| Rotifers are a group of micro metazoans.As basic consumers,rotifers play an important role in aquatic food webs.Rotifers have a large number of species and are widely used in aquatic product breeding.At the same time,rotifers,as an important component of zooplankton in rivers,lakes and ponds,also play an important role in environmental improvement and monitoring.Although rotifers have natural advantages in breeding,environmental improvement and monitoring,there are still some problems in their production and breeding.These problems mainly include that they have short life span,rapid population change and short population peak period.Present technology cannot meet the needs of rotifers in production.Therefore,it is urgent to study the influencing factors of rotifer senescence.For this,we put forward two hypotheses: first,by studying the life extension of individual rotifers,the whole life of rotifers will be prolonged and the population density will increase,and thus we can prolong the peak population density of rotifers.Second,the problem of high and stable rotifer production cannot be solved by ecological methods at present.Therefore,this experiment intends to study the biological mechanism of controlling rotifer senescence through molecular biological methods,so as to achieve high and stable rotifer production.Rapamycin is an inhibitor targeting TOR and has been widely recognized in the process of regulating human aging.m TOR is a high molecular protein kinase,which reacts and regulates many processes of biological cells,including cell nutrition supply and mitosis.Studies have shown that rapamycin can also affect the aging process of cells and organisms by producing reactive oxygen species ROS.Under normal circumstances,it is difficult to detect the presence of heat shock proteins in the body,but heat shock proteins can be detected when external pressure occurs.When m TOR is phosphorylated,it can promote the release of HSP90 complex and significantly reduce the synthesis ability of heat shock proteins including HSP90,but has no effect on HSP70.The specific regulatory mechanism needs further study.In this paper,the life span,total reproduction and prophase of Brachionus manjavacas were measured at different rapamycin concentrations.In this paper,the life span,total reproduction and prophase of B.manjavacas were measured at different rapamycin concentrations.We cloned The gene fragments of TORC1,P70S6 K and S6 were,and determined the gene expression difference of rapamycin at different time after different time;we determined the difference of heat shock protein HSP70 and HSP90 gene expression under different rapamycin concentration and time;also,we determined the enzyme activities of SOD and CAT under different rapamycin concentrations.1.Effects of rapamycin and rotifer life span and reproductionThe results showed that rapamycin with appropriate concentration had positive regulation on the life span and reproduction of B.manjavacas.The average life span of rotifers treated with rapamycin at concentrations of 1.0?M,2.0 ?M,4.0 ?M and 8.0 ?M was 39.8%,54.1%,42.1% and 15.3% longer than that of the control group,respectively.However,the average life span of rotifers treated with 16?M rapamycin was only 71.50±6.13 h,which was significantly lower than that of the control group(P<0.05).The results of the whole experiment showed a trend of increasing first and then decreasing,and finally it could even inhibit the life of rotifers.Compared with the control group,the prophase of rotifer treated with rapamycin at concentrations of 2.0 ?M,4.0 ?M and 8.0 ?M was prolonged by 18.0%,18.8% and20.4% respectively.In contrast,the duration of prophase of rotifer treated with rapamycin at 1.0?M concentration was higher than that of control group,but there was no significant effect(P > 0.05).Similarly,the rotifer treated with 16.0 ?M concentration had no significant difference from the control rotifer in the prophase of reproduction(P>0.05).The results of the whole experiment showed a slowly rising and then declining trend.The average reproductive capacity of rotifers treated with rapamycin at concentrations of 1.0 ?M,2.0 ?M,4.0 ?M and 8.0 ?M was significantly longer than that of the control group(P<0.05),increased by 33.3%,62.9%,49.6% and 31.3%,respectively,and was the highest under rapamycin treatment at concentrations of 2.0 ?M.However,when treated with 16.0 ?M rapamycin,the average reproductive capacity of rotifers was only 0.35±0.12,which was significantly lower than that of the control group(P < 0.05).The results of the whole experiment showed a trend of increasing first and then decreasing,and finally even inhibited rotifer reproduction.2.Expression of rapamycin and TORC1,P70S6 K and S6 genesThe gene fragments of TORC1,P70S6 K and S6 obtained from Gen Bank were detected by real-time fluorescence quantitative PCR.The results showed that the m RNA expression of TORC1,P70S6 K and S6 genes showed the same trend on the whole: first increased and then decreased.Results of TORC1 gene expression: when rapamycin was used to treat rotifers for 12 hours,the m RNA expression of rotifers in each concentration group was significantly lower than that in the control group(P<0.05).At 24 h and 36 h,the m RNA expression of rotifer in each concentration group was significantly higher than that in the control group(P<0.05).At 48 h,the m RNA expression of rotifers in the8.0 ?M and 16.0 ?M concentration groups was still significantly higher than that in the control group(P < 0.05),but compared with 36 h,the m RNA expression of rotifers in the two groups also decreased significantly and tended to return to normal level.Results of P70S6 K gene expression: when rapamycin was used to treat rotifers for 12 h,the m RNA expression of rotifers in the 2.0,4.0,8.0 and 16.0 ?M concentration groups was significantly higher than that in the control group(P<0.05).At 24 h and 36 h,the m RNA expression of rotifer in all concentration groups was significantly higher than that in the control group(P < 0.05).At 48 h,the m RNA expression of rotifers in the 8.0 ?M and 16.0 ?M concentration groups was still significantly higher than that in the control group(P<0.05),but compared with 36 h,the m RNA expression of rotifers in the two groups also decreased significantly and tended to return to normal level.S6 gene expression results: when rapamycin was used to treat rotifers for 12 hours,the m RNA expression of rotifers in 4.0 ?M and 8.0 ?M concentration groups was significantly higher than that of the control group(P<0.05),while at 16 ?M it was significantly lower than that of the control group(P<0.05),while there was no significant change in 1.0 ?M and 2.0 ?M(P>0.05).At 24 h and 36 h,the m RNA expression of rotifer in each concentration group was significantly higher than that in the control group(P<0.05).At 48 h,the m RNA expression of rotifers in the 8.0 ?M and 16.0 ?M concentration groups was still significantly higher than that in the control group(P<0.05),but compared with 36 h,the m RNA expression of rotifers in the two groups also decreased significantly and tended to return to normal level.3.Expression of rapamycin,HSP70,HSP90 genesHSP70,HSP90 gene fragments obtained from Gen Bank were detected by real-time fluorescence quantitative PCR.The results showed that at 12 h,the 1.0,2.04.0,8.0 and 16.0 ?M groups were significantly higher than the control group(P<0.05).At 24 h,compared with the control group,the m RNA level of rotifer in the full concentration group increased significantly(P<0.05).At 36 h and 48 h,m RNA levels in the 1.0,2.0 and 4.0 ?M groups were significantly higher than those in the control group(P<0.05),while there was no significant difference in other groups(P>0.05).On the whole,the expression of HSP90 gene in each group showed a trend of increasing first and then decreasing with concentration.However,rapamycin has no significant effect on HSP70 gene expression.Based on the fact that they have the same signal molecule 4E-BP,the experimental result shows that the effect of rapamycin on cells has nothing to do with 4E-BP pathway.4.Rapamycin and rotifer antioxidant activityAfter 24 hours of exposure to different concentrations of rapamycin,the activities of SOD and CAT in rotifer Brachionus manjavacas showed a trend of first increase and then decrease with the increase of rapamycin concentration.The SOD and CAT activities of rotifers in the 2.0 ?M and 4.0 ?M groups were significantly higher than those in the control group(P < 0.05).With the increase of rapamycin concentration,the SOD and CAT activities of rotifers showed a trend of increasing first and then decreasing.Among them,when rapamycin concentration is 2.0 ?M,rotifer SOD and CAT activities are the highest. |
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