基因CpMet、CpKr-h1后,分别检测CpAK1和CpAK2的mRNA表达水平。结果用20E蛹期注射处理,24 h后CpAK1的mRNA表达水平显著下调至19.77%,而CpAK2的mRNA表达水平在处理后6 h和12 h分别上调5.70和2.77倍。20E处理6 h后 CpAK1的蛋白表达水平显著下调而CpAK2的蛋白表达水平无明显变化。用烯虫酯蛹期注射处理,CpAK1的mRNA表达水平24 h后显著上调3.36倍,而CpAK2的mRNA表达水平在6 h和12 h分别下降至46.81%、39.34%。烯虫酯处理6 h和12 h后 CpAK1蛋白表达水平显著上调,而CpAK2的蛋白表达水平显著下调。干扰CpBR-C导致CpAK1的mRNA表达水平上调2.35倍,而 CpAK2的mRNA表达水平下调,仅为对照的 2.80%。干扰CpECR后CpAK1 表达水平显著上调 8.13 倍,而CpAK2 表达水平显著下调42.79%。CpMet的沉默导致 CpAK1 表达水平下调至 43.87%,而CpAK2表达水平显著上调3.05倍。干扰 CpKr-h1 基因导致CpAK1表达水平显著下调至 13.59%,而 CpAK2 表达水平显著上调 4.35 倍。结论 20E对CpAK1的表达负调控,对CpAK2正调控。烯虫酯对CpAK1的表达正调控,对CpAK2负调控。干扰CpBR-C和CpECR导致CpAK1显著上调,CpAK2显著下调。干扰CpMet和CpKr-h1基因后CpAK1显著下调,而CpAK2显著上调。淡色库蚊化蛹到羽化阶段,2种精氨酸激酶的表达水平受蜕皮激素和保幼激素调节,总体保持相对恒定,这种调节机制对于维持三磷酸腺苷（ATP）的稳态具有重要意义。

关键词: 淡色库蚊, 精氨酸激酶, 蜕皮激素, 保幼激素, 表达

Abstract: Objective To investigate whether the expression of arginine kinase genes (CpAK1 and CpAK2) in Culex pipiens pallens is affected by ecdysone and juvenile hormone and associated signaling pathways.Methods After being treated with 20-hydroxyecdysone (20E) and methoprene (a juvenile hormone analog) for different time durations, the mRNA and protein expression levels of CpAK1 and CpAK2 in Cx. pipiens pallens were measured by using reverse transcription real-time fluorescence quantitative PCR and western blot, respectively. After suppressing ecdysone signaling pathway-related genes (CpBR-C and CpECR) and ecdysone signaling pathway-related genes (CpMet and CpKr-h1) with RNA interference, the mRNA expression levels of CpAK1 and CpAK2 were measured.Results After 20E injection at the pupal stage, the mRNA expression level of CpAK1 was significantly reduced to 19.77% at 24 h, while the mRNA expression level of CpAK2 was up-regulated by 5.70 and 2.77 times at 6 h and 12 h, respectively; and 6 h after 20E treatment, the protein expression level of CpAK1 was significantly down-regulated, while the protein expression level of CpAK2 had no significant change. Methoprene treatment at the pupal stage significantly increased the CpAK1 mRNA level by 3.36 times at 24 h, and significantly decreased the CpAK2 mRNA level to 46.81% at 6 h and 39.34% at 12 h; and at 6 h and 12 h, the treatment significantly up-regulated the CpAK1 protein expression level, and significantly down-regulated the CpAK2 protein expression level. Suppressing CpBR-C caused a 2.35-fold increase in the mRNA expression level of CpAK1 and a decrease in the mRNA expression level of CpAK2 to only 2.80% of the control. After CpECR knockdown, the CpAK1 expression level was significantly increased by 8.13 times, while the CpAK2 expression level was significantly decreased by 42.79%. CpMet silencing led to a significantly down-regulated CpAK1 level to 43.87% and a significantly up-regulated CpAK2 level by 3.05 times. Suppressing CpKr-h1 resulted in a significantly down-regulated CpAK1 level to 13.59% and a significantly up-regulated CpAK2 level by 4.35 times.Conclusions 20E negatively regulates CpAK1 and positively regulates CpAK2, while methoprene positively regulates CpAK1 and negatively regulates CpAK2. Interfering with CpBR-C and CpECR significantly up-regulates CpAK1 and significantly down-regulates CpAK2. Interfering with CpMet and CpKr-h1 significantly down-regulates CpAK1 and significantly up-regulates CpAK2. During the pupation to eclosion of Cx. pipiens pallens, the expression of the two arginine kinases is regulated by ecdysone and juvenile hormone at relatively stable levels in general, and this regulatory mechanism is of great significance for maintaining ATP homeostasis.

Key words: Culex pipiens pallens, Arginine kinase, Ecdysone, Juvenile hormone, Expression

中图分类号:

R384.1

钱坤, 关晴晴, 覃盛锋, 王扬. 蜕皮激素和保幼激素对淡色库蚊精氨酸激酶基因CpAK1和CpAK2表达的影响[J]. 中国媒介生物学及控制杂志, 2024, 35(3): 281-286.

QIAN Kun, GUAN Qing-qing, QIN Sheng-feng, WANG Yang. Effects of ecdysone and juvenile hormone on arginine kinase genes CpAK1 and CpAK2 in Culex pipiens pallens[J]. Chinese Journal of Vector Biology and Control, 2024, 35(3): 281-286.

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