Construction Of WD0631 And WD0632 Transgenic Drosophila Strains And Their Relationship With CI

Wolbachia is a gram-negative bacterium that is widely found in arthropods.Wolbachia controls host reproduction in a variety of ways, such as parthenogenesis,killing male, inducing host cytoplasmic incompatibility ?CI?, and feminization.Cytoplasmic incompatibility ?CI? is one of the most common and studied phenotypes. CI is a phenomenon that when males infected with Wolbachia mate with females not infected or infected with different strain of Wolbachia, they can cause death of embryos,and having low hatching rate. The cytoplasmic incompatibility of oosperm causes the chromatin condensation defect from the male parent at the first mitosis. The developmental rate of the male pronucleus lags behind and forms the chromatin bridge,which causes the embryo development to stop and cause the embryo to die. Although many studies have proposed a variety of models to explain the CI phenotype,but Wolbachia induced CI molecular mechanism is still being explored.It has been reported that a 56kDa protein encoded by the Wolbachiagene wPip₀282 was detected in C.pipiens spermathecae as well as in ovarian tissues by SDS-PAGE and mass spectrometry. The Wolbachia protein homologous to wPip₀282 and its downstream co-transcribed gene wPip₀283 was found only in Wolbachia, which can induce CI, by homologous comparison with a number of different proteins encoded by Wolbachia. In wMel Wolbachia strain, only wMel₀631 and wMel₀632 were homologous to the proteins encoded by wPip₀282 and wPip₀283. The genes encoding wMsl₀631 and wMel₀632 proteins were WD0631 and WD0632. Therefore, in order to explore whether WD0631 and WD0632 are related to CI, we extracted the genome DNA of Dmel wMel. The CDS of Wolbachia gene WD0631 and WD0632 were amplified, and then used the amplified WD0631 gene ligated with pEASY-T1 vector, building into PEASY-T1-WD0631 plasmids. The plasmid pEASY-T1-WD0637 was digested with Bgl? and Kpn?, and ligated with the digested pUAST expression vector to form recombinant plasmid pUAST-WD0631. The recombinant plasmid pUAST-WD0631 and the transposase plasmid P ? 2-3 were mixed according to a certain proportion. The mixed plasmids were injected into the germ plasm of early embryo of w1118 Drosophila through embryo microinjection technique. The survived fruit fly can be successfully screened by the mini-white eye marker gene in the recombinant plasmid. The obtained flies with eye color were crossed with the balanced Drosophila melanogaster to know where the P?insertion? was, then, to get the homozygous transgenic flies. Used the amplified WD0632 gene ligated with pEASY-T1 vector, building into PEASY-T1-WD0632 plasmids. After pEASY-T1-WD0632 was digested with XhoI and XbaI, The purified WD0632 gene was ligated with the double-digested pUAST expression vector. However,after several attempts, none of them were successfully ligated. Finally, the recombinant plasmid pUAST-WD0632 was not obtained.In order to study whether the pUAST-WD0631 transgenic fruit fly would produce CI-related phenotype, I used nosGal4 and bamGal4 mated with the transgenic flies, so the WD0631 gene was driven to be overexpressed in the gonad and testis by the UAS/Gal4 system. The expression of WD0631 gene in Drosophila melanogaster was detected by RT-qPCR. The results showed that WD0631 gene was overexpressed in Drosophila melanogaster, which was significantly different from the control group ?p<0.01?. In order to further study the effect of WD0631 gene on fertility of male transgenic flies, I used actGal4, nosGal4 and bamGal4 driven WD0631 transgenic Drosophila melanogaster, 1day old offspring male flies were mated with 3 day old normal virgin flies to statistics the hatching rate of offspring embryo, the results showed that the hatching rate and egg production of offspring were not different from that of the control group ?p>0.05?, which indicated that WD0631 gene could not influence the fecundity of Drosophila alone. WD0631 gene and WD0632 are upstream and downstream genes, and co-transcription occurs, so the two genes may play a role in order to induce Drosophila CI.In this study, the Wolbachia gene WD0631 transgenic flies were successfully constructed .Our analysis presented that the Wolbachia gene WD0631 has integrated into the Drosophila genome absolutely can be overexpression. But the expression of the WD0631 gene is not enough to affect the offspring fecundity of Drosophila, suggesting that genes WD0631 and WD0632 work together may affect the fecundity of Drosophila and induce host flies producing CI-like phenotype.