Preliminary Researches On The Function Of PFBC Pathogenic Gene XPR1 Homologous CG10483 In Drosophila

Primary familial brain calcification（PFBC）is a rare and dominant disease of the nervous system.It is characterized by calcification of the basal ganglia of the brain,a disease that can manifest in a variety of clinical manifestations,including Parkinson’s disease,dementia,and cognitive disorders.Five PFBC pathogenic genes have been identified.SLC20A2,PDGFRB and PDGFB have been studied by many research groups since they were discovered,while the functions and effects of XPR1 and ISG15 are still unclear.We mainly focus on the functional research of XPR1 gene.The XPR1 gene was first identified in mouse as a retroviral receptor.It is known that the protein encoded by XPR1 is composed of 696 amino acids,and is a multiple pass transmembrane protein.In the subsequent studies,researchers have found that this protein controls the output of phosphate in cells.It is the only protein with a phosphorus export function found in humans.In the bioinformatics prediction,both ends of this protein are located in the cell,and two important domains are SPX and EXS.Among them,the SPX domain is relatively conserved and found in many eukaryotic proteins,and most of the XPR1mutations found by Legati et al in PFBC patients are located in this domain.Drosophila melanogaster has been widely used in biological research since Morgan with advantages of small size,clear genetic background and rapid reproduction.About 75%of known human disease genes have a recognizable match in the genome of fruit flies^[41]and50%of fly protein sequences have mammalian homologs.Therefore,Drosophila is often used as biological models to study the functional mechanisms of human related genes.In Drosophila,CG10483（dXPR1）is one of the XPR1 homologous genes,and its similarity with the human XPR1 gene is 55%.In the initial experiment,we used the RNAi of dXPR1 and the UAS-dXPR1 Drosophila to conduct a preliminary experiment.We used knock-down andoverexpression of dXPR1,and found that the calcium ion content in the brain of the third instar larvae of dXPR1 overexpression was significantly increased compared with wild-type,while konck-down of dXPR1 was opposite.Furthermore,we performed the RNAi screening using GCa MP technology and found two genes may interact with dXPR1.Six XPR1 mutation sites have been found in previous report.In the study we selected three representative XPR1mutation sites to identify its function.So we constructed three overexpressed lines with point mutations,via using P-factor insertion and site-specific recombination techniques.At the same time,we used CRISPR/Cas9 system to inject the two sgRNA into the Drosophila eggs expressing the Cas9 enzyme to generate the dXPR1^-mutant which deleted the large fragment of the dXPR1 gene.These constructions will provide a basis for further exploration of the dXPR1 functions.