The Functional Study Of Cep78 In Ciliogenesis And Spermatogenesis In Drosophila

Cilia are microtubule-based hair-like organelles that protrude from the cell surface.Defects in the structure and function of cilia can lead to the development of a variety of human genetic diseases.The occurrence of cilia is a dynamic process,which is closely related to the cell cycle.When cells exit the cell division phase and enter the quiescent phase,the mother centriole transforms into the cilia basal body,which initiates the formation of cilia.Cilia are transformed from centrosomes,and mutations in centrosomal proteins(CEPs)often lead to cilia-related diseases.Cep78(Centrosomal Protein of 78 kDa)is a CEP with a molecular weight of about 78 k Da in mammals,its mutation can lead to cilia-related diseases.However,at present,we still know little about the mechanism of Cep78 regulating cilia formation.Therefore,the study of Cep78 molecular function can provide reference for the diagnosis and treatment of ciliopathies,which is of great significance.Drosophila melanogaster is a classic model organism commonly used in cell biology and genetics research,and the CG7886 of Drosophila is the homologous gene of mammalian Cep78,and the function of CG7886 has not been reported.In this project,Drosophila melanogaster was taken as the object to analyze the role of Cep78 in cilia formation and spermatogenesis in order to disclose the molecular function of Cep78.This study is divided into three main parts:Part Ⅰ: Construction of Cep78 transgenic Drosophila melanogaster and subcellular localization analysisThe Cep78 transgenic Drosophila strain was constructed by att P-Phi C31 fixed-point insertion technology.Firstly,the recombinant plasmid p JFRC2-Cep78-GFP containing the att B site and Cep78 sequence was constructed,injected into the Drosophila embryo harboring the att P site.With the action of Phi C31 integrase,the Cep78-GFP sequence was integrated into the second chromosome of the Drosophila,and finally the red-eye Cep78 transgenic Drosophila strain was obtained.After that,the subcellular localization of Cep78 was observed by laser confocal microscopy by dissecting Cep78 transgenic Drosophila and immunostaining its antennae and testis.The results showed that Cep78 was localized at the base of the cilia in the cilia of sensory neurons,and Cep78 was localized at the distal centrioles,transition zone and sperm flagella in the testis,respectively.Part Ⅱ: Construction of cep78 knockout mutant Drosophila strain and phenotypic analysisThe cep78 knockout mutant Drosophila strain was constructed by CRISPR-Cas9 gene editing technology.Firstly,a pair of recombinant plasmids p EASY-Blunt-g RNA with target sequences was constructed,injected into Drosophila with Cas9 background.The large fragment deletion mutant Drosophila strain was obtained through Drosophila hybridization and genotype identification.Gene sequencing and peak-map alignment showed that the deletion of the base at position 277 th led to Cep78 to produce a frameshift mutation,but its heterozygous male mutant was sterile and it was difficult to obtain a homozygous mutant.To avoid the possible dominant negative effect,a complete knockout mutant was constructed in the same way,which retained only 3 amino acids.Secondly,the phenotypes of cep78 knockout mutation were analyzed.It was found that the reproductive ability of the mutant phenotype was completely lost,the crawling ability was also reduced.It was speculated that the cep78 mutation led to the destruction of the cilia structure and could not produce mature sperm.So,the ultra-microstructure of the cilia/flagella of the mutant was analyzed by transmission electron microscopy(TEM),and the results showed that the sperm axoneme of the mutant was destroyed and the mitochondrial derivatives were abnormal,sperm could not be individualized normally and it resulted in the infertility of male mutant Drosophila,but its sensory neuronal cilia structure was not found abnormal.So,Cep78 plays an important role in spermatogenesis.Part Ⅲ: Mechanism analysis of Cep78 in regulating basal body extensionAn important phenotype of the cep78 mutant is that the spermatocyte centriole is significantly shortened,indicating that it plays an important role in basal body elongation and maturation.Given that the unc(Uncoordinated)mutant and the cep120(Central Protein of 120 k Da)mutant of Drosophila have similar phenotypes,we studied the relationship between them through Drosophila hybridization and immunofluorescence.The results showed that: 1)when Unc was knocked out,the localization of Cep120 was significantly weakened and Cep78 disappeared completely;2)when Cep120 or Cep78 was knocked out,the positioning of Unc was not affected;3)when Cep120 was knocked out,the localization of Cep78 was significantly weakened,and the localization of Cep120 was also significantly weakened in cep78 knockout mutant.These results indicated that Cep120 and Cep78 were located in the downstream of Unc and co-regulated basal body extension.In order to verify whether there is an interaction between Cep78 and Unc,Cep78 and Cep120,GST pull-down and Co-IP analysis were performed and the results showed a direct interaction between Cep78 and Unc,Cep78 and Cep120.In this study,the localization of Cep78 in cilia was studied in Drosophila,and the changes of cilia/flagella ultra-microstructure in cep78 knockout mutant were analyzed,which revealed the function and specific mechanism of Cep78 in basal body extension during sperm formation.It provides reference value for further research on the strict control of centriole length.It lays the foundation for a comprehensive and thorough understanding of cilia formation and spermatogenesis mechanisms.At the same time,it also provides reference for the research and treatment of ciliopathies.