The Cloning Of Myosin 18a Gene And Its Role In Acrosome Development In Gampsocleis Gratiosa

Gampsocleis gratiosa belongs to Orthoptera,which is an important model insect of Orthoptera,easy to feed and use,and its spermiogenesis process has been studied more clearly,which provided conditions for the study to investigate the role of Myosin in the acrosome development of Gampsocleis gratiosa.For the traditional model animals,for example,there have been some studies of acrosome development in Drosophila.However,for Orthoptera insects,the model of acrosome development in Drosophila couldn't solve some problems in the acrosome development of Orthoptera.Thus,exploring the process of acrosome development provides a good reference for the study of Orthoptera insects and also lays the foundation for further researches.In the early stage of this laboratory,the adults of Gampsocleis gratiosa were handed over to BGI·Tech,BGI·Tech measured the male and female adults of transcriptome data and established transcriptome database by the Illumina Hiseq platform.In this study,the myosin gene sequences were retrieved from the Gampsocleis gratiosa adult transcriptome database,the retrieved genes were compared in NCBI,and according to the results of the comparison,the Gg Myo18 a gene was identified in this study.According to the analysis of the differential transcriptome data and the relative expression in different tissues in Gampsocleis gratiosa,this study found that the expression level of Gg Myo18 a gene in the testes was significantly higher than that of other tissues.Therefore,this study supposes that Myosin 18 A protein plays an important role in the acrosome development of Gampsocleis gratiosa.In this study,we amplified the open reading frame sequences of the Gg Myo18 a gene firstly and conducted bioinformatics analysis.Later,we designed several interference fragments and synthesized ds RNA for injection in vivo to determine the best interference time.After interference,the m RNA expression of myo18 a gene in the testis was down-regulated by about 79%,the most obvious interference effect on the 6th day.Lastly,the Gampsocleis gratiosa testis after interference were taken as sperm smear.The PAS staining technique,immunofluorescence staining technique,paraffin section technique,frozen section technique,and transmission electron microscope technique were used to observe the changes of different structures of the Gampsocleis gratiosa acrosome development.A series of changes such as the reorganization and deformation of nuclear material,the formation of microfilaments and microtubules emerge in the acrosome development of the Gampsocleis gratiosa.PAS staining is used to observe the changes of polysaccharides in the acrosome complex.Immunofluorescence staining is used to observe microtubules,microfilaments.DAPI staining is used to observe changes in nucleus.Transmission electron microscopy is used to observe changes in organelles.This study explored the function of Myosin 18 A protein in acrosome development due to the different observations.The results showed that,the Golgi apparatus after interference changed from normal bands to compact irregular shape.The surface of the pro-acrosome changed from regular spherical shape to irregular uneven surface.The polysaccharides in the pro-acrosome changed from compact spherical distribution to loosely irregular shape.The microfilaments in the acrosome development appeared to be "broken".By comprehensive analysis of these results of the microstructure and ultrastructure after interference with the myo18 a gene,the conclusions were as follows:1.The Golgi apparatus changed from a band to a compact irregular shape,and irregular changes also occurred on the surface of the pro-acrosome after the interference.The accumulation of polysaccharides in the pro-acrosome was also affected.This indicates that Myosin 18 A protein may participate in the maintenance of the normal morphology of the Golgi apparatus and the transport of material between the nucleus and the pro-acrosome in the early stage of acrosome development.2.The structures formed by microfilaments appeared to be "broken" to varying degrees due to the assembly being affected.It indicates that Myosin 18 A protein may participate in the assembly of microfilaments during acrosome development.In short,this study considers that Myosin 18 A protein is a relatively special protein.Myosin 18 A protein not only functions as a "cargo motor" to transport substances,but also participates in the maintenance of the Golgi morphology.Myosin 18 A protein indirectly affects acrosome development by affecting the normal morphology of the Golgi apparatus.