Effects of ecdysone and juvenile hormone on arginine kinase genes CpAK1 and CpAK2 in Culex pipiens pallens
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.
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