Identification And Functional Study Of The Sterility Genes Armi-cycJ And Cher In Drosophila Melanogaster

Objective: Sterile genes of flies were screened from the laboratory mutant library to further identify mutant genes of female sterile lines mf949,mf2150 and mf3392 and to study their gene functions.Methods: Male and female flies hybridized with homozygous mutant strains were selected for fertility verification,egg laying and whether offspring were produced,ovaries were dissected under a microscope and morphology was observed.Hts was used as the primary antibody,and the changes of adult ovarian germ cells and somatic cells were determined by immunofluorescence staining.Genomic DNA of fruit flies was extracted,reverse PCR was performed and PCR products sequenced.Then,through the BLAST fruit fly genome,the site and direction of Minos insertion were found and the genes affected by Minos insertion were confirmed.In addition,according to the BLAST results,complementary experiments were performed with the mutation of the gene to further verify the consistency of the phenotype and the mutated gene.Knock down or overexpress the gene in its somatic cells or germ cells,analyze the range of action of the sterility gene,and perform immunofluorescence staining of the specific target protein to verify the specific role of the gene according to the specific information of the gene.Finally,this project also uses hybridization experiments to remove transposons from the genome of the mutant strain,and re-detect fertility and ovarian changes,verify the screening results,and exclude the influence of genetic background.Results:(1)After the mutant strain mf3392 passed the fertility test,it was determined that the female was sterile and did not lay eggs;The adult worms that dissected mf3392 found that their ovaries could normally form terminal fibroblasts but the development defects of the egg ventricles,and then further explored the changes of larvae and pupae,and found that there was no obvious change in the morphology of larval gonads,but the ovaries in the pupal stage could be clearly seen with developmental abnormalities.Hts immunofluorescence staining of adult mf3392 showed an increase in differentiated germ cells,and Hts-RC staining showed accumulation of annular ducts in small pieces of ovaries and an increase in part of the egg chamber.(2)mf3392 and Armitage’s two mutations,armi1 and armi72.1,did complementary experiments,and found that the hybrid offspring ovaries had dorsal limb-related phenotypes,which were consistent with the reported phenotype of Armitage,but inconsistent with the phenotype of mf3392,and it was possible that the transposon quadratic jump produced a background;and mf3392 was hybridized with the corresponding deficiency line,and the latter representative type was consistent with mf3392.It shows that the background of the second jump is in the area corresponding to the deficiency line;Sequencing results and reviewing relevant literature found that mf3392 is a double mutant armi-Cyc J of the biogenic factor armitage and cyclin Cyclin J in the PIWI signaling pathway.(3)After the mutant strains mf949 and mf2150 passed fertility testing,it was determined that they were both female sterile and infertile with eggs;Dissection of adult ovaries with unclear adhesions in the egg chambers,producing eggs with shorter axes and shorter dorsal limbs;Further dissection of the larvae and pupals showed no significant changes in the larval gonads of mf949 and mf2150,and no obvious changes in the ovaries in the pupal stage.(4)Immunofluorescent staining of mf949 and mf2150 with primary antibody Hts-RC,ring canal is present only before stage 4 of the egg chamber and cannot persist until later;The complementary experimental results between the two strains of mf949 and mf2150 confirmed that mf949 and mf2150 are mutant strains of the same gene.(5)The sequencing results showed that mf949 and mf2150 were new mutants that affected two different sites of the Drosophila silk protein gene cheerio in material transport.The fertility and phenotype of the established lines were restored after the mf949 and mf2150 transposon skipping,which confirmed that the phenotype and sterility of the mutant lines mf949 and mf2150 were caused by cheerio mutations.Conclusions: We have obtained a new mutant for armitage,a biogenic factor in the piwi pathway,and two new mutants for cheerio,a drosophila silk fibroin gene associated with material transport.This study provides a new and effective genetic mutation resource for further study of the regulation of PIWI signaling pathway and the function of cheerio in material transport.