Effect Of Bmovo-1Overexpression On The Ovary Development Of Silkworm And Secretory Expression Of HICF-I In Silk Gland Biological Factory

To understand the effect of regulating Bmovo gene expression on ovariandevelopment and silk protein synthesis in the silkworm (Bombyx mori),In this study, theexpression pattern analysis of the four kinds of isoforms of Bmovo gene, Bmovo-1(GU477588), Bmovo-2(HQ831344), Bmovo-3(HQ831345) and Bmovo-4(JQ665224)were firstly investigated by qPCR, and found that the Bmovo genes transcription weremainly in the silkworm gonad;Western blot analysis shows that both BmOVO-1andBmOVO-2could be detected in the ovaries and testes, while BmOVO-3was mainlypresent in the ovary and BmOVO-4was mainly present in the testis;The results fromimmunofluorescence showed that BmOVO could be detected in the follicular cells,nurse cells, primary oocyte.To understand the Effect of Bmovo-1gene overexpressionon the ovary development and silk protein synthesis in silkworm, the Bmovo-1overexpression transgenic vector pigA3GFP-IeNeo-VlgBmovo-1was then introducedinto the eggs of silkworm by sperm-mediated method. Transgenic silkworms wereobtained by screening with G418, fluorescence observation, further corfirmed by PCRand Dot blotting. qPCR detection results showed that the transcription level of Bmovo-1in the transgenic silkworm ovaries, testes was2.23and1.85times comparing with thatof the non-transgenic silkworm, respectively. The results of the economic traitsinvestigation showed that The weight of the pupa and cocoon shell of the femaletransgenic silkworm respectively decreased by10.34%and14.59%, those of the maletransgenic silkworm respectively decreased by12.24%and21.72%, and the number ofeggs laid increased by13.39%owing to the overexpression of Bmovo-1gene. Moreover,the ovarian weight of the transgenic silkworm increased, the testis weight decreased inthe different development stages comparing with non transgenic silkworms.The contentof trehalose in the blood and in ovaries of transgenic silkworm was increased and therewas not obvious difference in the trehalase activity in the blood between transgenicsilkworm and non-transgenic silkworm.To understand the mechanism of ovarian development and oogenesis in the molecular level, the mRNAs were respectivelypurified from the total RNA isolated from the ovaries of the3-day larvae and6-daylarvae of5th instar, and the2-day pupae, RNA-Seq was performed after the mRNAswere mixed.6,306,078reads was obtained, the reads was100bp averagely in size, inwhich the clean reads were up to98.87%, suggesting the sequencing saturation wasquite well and the dates was realistic and believable. The clean reads were mapped to9606genes. The log2(Fold_change) of the genes relative to actin A3gene werecalculated, the66high expressed genes (log2(Fold_change)＞2) were further analyzedby KEGG. The results showed that these genes were mainly involved in the energymetabolism, translation and signal transduction. Results preliminary clarified that therelationship between ovarian development, oogenesis and gene expression, andinteraction of ovarian development with silk protein synthesis, and also provided newclues to improve the number of eggs laid and the silk production.Meanwhile, the research on the secretory expression of insulin-like growth factor(hIGF-I) in transgenic silk gland were also carried out. The transgenic vectorpiggyA3GFP-serHS-IGF-I-ie-neo was introduced into the eggs of silkworm bysperm-mediated method. Transgenic silkworms were obtained by screening with G418,fluorescence observation, and further confirmed by PCR and Dot blotting. and werereared to the G8generation. The degummed solution of the transgenic silkworm cocoonof G8generation was precipitated by adjusting pH to isoelectric point, and the contentof the hIGF-I estimated by ELISA was approximately162.7ng/gram.