Studies On Enzymatic Properties,expression Regulation And Function Of Two Arginine Kinases From Culex Pipiens Pallens

Culex pipiens pallens is the dominant mosquito species in China,which has the characteristics of wide distribution and great harm.However,the long-term use of chemical insecticides has led to serious resistance of mosquitoes,so it is urgent to develop mosquito control insecticides for new target.Arginine kinase(AK)is the only phosphokinase in invertebrates,which is a key kinase in energy metabolism by catalyzing the reversible reaction of arginine and ATP to produce arginine and MgADP.Since AK does not exist in vertebrates,it can be used as a target to develop pesticides with high selectivity and safety to vertebrates.Based on the full-length cDNA of two arginine kinase genes CpAKl and CpAK2 of Cx.pipiens pallens obtained in the previous study,and we study the subcellular localization,enzymatic properties,expression regulation mechanism,and function in different developmental stages of CpAK1 and CpAK2 by prokaryotic expression,quantitative real-time PCR,Western blot(WB)and RNA interference.The subcellular distribution of CpAKl and CpAK2 was detected by WB of different cell fractions,while CpAKl was founded in mitochondria and cytoplasm,and CpAK2 only existed in cytoplasm.Recombinant CpAK1 and CpAK2 proteins molecular weights were about 48 kDa by expressed in The Kcat values of CpAK1 and CpAK2 for L-arginine were147.5 ±4.817 s-1 and 17.7±36.45 s-1,respectively,and the Kcat value of CpAK1 was much higher than that of CpAK2.Substrate specificity analysis showed high specificity of CpAK1 and CpAK2 for L-arginine,and the lowest specificity of substrate was L-Canavanine among guanidino substrates investigated.Compared with substrate L-arginine,the reaction rates decreased by 80.01%and 86.39%,respectively.The transcriptional regulation of CpAKl and CpAK2 by hormone treatment and RNA interference.Pupal injection of 20E resulted in a was significantly down-regulated of CpAKl mRNA level to 19.77%at 24 h,while CpAK2 mRNA level was up-regulated by 5.70-fold and 2.77-fold at 6 and 12 h,respectively.The protein levels of CpAK1 had no significant change,while the protein expression of CpAK2 was significantly up-regulated after 20E treatment for 6 h and 12 h.Pupal injection of juvenile hormone analogue methoprene resulted in a was significantly up-regulated of CpAK1 mRNA level by 3.36-fold at 24 h,while the CpAK2 mRNA was significantly decreased to 46.81%and 39.34%at 6 h and 12 h,respectively.CpAKl protein level was up-regulated and CpAK2 protein level was significantly downregulated 12 h after methoprene treatment.Interference with ecdysone signaling pathway related genes CpBR-C and CpECR resulted in the up-regulation of CpAKl and the downregulation of CpAK2.Interfering with Juvenile hormone signaling pathway related genes CpMet and CpKr-h1 genes,CpAK1 was significantly down-regulated,while CpAK2 was significantly up-regulated.The effects of temperature and humidity stress on the mRNA and protein levels of CpAK1 and CpAK2 were examined using RT-qPCR and western blot(WB).The results showed that the expression level of CpAK1 mRNA increased,which was 3.46-fold and 3.53-fold at 6 h treatment at 38? and 4?,respectively,and expression of CpAK2 mRNA was significantly down-regulated.The protein expression levels of CpAKl and CpAK2 were basically consistent with the mRNA expression levels under high and low temperature stress.The CpAK1 mRNA was significantly up-regulated at 6 h,and the CpAK1 mRNA was downregulated to 45.45%at 12 h under low humidity(RH20%)stress.Under high humidity(RH100%),the CpAK1 mRNA had no significant change,while the CpAK2 mRNA was significantly down-regulated.The protein expression levels of CpAKl and CpAK2 were basically consistent with the mRNA expression levels under high and low humidity stress.Comparative analysis of functions of CpAK1 and CpAK2 in the growth,reproduction and blood sucking behavior of Cx.pipiens pallens used the RNA interference of CpAKl and CpAK2 at pupal and adult stages was performed by microinjection technique.RNA interference at pupal stage,the eclosion rates of dsCpAK1 group and dsCpAK1+dsCpAK2 group decreased significantly to 29.33%and 26.67%,respectively.The pupal survival rates of dsCpAKl and dsCpAK1+dsCpAK2 groups were significantly decreased to 82.00%and 80.66%,respectively.The survival rates of dsCpAKl,dsCpAK2 and dsCpAK1+dsCpAK2 groups at adult stage were significantly decreased to 56.35%,76.50%and 15.79%,respectively.The average number of eggs in the group of simultaneous injection of dsCpAK1 and dsCpAK2 in pupal stage decreased significantly to 73.93%.There was no significant difference among the injection groups in adult stage.The hatching rate of dsCpAKl and dsCpAK2 co-injection group decreased significantly in pupal and adult stages,which were 58.68%and 66.50%respectively.There was no significant difference in the blood sucking rate of the interference injection group in pupal stage,but the blood sucking rate of the adult injection group and the simultaneous injection group decreased significantly to 19.19%and 30.30%respectively.The blood engorgement rate of dsCpAK1 and dsCpAK2 co-injection group at the pupal stage decreased significantly to 36.44%,and that of the group injected with dsCpAK1 and dsCpAK2 at the adult stage decreased significantly to 58.33%and 51.85%respectively.