Bioinformatics Analysis And Functional Verification Of 5-HT Receptor Gene Family In Aedes Aegypti

Serotonin（5-HT）plays a vital role in many physiological processes of insects,regulating physiological activities such as growth and movement through multiple 5-HT receptors.It is also a potential target of new insecticides.However,the current research on the function of 5-HT receptors in Aedes aegypti is still unclear.We constructed the 5-HT receptor 3D structure of A.aegypti through the homology modeling based on the human 5-HT receptor template and made the phylogenetic tree with bioinformatics and phylogenetic analysis.By RT-PCR,we studied the expression of 5-HT receptors in the different tissues（head,thorax,abdomen,limbs）and the various stages（larvae,female,male）.Finally,knocked out the 5-HTR_7A gene and screen the mutant,so as explore the functions of 5-HTR_7A gene in physiological behavior and other aspects of A.aegypti.Six 5-HT receptor genes of A.aegypti obtained from NCBI and Vector Base.Bioinformatics analysis showed that all the receptor structures had seven transmembrane regions,among whichα-helix accounted for a high proportion in the secondary structure of the receptors.The evolutionary analysis showed that A.aegypti 5-HT receptors were located in three different branches,each of which had high homology with the 5-HT receptors of Drosophila melanogaster.The expression profile showed that the expressions of 5-HT receptors were the highest in males compared with all the larvae and females.There were significant differences in the tissues of the female.The 5-HTR_1B,5-HTR_2B,and5-HTR_7B were all expressed in the head,thorax,abdomen,and limbs.However,the expression abundance of the 5-HTR_7Awas the highest in the limbs compared with other tissues.The 5-HTR_1A was expressed in all tissues except limbs.Using the CRIPSR-Cas9technology to knock out the 5-HTR_7A gene of A.aegypti,we got mutant and found its sequence lost two bases near the PAM area.The transmembrane area structure was changed,from seven times reduced to four times.The sites were lost which combined with extracellular 5-HT and intracellular G protein loci,respectively.Morphological observation of mosquitoes showed that the mutant had abnormal phenotype in larval stage,the mutants had the abnormal head-to-chest ratio and decreased in motor activity.The pupal could not normal development and death.By using external stimuli to larvae and pupae in abnormal phenotypes,we also found the mutants G1 and G2 generations response to external stimuli longer than the wild type.Due to the higher mortality of larval and pupal of the mutants,the number of generations is less than the wild type.We also found that the egg of mutants G1 and G2 generations hatching rate of the offspring of adult mosquito mutants was lower than that of the wild type.We also used RT-PCR to detect the expression level of the mutant 5-HTR_7A.Then we found that the expression level of this gene decreased by about65%compared with the control group after knockout.In addition,software was used to perform micro RNA target prediction analysis on this gene,and the developmental expression profile of mi RNA was constructed.CRIPSR-Cas9 knocked out the mi RNA to further explore the relationship between the gene and mi RNAs.The A.aegypti 5-HT receptor family gene have high homology with the Drosophila receptor in evolution and has different spatiotemporal expression patterns.The results of knockout the gene of5-HTR_7A suggested that it may be related to the abnormal development and motor ability of A.aegypti.And it also affected the normal egg hatching.The deletion of mi R-281-3p could lead to heterozygous death.We detected mutations in the egg that may play an important role in the development of the whole egg stage to the larva.