| Rapamycin is a TOR targeting inhibitor,which has been widely recognized in regulating the body's aging process.mTOR is a high molecular weight protein kinase that responds to regulating many processes in cells including nutrient supply and mitosis.Studies have shown that rapamycin can also cause ROS to affect the body's aging process.Heat shock protein cannot be detected in vivo only when the external pressure can be detected in normal circumstances.Phosphorylation of TOR promotes the release of Hsp90 chaperone complex,accompanied by a significant reduction in the ability of heat shock protein synthesis.Specific regulation and its interaction mechanism needs to be further explored.TOR,S6K and S6 gene fragments were cloned and the differences of gene expression levels were measured at different concentrations of rapamycin at different time.The experiments showed that the mRNA expression of TOR,S6K and S6 genes were determined by different rapamycin concentrations.The differences on MnSOD,CAT,CuSOD and Hsp90 gene expression with different rapamycin concentrations and different time.It will provide a theoretical basis for understanding the mechanism of rotifer in future.1.Life span and reproduction studies of rotifers by rapamycinThe results showed that rapamycin could prolong the life span of rotifers,which was related to the total reproductive quantity and the pre-reproductive period.2.0?M and 4 ? M treatment groups were extended life expectancy of 15%and 24%(P<0.05).In the test concentration range rotifer life increased first and then decreased,the final life was inhibited.There was no significant difference in the mean number of B.calyciflorus treated with rapamycin concentrations of 0.5,1.0,2.0 and 4.0?M compared with the control group(P>0.05).and the number of progeny was inhibited in the 8.0 and 16.0?M treatment groups compared with the control(P<0.05).With the increasing concentration of rapamycin,the time required for the pre-reproductive period of each experimental group showed a decreasing trend(P>0.05),but there was a significant difference between the 2.0?M and 4;0?M rapamycin treatment groups(P<0.001)in the 0.5 and 1.0?M treatment groups compared with the control(22.69 h)).2.The studies of rapamycin and TOR,S6K and S6 gene expressionThe TOR gene fragment was 375bp,encoding 125 amino acids.The S6K gene fragment was 951bp,encoding 317 amino acids,and the S6 gene fragment was 735bp,encoding 245 amino acids.The changes of TOR,S6K and S6 gene mRNA expression were detected by Real time PCR.The mRNA expression of TOR,S6K and S6 showed a tendency to rise first and then decrease in these tests.mRNA expression of TOR:at 12h,rapamycin(0.5?M)increased the mRNA expression 1.7-fold with control(P<0.01).The concentrations(2.0,4.0,8.0 and 16.0?M)were significantly inhibited(P<0.05),and the 1?M treatment was significantly higher than the control group(P<0.05).At 48h,the expression level of gene was decreased to normal level in 8.0 and 16.0?M treatment groups.There was no significant difference between the two groups(P>0.05).S6K mRNA expression:The mRNA gene expression with 0.5,4.0 and 8.0?M rapamycin treatment was significantly higher than the control at 12h(P<0.05)The mRNA gene expression in 2.0?M and 16.0?M concentration group was significantly inhibited relative to the control group(P<0.05).S6 mRNA expression:the mRNA expression was significantly higher than control group(P<0.05)except 4.0 and 16.0?M at 12h.At 24h,the mRNA expression level was significantly higher than the control group(P<0.001)except 16.0?M treatment group.At 36h,the expression level of all the experimental groups treatments(0.5,1.0,2.0,8.0 and 16.0?M)was significantly higher than the control(P<0.05).The expression level of all the experimental groups was normal(P>0.05).3.The studies of rapamycin,antioxidation and Hsp90 genes expressionThe mRNA expression of antioxidant genes and Hsp90 was changed by rapamycin.MnSOD mRNA expression:the mRNA expression level of rapanycin(2,4.0 and 8.0?M)were all significantly higher than that with the control(P<0.05)at 24h.There was no significant difference in the mRNA expression in rapanycin(2.0 and 4.0?M groups)(P>0.05)and returned to normal level at 48h.CA T mRNA expression levels:The mRNA expression in rapamycin(2.0 and 16.0?M)groups were notably higher than control(P<0.001)at 24h.The mRNA expression in the rapamycin treatment groups(2.0,4.0 and 8.0?M)was dramaticlly higher than control group at 48h(P<0.001).CuSOD mRNA expression:The mRNA expression levels in each treatment group were all significantly increased(P<0.001)at 24h.The mRNA levels in the treatment 2.0,4.0 and 8.0?M groups were significantly higher than control group(P<0.001)at 48 h.The mRNA expression level was significantly inhibited by rapamycin(16.0?M)(P<0.001).Hsp90 mRNA expression levels:the mRNA expression level was markedly increased(P<0.001),and was not observably different from that in the control group in the 4?M treatment(P<0.001).The mRNA expression level in 2.0?M and 4.0?M treatment groups was prominently higher than that with control(P<0.001). |
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