The Role Of PI3K-Akt Signal Transduction Pathway In Diapause Of Silkworm,Bombyx Mori

Using silkworm eggs variety 932,Xianghui and Dazao as materials in this study to explore the molecular mechanism of diapause and diapause termination of silkworm.The differences in carbohydrates in silkworm eggs of different streatments and development stage were detected by GC-MS.The methods such as bioinformatics and qPCR were used to screen the PI3K-Akt signaling pathway correlative gene in diapausing eggs,non-diapause eggs,instant pickled eggs and red bean color pickled eggs.The expression of related proteins was detected using ELISA and Western Blot.Activity changes to further explore the regulatory mechanism for the eliminating diapause of silkworm.The results obtained are as follows:（1）According to results of GC-MS,there were 5 kinds of monosaccharides,including glucose,fructose,maltose,trehalose and sorbose,were detected in diapausing eggs,non-diapause eggs,instant pickled eggs and red bean color pickled eggs.The results of internal standard normalization with ¹³Cribitol indicate that trehalose in diapausing eggs was less than 1.0.The trend of lower increase at the 4^th and 6^th day later showed a downward trend after pickled in the red bean color pickled eggs and a pick-up to 1.0 on the6^th day after pickled,only in diapause eggs and red bean color pickled eggs were significantly different.Sorbose showed a tendency to increase with the increase of egg age in diapausing eggs,and the content was very high at close to 100.This occurred both in non-diapause eggs and in instant eggs.The trend that sorbose was higher at the 1^st and 2ⁿdd day after laying,then lightly increased around 140 at the 4^th and 6^th day,at last dropped sharply below to 0.5285.The content of glucose and fructose in each state egg was very low and the difference was not significant.The content of maltose in leaching eggs was less than 0.07,and the difference was significant between 0.05 and 0.16 in diapause eggs.From the above results,the changes caused by acid leaching can regulate the initiation of embryo development and the termination of diapausethe.（2）The quantitative analysis of gene expression profiles of four candidate genes in the PI3K-Akt signaling pathway,BmAkt,BmGsk-3,BmGs,and CaMKII,were performed by qPCR technique in different developmental stage of silkworm eggs such as diapausing eggs,non-diapause eggs,instant pickled eggs,and red bean color pickled eggs.Firstly,quantitative analysis of stage expression levels showed that BmAkt expression was extremely low in non-diapause eggs,instant pickled eggs,and maintained at a high level in diapausing eggs,but it was lower in the red bean color eggs.After the acid treatment,the trend of the first up-regulation to the 5^th day after laying was down-regulated.Secondary,the expression levels of BmGsk-3 in diapausing eggs,non-diapause eggs and instant acid-exposed eggs were very low,and they were only expressed in pickled eggs and reached the highest level on the 2^nd and 3^rd days after oviposition.Thirdly,the level of BmGs expression in diapause eggs,non-diapause eggs,and instant in pickled eggs is very low.The red bean color eggs reached the highest expression on the 2^nd day after pickled and then down-regulated and re-introduced on the first two days of incubation,indicating that the BmGs were at a higher expression level in the stage of the diapause termination of the silkworm and early hatching.Fourthly,the relative expression of CaMKII in silkworm eggs is only significantly different between diapausing and red bean color eggs.The results showed obvious tendency of up-regulation in non-diapausing eggs and instant dipping in the first two days of egg hatching in acid rinsed egg and red bean color bean.（3）The expressions of BmAkt and Bmp-Akt proteins were detected by Western Blot in932 strains of silkworm diapause eggs and red bean color pickled eggs at different stages.The results showed that there were two bands at 51 kDa and 61 kDa in BmAkt.Bmp-Akt has a band at 51 kDa.By comparing the changes of p-Akt/Akt ratio,it reflected the activation of PI3K-Akt signaling pathway.The ratio of diapause eggs to adipocytes at the3^rd to 6^th day was similar.However,the ratio of p-Akt/Akt in red bean color pickled eggs was significantly higher than that of diapausing eggs after the 7^th day,and it was then down-regulated on the 10^th day after hatching indicating that the activation degree of PI3K-Akt signaling pathway in the earlier termination stage of diapausing eggs and red bean color eggs is low,while it is relatively high during embryonic development after the release of diapause.（4）GSK activity of glycogen synthase kinase was determined in diapause eggs and red bean color pickled eggs.The activity of GSK was no more than 13.00 U/L.Pro in diapausing eggs,but it was immersed in red bean color bean eggs.It was 32.71 U/L.Pro after acid treatment,and followed by a decreasing trend to the lowest value of 17.39U/L.Pro on the 4^th day after pickled.It then increased to the maximum value of 89.29 U/L on the 1st day before hatching.The activity of GSK was significantly higher in red bean color eggs than in diapausing eggs.Acid treatment increased the GSK activity in silkworm eggs.（5）The changes of Mg,Fe,Na,K and Ca metal elements in diapause silkworm eggs before and after pickling were compared by atomic absorption spectrometry.There were no significant differences in Mg,Fe,Na and K changes before and after acid pickling in silkworm eggs.The content of element was downgraded to 83.84 after pickling,and the change was significantly reduced.