CDNA Cloning And Expression Of Two Arginine Kinase Genes In Culex Pipiens Pallens And Their Effects On Growth And Blood-sucking Behavior

Arginine kinase is one of the crucial enzymes involved in energy metabolism during insect life activities.Since arginine kinase exists only in invertebrates and its energy metabolism pathway is completely different from which in mammals,arginine kinase can be used as a good target for insect control.In this paper,Culex pipiens pallens,an important city health pest,was used as the research object.We found and successfully cloned the full-length sequences of two arginine kinase genes in Culex pipiens pallens,CppAKl and CppAK2,and carried out sequence homology,phylogenesis and domain analysis of CppAK1 and CppAK2.We also analyzed the expression patterns of CppAKl and CppAK2 in different tissues and developmental stages of Culex pipiens pallens and their effects on growth,development and blood-sucking behavior by quantitative real-time PCR,in vitro expression,RNA interference and other molecular biological techniques.The results of this study have important theoretical basis and practical value for the development of new mosquito control chemical with AK as target.The main results are as follows:The full-length CppAKl and CppAK2 are cloned,of which CppAK1 contains 1454 nucleotides with an ORF encoding 410 amino acid residues and the total length of CppAK2 is 1799 bp,encoding 466 amino acids in its ORF.An amino acid sequence alignment shows that the consistency of CppAK1 and CppAK2 amino acid sequences was only 64.76%Phylogenetic analysis also showed that the AK genes of the Diptera species,such as Aedes aegypti,Aedes albopictus,Drosophila melanogaster and so on,first clustered into one group.Most of the AK2 genes in the mosquito family are separated from many AK1 and show different groups.The prokaryotic expression of CppAKl and CppAK2 is carried out in vitro and the results showed that the enzyme catalytic activities of the two kinds of arginine kinase were quite different,and the parameters of CppAKl were better than those of CppAK2.At different temperatures,the catalytic rate of CppAKl to substrate L-arginine was generally higher than that of CppAK2.CppAK1 has a high catalytic efficiency in the temperature range of 30-45 ?,and the optimal catalytic rate of CppAK2 was lower than that of CppAK1.In different pH environments,both CppAK1 and CppAK2 reached the highest catalytic rate at pH 7.5,indicating that temperature and pH value were important factors affecting the activity of Culex pipiens pallens AKsThe expression levels of CppAKl and CppAK2 in different developmental stages and tissues of Culex pipiens pallens were detected by fluorescence RT-qPCR and the results showed that CppAKl and CppAK2 are expressed at all developmental stages,and the expression level of CppAK1 was significantly higher than that of CppAK2.The expression level of CppAK1 in the egg stage was the highest which was great significantly different from other ages and the expression level of CppAK2 in egg stage and pupal stage is the significantly higher than other developmental stages.The anatomy of adult adults of Culex pipiens pallens showed that CppAKl and CppAK2 were distributed in all the different tissues.and the expression level of CppAK1 was great significantly higher than that of CppAK2.The CppAK1 expression levels from the highest to the lowest are chest,Post-developmental ovary,head,ovary before blood-sucking,Malpighian tube,fat body,midgut and salivary glands.CppAK2 is most expressed in ovary before blood sucking,followed by fat body,and the expression level is from high to low ovary before blood-sucking,fat body.Post-developmental ovary,chest,head.Malpighian tube,salivary gland,midgut.The effects of CppAK1 and CppAK2 on the growth and development of Culex pipiens pallens were studied.Micro-injection was used to perform RNA interference on the female adults of Culex pipiens pallens which did not suck blood.The results showed that the survival rate of female mosquitoes after dsCppAK1 injection was 56.4%,which was significantly different from that of the control,though the survival rate of female mosquitoes after dsCppAK2 injection was 76.5%.The lowest survival rate of female mosquitoes was 25.7%after injecting dsCppAKl and dsCppAK2 at the same time.One day after the injection,the female mosquitoes were allowed to suck blood,the egg production,average egg production and hatching rate were observed follow.The number of eggs per egg rafts which laid by mosquitos injected dsCppAK1 was found dropped by 21.3%,the dropped average number of injected dsCppAK2 eggs was 24.9%,the number of injecting dsCppAKl and groups dropped by 27.7%.Compared with the hatching rate,the hatching rate of the dsCppAK1 injection group was 89.9%the same with the control group.The hatching rate of the dsCppAK2 injection group was reduced to 85.0%,group's hatching rate which simultaneously injected both CppAKl and CppAK2 was significantly reduced to 59.9%,which was 33.3%lower than the control groupThe effects of CppAK1 and CppAK2 on blood-sucking behavior of Culex pipiens pallens were studied.The relative expression of CppAKl and CppAK2 in female mosquitoes was detected without blood sucking or after blood sucking 3 days ago.The result showed that the expression of CppAKl and CppAK2 in female mosquitoes increased significantly after blood sucking,and there was significant difference in the expression of CppAKl before and after blood sucking.Micro-injection was used to perform RNA interference on the female adults of Culex pipiens pallens,the results showed that the blood-sucking response time of female mosquitoes increased significantly after injection of dsCppAK1,and the blood-sucking rate was reduced by 28.7%with dsCppAK1 and 35.4%with dsCppAK2;the suck blood volume of both injected mosquitos was reduced by 63.5%.The blood satiety rate of female mosquitoes in the dsCppAK1 injection group and the simultaneous injection group also showed similar changes,in which the satiety rate of female mosquitoes in the simultaneous interference group decreased by about 36.8%,showing a significant difference.There was no significant difference in blood sucking response time,blood sucking rate,blood sucking volume and blood satiety rate between the group injected with dsCppAK2 and the control group.