Ectopic Expression Of Male Determinant Initiation Factor AalNix And Regulatory Pathway Of Doublesex In Aedes Albopictus

Background:Ae.albopictus is considered to be the most invasive and invasive species in the world.It is one of several globally important arboviruses,including dengue virus(DENV),Zika virus(ZIKV),yellow fever virus(YFV)and Chikungunya virus(CHIKV).The most effective way to prevent mosquito-borne infectious diseases is to control the source of infection and reduce the transmission vector.However,due to the lack of effective vaccines for most mosquito-borne infectious diseases,the control of mosquito-borne populations is still the most effective measure for the prevention and control of mosquito-borne infectious diseases so far.The previous research of our research group has confirmed that the AalNix gene of Ae.albopictus is a male-determining gene and has a complex structure.However,whether AalNix can be used as an apex gene to initiate male development still needs further exploration.Doublesex(dsx)gene is the central gene of insect sex determination and a key factor in regulating sex differentiation.Studying the regulation pathway of dsx not only helps to clarify the sex determination mechanism of mosquitoes,but also has positive significance for the biological control of mosquitoes.Objectives:1.Construct a transgenic mosquito strain,insert the expression vector containing AalNix elements into the Ae.albopictus genome,and explore whether the ectopic expression of AalNix can initiate male development,to further clarify the role of AalNix's male determination and its possible application prospects.2.Explore the role and mechanism of doublesex in the sex determination pathway,find the downstream target genes of dsx,and clarify whether its upstream is directly regulated by AalNix.Methods:1.The promoter sequence of AalNix itself was cloned,and the expression vector of 4 main isoforms of AalNix gene of Ae.albopictus was constructed.Using PiggyBac-mediated insertion,the expression vectors containing AalNix expression elements were inserted into the genome of Ae.albopictus to construct transgenic lines.Observe whether the offspring of mosquitoes can become male or masculinize by stereomicroscope,count the number and ratio of various phenotypes of the transgenic offspring,and detect the expression level of the male and female splicing of the downstream effect genes dsx and fru.2.Using the Ae.aegypti female-specific DSXF(GenBank:ACY78700)amino acid sequence as the query sequence,use tBLASTn to search the Ae.albopictus genome and transcriptome database.The full length of the Aalbdsx gene was cloned by 5' and 3'-RACE,and its expression and splicing at different developmental stages were tested.Using RNAi and high-throughput sequencing combined with ChIP-qPCR methods to identify Aalbdsx downstream target genes.AalNix isoform 1 was overexpressed in the C6/36 cell line,and after successful expression,it was tested by RIP-qPCR/PCR to determine whether its downstream directly regulates dsx.Results:1.The promoter sequence of Ae.albopictus AalNix was cloned,and a transgenic line for each isoform was successfully established.Among them,the transgenic line with ectopic expression of AalNix isoforml and isoform2 cannot change the sex of the female.The transgenic lines of AalNix isoform3&4 can completely reverse part of females to males,and sex-reversed males can mate with WT females to produce males that do not contain M-locus(mm;?);and some females have a masculinized phenotype.Because of the dense tentacles and the appearance of male-like reproductive process basal ganglia and reproductive spines,the downstream effect genes dsx and fru also progressed towards male splicing.2.Using the amino acid sequence of Ae.aegypti Aaedsx to identify the Aalbdsx gene in the Ae.albopictus database,using 5'and 3'RACE combined with RT-PCR and PCR to obtain the full length and gene structure of the Aalbdsx gene.The gender expression mode and temporal and spatial expression characteristics of dsx were clarified,and confirmed that AalbdsxM is the default splicing mode of this gene.The RNAi method was used to identify the direct target genes of DSX,proving that Aalbdsx mainly affects the development of the ovary through the vitellogenin receptor(vgr)gene.It reveals that AalNix does not directly combine with Aalbdsx to play a role in the sex determining pathway.Conclusions:1.The male-determining gene AalNix isoform3&4 of Ae.albopictus can initiate male development by regulating the sex-specific splicing of downstream dsx and fru.Therefore,AalNix can be used as the apex gene of sex determination to play a regulatory role on the sex determination pathway,and has the application prospect of genetic control of mosquito vector populations.2.There are one or more splicing factors between AalNix and Aalbdsx to regulate the sex determination pathway.Aalbdsx,as the downstream effector gene of the sex determination pathway,has multiple target genes,including vg and vgr,while Aalbdsx affects vgr,the direct regulation of vgr by Aalbdsx is essential for the ovarian development of Ae.albopictus.