Studies On The Role Of BmSTAT In Diapause Of Silkworm,bombyx Mori

In order to explore the molecular mechanism of diapause and diapause termination in silkworm,Bombyx mori variety 932,Xianghui and Dazao were used as materials in the study.Bioinformatics,Real-time quantitative PCR,Western Blot,and yeast two-hybrid methods were used to select correlative proteins in diapause or diapause termination of silkworm eggs.Transcriptional expression and regulation in cell division n cycle of BmSTAT in instant pickling eggs,postnatal 3rd day pickling eggs,diapause eggs,and non-diapause eggs were also analyzed.Then we get the following results:(1)According to the protein data of the 932 cultivars detected by the shotgun on the 3rd day pickling eggs,diapause eggs and non-diapause eggs,compared by classification,162 proteins were found common in the diapausing eggs of the postnatal 1st and 3rd,and 123 proteins could be annotated.In the non-diapause eggs,there were 224 proteins were common on the postnatal 1st and 3rd,and 187 proteins could be annotated.Comparing the difference proteins of the 3rd day pickling eggs with diapause eggs,and non-diapause eggs of the postnatal 1st and 3rd,the results showed that there were 154 unique proteins on the 3rd day pickling eggs,and 117 proteins could be annotated.Classified by GO,these proteins are mainly located in cells and organelles.In the classification of molecular functions,most proteins have binding and catalytic activity,and they are involved in biological processes mainly including cells and metabolic processes.A BmSTAT protein closely related to the diapause of silkworm eggs was selected out,cloned and expressed.(2)The BmSTAT gene was cloned by molecular biological techniques.Real-time quantitative PCR was used to detect the expression of BmSTAT in 3rd day pickling eggs and diapause eggs.The expression profiles of BmSTAT of silkworm eggs from the 1st to 4th day after treatment were not significant.The expression level between 0.1~1,but the 5th to 8th days,the expression gradually decreased until the 8th day,and the value was 0.042.In comparison with the diapause eggs,the expression of BmSTAT gene was stable at postnatal 3rd to 10 th days,and the value between 0.40 and 3.92.In the instant pickling eggs of Xianghui,the expression level of BmSTAT gene was significantly down-regulated on the 2nd day after pickling,which decrease to 99.9% from the 2 hours after the pickling until the silkworm eggs became turn-green and the expression level was below 0.1,while the expression level in diapause eggs between 0.96 and 5.4.In the non-diapause eggs produced by Dazao,the expression level of BmSTAT gene was lower than 0.003 from the eggs after laying down to the turn-green stage at a low level of expression.From the changes of BmSTAT gene expression in pickling eggs,diapause eggs and non-diapause eggs,the expression of BmSTAT gene was down-regulated after silkworm eggs were developed.(3)Western Blot detection of BmSTAT in the 3rd day of pickling eggs and diapause eggs of 932 cultivars,BmSTAT was found existing with monomer and dimer form.The relative expression level of monomer of BmSTAT was increased gradually on the 2nd day after pickling,and reached the maximum on the 4th day.The amount was 1422596,then decreased to the 8th day at 964276.The relative amount of dimer expression also gradually decreased after pickling.In general,when the pickling eggs were developed,the relative expression levels of monomer and dimer BmSTAT were lower in pickling eggs than that in unpickling eggs.(4)The yeast two-hybrid method was used to detect the interaction between BmSTAT and CyclinB.The results showed that BmSTAT and CyclinB can interact to form a fusion protein.It is speculated that BmSTAT will affect the role of CyclinB in the cell cycle and then affect the process of cell division cycle.