Functional Analysis Of The F-box Gene Ppa In Drosophila

Ubiquitination is essential for the homeostasis of intracellular environment.Ppa encodes an F-box protein,which is one of the components of SCF ubiquitination complex.It was reported that Ppa was involved in the regulation of body segmentation and centromere localization in Drosophila,however,it has not been reported in lipid metabolism and spermatogenesis.This study found that Ppa could promote lipid storage and regulate spermatogenesis.The results are as follows:(1)PPA has three isomers.The coding sequence of Ppa was amplified by PCR,the expression vector and fluorescent fusion protein expression vector of target gene were constructed respectively,and the transgenic flies were obtained.The whole-body expression of PPA-PA/PC-GFP protein was driven by da-Gal4,and it was found that PPA-PA/PC was located in the salivary glands and fat bodies of the 3rd instar larvae,as well as in the seminal vesicle and ejaculatory duct of male testis.(2)The transgenic flies with two sgRNAs was constructed.The Ppa-sgRNA flies was crossed to Cas9 flies.Six deletion mutants of Ppa were successfully obtained by PCR,namely Ppa¹,Ppa²,Ppa³,Ppa⁴,Ppa⁵ and Ppa⁶,which missed 445 bp,454 bp,445 bp,453 bp,576 bp and 446 bp,respectively.The lipid droplets of the 3rd instar larvae staining by BODIPY 493/503 and DAPI,and it was found that the deletion mutants showed smaller lipid droplets when compared with w¹¹¹⁸.On the other hand,the trans-heterozygotes mutant Ppa¹/Ppa² also showed decreased lipid droplets,while Ppa¹showed individualization defects in male testis.(3)In the double mutant of Ppa RNAi and DGAT overexpression,it was found that Ppa could enhance the ectopic fat accumulation phenotype in the salivary glands.The overexpression of Ppa driven by da-Gal4 led to the increased size of lipid droplets.Overexpression of Ppa in Ppa¹ could restore the lipid droplets to normal state.In summary,this study clarified that Ppa could promote lipid storage in Drosophila,and found that Ppa might participate in the individualization of spermatogenesis,laying the foundation for further revealing its molecular mechanism.