Study On The Low-Complexity Domains Of Boule Protein Involved In Spermatogenesis Of Drosophila

A large number of studies have shown that RNA-binding proteins play a vital role in the growth and development of organisms and the formation of gametes.The DAZ gene family is essential for the regulation of human reproduction and development.The product of it's Drosophila gene homologue is an RNA-binding protein,which specifically regulates the development of germ cells and sperm formation.The change of boule gene expression or the lack of BOULE protein can affect many activities in Drosophila,such as participating in neural network signaling pathways,germ cell development,and gamete cell formation,et al.This paper mainly studies the regulation of Low-complexity domain(LC domain)of boule(BOL)gene on the process of spermatogenesis in Drosophila melanogrosa,as well as the effect of deletion of LC domain on protein aggregation.The main research results are as follows:1.Phenotypic analysis and RNA level analysis of boule^?LCmutant Drosophila were performed by Western Blot and immunofluorescence staining.It was found that sperm cells of boule^?LCmutant male Drosophila(BOULE protein deficient LC domain)were reduced and scattered,and male sterility was observed;The deletion of LC domain affected the RNA expression level of boule gene.2.Transgenic flies of boule gene,including boule-Re,boule-Rb and boule-RF(splicosomes of boule gene),were obtained by microinjection technique.Different specific promoter subsystems were used to rescue the meiotic block phenotype caused by bol1mutant,and it was found that systemic overexpression of BOULE protein was lethal.Overexpression of boule-RF using the testis specific promoter system(?₂-tublin)could save the mutant boule gene phenotype until the formation of sperm cells,while boule^?LCcould not save the mutant phenotype at all.3.The expression of TWINE protein in boule^?LCmutant Drosophila was detected by preparation of TWINE antibody.The deletion of the LC domain did not affect the expression of TWINE protein,which confirmed that the LC domain may be related to the process of sperm cell maturation and differentiation.4.Human 293T cells were transfected with overexpressed boule and boule^?LCplasmids to verify the protein aggregation in vitro.By semi-denaturing gel electrophoresis,we found that the LC domain of BOULE protein greatly affected the protein aggregation in cells.