Functional Study Of ?-4GT1 Gene In Drosophila Melanogaster

Ceramide is composed of sphingosine and long-chain fatty acid,and it is usually glycosylated to form glycosylsphingolipid(GSL),which is located in the cell membrane.It has been reported that glycosylated ceramides participate in mediating many biological processes,including development and immunity.The fruit fly,Drosophila melanogaster,has well-known genetic background and many available genetic manipulation approaches,thus,it is one of the best model organisms for studying development and immunity.In Drosophila,the first step in the glycosylation of ceramide is to form glucosylceramide(GlcCer),and then several glycosyltransferases are involved in the following glycosylation process,including ?-1,4-N-acetylgalactosaminyltransferase 1(?-4GT1)that participates in the final step.Previous work focuses more on glycosyltransferases involved before the final step,and less is known about the functions of ?-4GT1 gene in the glycosylation of ceramide.In this study,we aimed to investigate whether mutation or knockdown expression of ?-4GT1 gene will affect immunity and gonad development in Drosophila.By injecting lipopolysaccharide(LPS)and peptidoglycan(PGN)into wild-type w1118,?-4GT1,Toll-1 and Toll-7 mutant flies,we analyzed expression of antimicrobial peptide(AMP)genes after LPS and PGN stimulation.By comparing the phenotypes of ovary and testis in the ?-4GT1/Cyo mutant,bamGal4 driven ?-4GT1 RNAi and control flies,we analyzed the role of ?-4GT1 in the development of gonad in Drosophila.The main results of this study are as followings:(1)In the female wild-type(w1118)flies,pricking(injury)up-regulated the IMD pathway mediated Diptericin(Dpt)gene-4-fold,injection of water induced Dpt expression-10-fold and also up-regulated the Toll pathway mediated Drosomycin(Drs)gene?2-fold.In the male w1118 flies,pricking or injection of water did not significantly change the expression of Dpt,but up-regulated Drs gene?10-fold.(2)In w1118 and ?-4GT1 mutant flies,injection of PGN,but not LPS,induced expression of both Drs and Dpt.In the Toll-1 and Toll-7 mutant flies,injection of both PGN and LPS up-regulated Drs and Dpt genes.Also,the females and males of Toll-1 and Toll-7 mutant flies responded differently to both PGN and LPS stimulation.(3)In w1118 flies,expression of ?-4GT1 gene was relatively high during early embryo development,pupation stage,female and male flies,and the hatching rate was significantly lower in the ?-4GT1/Cyo mutant flies than in the control flies.In the ?-4GT1/Cyo mutants,about 20%of the ovary was abnormally smaller in size,but apoptosis was not enhanced in these testes.In addition,in the ?-4GT1/Cyo mutant testis,the arrangement of some sperm bundles was loose and the number of mature sperms in the seminal vesicle was reduced.In the bamGal4 driven ?-4GT1 RNAi flies,similar phenotypes were observed in the testis as those of the ?-4GT1/Cyo mutants.Our combining results suggest that ?-4GT1 gene may participate in the development of gonad in Drosophila.