CG6015基因在果蝇睾丸生殖干细胞生命维持中的功能研究

Function of CG6015 inregulating germline fate in the Drosophila testis

目的CG6015基因作为RNA剪接体的调控因子,是否参与调控果蝇睾丸生殖干细胞(GSCs)尚不明确。文中旨在探讨CG6015基因在果蝇睾丸GSCs生命维持中的功能,并探索其中潜在的调控机制。方法利用UAS-Gal4系统实现CG6015基因在果蝇睾丸GSCs中的特异性敲减。以W1118品系为对照组,Nos>CG6015 RNAi为实验组,采用免疫荧光染色技术分别观察2组睾丸的形态和GSCs的改变情况。在果蝇S2细胞中,应用基因沉默技术分别敲减阴性对照(NC)与CG6015基因,作为NC组与CG6015 siRNA组,利用qRT-PCR检测CG6015基因及RNA剪接体亚基Prp8、SmG和U2A的mRNA表达水平。结果与对照组小睾丸的比例(0)比较,实验组(100%)明显升高(P<0.001)。与对照组睾丸中的生殖融合体数量比较,实验组明显减少[(19.67±0.88)个vs(0.00±0.00)个,P<0.001]。与对照组比较,实验组Eya阳性细胞数量明显增加[(9.67±0.67)个vs(25.67±5.21)个,P<0.05]。与NC组Prp8、SmG基因的mRNA相对表达水平(1.00±0.00)比较,CG6015 siRNA组Prp8基因(0.39±0.03)明显下调,SmG基因(1.60±0.07)明显上调(P<0.05)。结论CG6015基因在果蝇睾丸中可以通过介导RNA剪接体来调控GSCs的生命维持,为男性不育症的致病机理提供了新思路。

Objective Whether CG6015,as a regulatory factor of RNA splicing,is involved in the germline fateremains unclear.This research systematicallyinvestigates the role of CG6015 in maintaining germline fate in theDrosophila testis and explores the underlying mechanism.Methods In Drosophila,UAS-Gal4 system was used to knock down CG6015 in germline stem cells.Immunostaining of control(W1118)and experimental group(Nos>CG6015 RNAi)was performed to estimate the change oftesticularmorphologyand germline fate.Besides,small interfering RNA(siRNA)was used to knockdown negative control andCG6015 in Drosophila S2 cells,as NC group and CG6015 siRNA group.The mRNA expression of CG6015 and spliceosomesubunitsPrp8、SmG and U2 Awas assessed through qRT-PCR.Results Immunostaining results showed that compared with the control group,the normal morphology of the Drosophila testes disappeared in experimentalgroup,presenting a small testicular phenotype(100%,n=58).Moreover,in comparison with control group,neither Vasa positive cells nor1 B1 positive signals[(19.67±0.88)vs(0.00±0.00),P<0.001]were detected in experimental group.Zfh1 positive cells were scattered while Eya positive cells showed obvious accumulation,comparing to control group[(25.67±5.21)vs(9.67±0.67),P<0.05].Additionally,the mRNA expression level of Prp8(0.39±0.03)in CG6016 siRNA group was downregulated(P<0.05),compared that in NC group(1.00±0.00).While the mRNA expression level of SmG(1.60±0.07)in CG6015 siRNA group wasupregulated(P<0.05)in comparation with that in NC group(1.00±0.00).Conclusion CG6015 is required for the maintenance of Drosophila germline stem cells via regulating spliceosome.