Study On The Function Of The CP Gene In Drosophila Melanogaster

Chorion protein(CP)gene is an important gene for the construction of egg shells.The egg shell protein encoded by the family of egg shell protein genes accounts for more than 95% of the dry weight of Drosophila melanogaster egg shells.The egg shell function is extremely important,it wraps in the egg surface to protect the egg from the external environment the stress,carries on the essential survival material exchange(like moisture,gas,nutrition and so on)as well as the sperm and the egg binding channel and so on the important physiological function.The formation of egg shell occurs at the end of 14 stages of egg development,and the formation or disappearance of mRNA expressed by CP gene controls the expression of corresponding egg shell protein.The whole process is very complex,with strict time and space specificity.This article in the first FlyBase filter shell protein genes,build CP gene UAS RNAiGal4 gene interference hybrid system(RNA interference,RNAi),through the statistical analysis of the hybrid strains of laying eggs and egg hatching out more significant hybrid strain,and through the real-time fluorescent quantitative PCR(RT-qPCR)to confirm whether the gene expression of success.Then decrease shooting ability to observe hatching significant strain eggs surface microstructure and morphology,spawning ability reduce significantly strains of ovarian and ovarian tubule development status,and electrophoresis separation spawn reduce significant strain of ovarian total protein and hatchability reduced significantly strain the eggs of total protein,compared with the CP genes RNAi protein expression differences between strain and parents.Finally,the development process of pupation,eclosion and life span of CP gene RNAi lines screened with the incubation rate lower than 50% and higher than 20% was explored.The specific results are as follows:(1)construction of UAS-RNAi/Gal4 hybridization system of Chorion protein protein-related gene,expression of target gene and screening results of positive gene.Under act-Gal4 maternal condition,the oviposition ability of CP7 Fb,CP16-1and CP19 increased after interference(P<0.05),and that of CP7 Fa,CP15,CP16-2,CP18-1,CP18-2,CP38,Knk and Mnn1 decreased after interference(P<0.05).The oviposition ability of CP7 Fa,CP7Fb,CP15,CP16-1,CP16-2,CP18-1,CP18-2,CP19,CP38,Knk,Mnn1 and Cysp was decreased under the experimental condition of Byn-Gal4 as the mother(P<0.05),especially CP7 Fb,CP18-2,CP38,Mnn1 were significantly decreased(P<0.05).Under the experimental condition of Cad-Gal4 as the mother,the egg-laying ability increased after CP18-1 interference(P<0.05),and the egg-laying ability decreased after CP7 Fa,CP7Fb,CP15,CP16-1,CP16-2,CP18-2,CP19,CP38,Knk,Mnn1,and Cysp interference(P<0.05),especially CP7 Fb,CP18-2,CP38,and Mnn1 interference(P<0.05).The oviposition ability of CP7 Fa,CP7Fb,CP15,CP16-1,CP16-2,CP18-1,CP18-2,CP19,CP38,Knk and Mnn1 were decreased after Da Gal4 as a mother(P<0.05).Under the condition of Lsp2-Gal4 as the mother,the oviposition ability of CP7 Fb and CP18-2 was slightly decreased after interference(P<0.05),and that of CP7 Fa,CP15,CP16-1,CP16-2,CP19,CP38,Knk,Mnn1 was decreased after interference(P<0.05).Under the experimental condition of NGT40-Gal4 as the mother,the oviposition ability of CP15,CP18-2 and Mnn1 increased after interference(P<0.05),and the oviposition ability of CP15 and Mnn1 increased significantly after interference.The oviposition ability of CP7 Fa,CP7Fb,CP16-1,CP16-2,CP18-1,CP19,CP38 and Knk decreased after interference(P<0.05).The oviposition ability of CP18-2,CP38 and Knk increased after CP18-2 interference(P<0.05),and increased significantly after CP18-2 interference.The oviposition ability of CP16-1 and CP16-2 did not change after CP7 Fa,CP7Fb,CP15,CP18-1,CP19 and Mnn1 interference decreased after CP7 Fa,CP7Fb,CP15,CP18-1,CP19 and Mnn1 interference.(2)egg morphology,cell apoptosis and surface microstructure were significantly decreased after gene interference.After the interference of CP gene,the eggs develop abnormally and continue to shrivel until completely inactivated and unable to hatch larvae normally.The structure of the egg surface is abnormal,and the hexagon structure on the normal egg surface continues to become irregular shape,which affects the egg respiration and the exchange of water and nutrients.Cell apoptosis could not be observed by nuclear staining due to the obstruction of Chorion protein,but the fluorescence staining showed that the structure was consistent with that observed under the body microscope.In view of the experimental results in this chapter,it is speculated thatCP gene mutation can cause abnormal egg development because of abnormal egg structure.Abnormal development of the surface structure of the egg causes the egg to be unable to breathe and exchange water and nutrients,which results in abnormal development inside the egg,resulting in the inactivation of the egg and the inability to complete the hatching process.(3)morphology and structure of ovaries of hybrid lines with significantly reduced ovipositing ability during development.After the interference of CP gene,ovarian development was abnormal,and the ovarian volume was relatively small compared with that of wild type and hybrid parents at the same period,and the normal number of ovarian tubules could not be observed,and there were no mature eggs in the ovarian tubules.It is speculated that CP gene mutation leads to abnormal ovarian development,resulting in abnormal structural development,reduced number of ovarian tubules,and inability to provide an appropriate environment for egg development,thereby decreasing the spawning ability of corresponding hybrid lines.(4)the differential proteins between the expression of ovary and total egg protein and the parents,especially the decreased ovipositing ability and egg hatching abilityThe difference protein was extracted from ovary and total egg protein,and it was found that the expression of yolk polypeptide in ovary of CP gene mutant lines was decreased.Yolk polypeptide is the main substance in the development of egg and ovary.The decrease of its expression content directly leads to the abnormal development of egg and ovary,which makes it unable to produce enough normal eggs.This paper speculates that there are more proteins affecting this process,and hopes to continue to explore.(5)pupation,eclosion and life span of the ovipositing and hatching ability decreased significantlyCP gene mutation can affect the development of eggs,so it directly affects the pupation of eggs.The results showed that the pupation time and the average pupation rate of eggs were significantly reduced,the pupation time and the pupation rate were significantly reduced,and the life span of fruit flies was not significantly affected.Itwas speculated that due to CP gene mutation,normal vitelloin could not be expressed,resulting in abnormal ovarian development and failure to develop normal number of healthy eggs.Abnormal substances in the eggs could not normalize pupation naturally,so pupation time increased and average pupation rate decreased.But adult worms that can eclosion show that their environment is normal and their lifespan unaffected.To sum up,CP gene mutation can reduce the egg-laying ability,egg hatching ability and ovarian development abnormalities of fruit flies.Combined with the sequencing results of ovarian protein of the mutant lines and quantitative PCR results,it was found that CP gene mutation would lead to decreased expression of yolk polypeptide,and we speculated that the reduced expression of yolk polypeptide in the mutant lines was related to reproductive defects.