Construction Of Drosophila Testis Gene Knockout Mutation And Analysis Of Male Reproductive Related Phenotypes

The development and function of the male reproductive system rely on the cooperation of many genes,which are expressed in reproductive system.However,for many of these genes,little is known about their specific functions and mechanisms.The development of CRISPR/Cas9 system is a milestone of the history of gene editing,which makes it possible for rapid,efficient and accurate gene modifications in many organisms,including Drosophila melangaster,a classical model system,CRISPR/Cas9,combined with the homology directed repair?HDR?,can generate knockout and knockin mutant alleles according to the design.In this research,to study the function of testis genes in male reproductive system in Drosophila,we generated the homozygous knockout mutant for each gene with the help of CRISPR/Cas9 system,and analyzed their male-related phenotypes.Base on the information in database,eight candidate genes,including CG4161,CG11475,CG2921,CG10541,CG7276,CG3800,CG8117 and CG16779 were selected according to the following criteria:?1?expression in testis;?2?having homologous gene in humans?Homo sapiens?and mouse?Mus musculus?;and?3?unreported functions in male reproductive.Firstly,RT?reverse transcription?-PCR was used to verify the expression of these genes in different tissues in Drosophila and mouse,which showed consistent results with that of database.Secondly,knockout mutants were generated with the help of CRISPR/Cas9 and verified by PCR.Thirdly,in order to examine the role of these genes in male reproduction,we checked the male fertility of these eight mutants.The result showed that CG3800^-/-and CG7276^-/-were partially sterile,and the offspring number was significantly lower than wild type,while other gene knockout strains were normal and showed no significant differences.Lastly,by observing the testis of CG3800^-/-and CG7276^-/-sterile male mutants,we found that they showed different types of abnormities,including seminal vesicle atrophy,spermatogonia stem cells reduction and cellular distribution disorder.The DNA and F-actin staining results also indicated that CG7276 and CG3800 played important roles in spermiogenesis.The generation of these mutants provided useful tools to unravel the function and mechanism of these genes.