The Study Of Embryonic Development And Immunolocalization Of BmMago Based On Paraffin Section In Silkworm(Bombyx Mori)

To obtain embryonic microphotographs of representative developmental stages, a paraffin section technical method for silkworm (Bombyx mori) eggs need be explored and established. Then, these microphotographs can conform and enrich the ancestral literal details, schematic diagrams, hand-drawing pictures on this study. In addition, it is believed that these microphotographs may also provide morphology reference for embryonic developmental gene function research in silkworm. To immunolocalize protein in silkworm testis, immunohistochemical staining method must be explored and applied, for the purpose of providing a valuable basis on further gene functional research on tissue and cell level.To find the best softening time of silkworm eggs using KOH as softening fluid, concentration gradient and softening time gradient have been tested. The method of fixation, clearing and staining have also been tried. Based on the established paraffin section technical method, a lot of embryonic microphotographs were gained from eggs of N4, a Japanese non- diapause strain. The Immunolocalization of BmMago was completed by immunohistochemical(IHC) staining method, which was explored and established with testis on day 3 of fifth larva, using IgG of BmMago as primary antibody. Some results of the study is as follows:1. Establishment of paraffin section technical method for silkworm (Bombyx mori) eggs. In details, with the eggs of Japanese non- diapause strain named N4, softening with 10% KOH for 5 min, fixing with Smith fixative, de-hydrating and clearing with Terpineol.2. A lot of embryonic microphotographs from representative developmental stages have been obtained, including stages of blastderm formation, germband formation, mesoderm differentiation, appearance of gnathal and thoracic appendages, appearance of abdominal appendages, early blastokinesis, middle blastokinesis, final blastokinesis, completion of organs and tissues. Moreover, process of yolk cleavage was surveyed; cellular blastoderm and syncytial blastoderm were distinguished; embryonic pictures were captured from different tissue eg. mid-gut, dorsal vessel, nerve cord, gonad, silk gland. The primordial germ-like cells were observed in mesoderm by serial cross section.3. There were many factors contribute to IHC, such as purity of primary antibody, dilution degree of primary antibody and secondary antibody, time used in the process of retrieval, block, rinse, antibody incubation, coloration. The best dilution degree in this study was 1:1000 of primary antibody and 1:500 of secondary antibody.4. The Immunolocalization of BmMago revealed that BmMago protein was expressed in all the cells of the silkworm testis, where it was predominantly localized to the nuclei.In conclusion, a new paraffin section technical method for silkworm {Bombyx mori) eggs has been established, which meet developmental gene functional research, has the advantages of simple, fast, integrity, high throughput. A lot of embryonic microphotographs from representative developmental stages were gained, which conformed and enriched the ancestral literal details, schematic diagrams, hand-drawing pictures on the research field. A immunohistochemical(IHC) staining method has been explored in silkworm, in which key steps have been tested and standardized. BmMago has been immunolocalized in silkworm testis.