| Brachionus calyciflorus Pallas belongs to the Phylum Rotifera,Monogononta,Ploima,Brachionidae,and it is the most common freshwater zooplankton.Due to its small size(about 100-500?m),short life cycle(5-7d),and easy to culture in the laboratory,and obvious aging characteristics,Therefore,it is considered to be one of the most valuable model organisms for aging biology research.mTOR is a high molecular weight protein kinase that responds to many regulatory processes within the cell,including mitosis and nutrient supply,with the S6 K and S6 genes being downstream substrates for the TOR signaling pathway.Heat shock proteins(HSPs)are small molecular weight proteins that are induced and have a stress effect when the body is stressed.Phosphorylation of mTOR promotes the release of the Hsp70 chaperone complex,accompanied by a significant decrease in the ability to synthesize heat shock proteins.However,the regulatory mechanisms and their interactions between the two have rarely been reported in the aging process of rotifers.In this paper,the effects of different temperatures on the lifespan,reproductive capacity,pre-fertility and late reproductive stage of B.calyciflorus were studied.The TOR,S6 K,S6 and HSP70 gene fragments were cloned and studied at different temperatures and different time points.The effects of temperature on the expression of MnSOD,CAT and CuSOD genes under different temperature and low temperature treatments were studied at different temperatures and time,which provided a scientific basis for rotifer biology.1.Effect of temperature on life span and reproductive capacity of B.calyciflorusThe light intensity was 4000 lx,the day length ratio was(L:D)16:8,and the temperature was set to 15 ° C,20 ° C,25 ° C,30 ° C and 35 ° C,of which 25 ° C was the optimum temperature for cultivating rotifers.The results showed that the average life expectancy of rotifers was 165.84±5.28 h when rotifers were cultured at 25°C,and the average lifespan of rotifers under treatment at 15°C was 275.76±6.52 h.Compared with the treated group at 25°C,the life extension effect was significant(66.28%,P<0.01).The pre-fertility and late reproductive stages were extended by 92.09% and 65.09%,respectively(P<0.01).The average lifespan of rotifers treated at 20 °C was 198.72±5.21 h,which was 19.82% longer than that of 25 treatment group.The average lifespan of the rotifers was 75.36±2.32 h and 67.2±1.78 h after treatment at 30°C and 35°C.Compared with the 25°C treatment group,the lifespan was shortened by 54.55% and 59.48 % respectively.(P<0.05),the lifespan was shortened by 54.55% and 59.48%,respectively(P<0.05).The pre-fertility and late reproductive stages of the 30°C rotifers were shortened by 62.63% and 12.17%,respectively;at 35 °C,they were shortened by 77.43% and 53.77%(P<0.05).The results of the rotifer's reproductive yield showed that there was no significant difference in the total reproductive yield of rotifers at 15 °C,20 °C,30 °C and 35 °C compared with the 25 °C treatment group(P>0.05).2.Effects of temperature on the expression of TOR,S6 K and S6 and HSP70 genes in B.calyciflorusThe TOR gene fragment obtained from the transcriptome is 375 bp,which encodes 125 amino acids;the S6 gene fragment is 735 bp,which encodes 245 amino acids;the S6 K gene fragment is 951 bp,which encodes 317 amino acids;the HSP70 gene fragment is 2152 bp,which encodes 656 Amino acids.Real-time quantitative PCR was used to detect the changes in gene expression of TOR,S6 K and S6 and HSP70.The results showed that the trends of gene expression of the first three were basically the same,and the gene expression changes with HSP70 showed obvious mutual inhibition trend at 15 °C,20 °C,30 °C,and 35 °C.The specific performance was as follows: the highest expression of S6 K gene was the highest in the first three at 15 °C for 2 h,about 0.65 times that of the control group.At this time,the gene expression of HSP70 was about 6.5 times that of the control group,and the difference was extremely significant(P<0.001);at 20 °C for 3 h,the change of S6 gene expression in the first three was the highest in the first three,about 0.9 times of the control group.At this time,the gene expression of HSP70 was about 5.3 times that of the control group,and the difference was extremely high.Significantly(P<0.001);at 25 °C for each time,there was no significant difference in gene expression between the first three and HSP70(P>0.05);at 30 °Cfor 6h treatment,the expression of TOR gene was the highest in the first three,about 0.46 times that of the control group.At this time,the gene expression of HSP70 was about 4.4 times that of the control group,and the difference was extremely significant(P<0.001).At 35 °C for 3 h,the expression of S6 K was the highest in the first three,about 1.4 times that of the control group.At this time,the expression of HSP70 was about 12 times that of the control group,and the difference was also significant(P<0.001).3.Effects of temperature on the expression of CuSOD,CAT and MnSOD genes in B.calyciflorusIn this experiment,the expression changes of CAT,MnSOD and CuSOD genes in Brachionus plicatilis under low temperature 15 °C and high temperature 35 °C were studied.At 15 °C,the mRNA expression of CAT was the highest when treated for 1 h,which was 2.2 times higher than that of the control group(P<0.001),and then decreased.At 4 h,the gene returned to normal level.When treated at 15 °C for 5 h,the expression of CuSOD and MnSOD was the highest,which was 2.7 times and 3.4 times of the control,respectively(P<0.001).The gene expression of CuSOD increased in the first 5 h,and decreased slightly at 6 h.However,it was still higher than the control.The gene expression change of MnSOD also increased in the first 5h,and returned to normal level at 6h.At 35 °C,the gene expression changes of CAT was the highest at 1 h and 2 h,about 5.9 and 4.3 times of the control group(P<0.001),and returned to normal level at 5 h,and the overall trend showed a gradual decline.When treated at 35 °C for 2 h,the gene expression of CuSOD was 4.4 times higher than that of the control group(P<0.001),while the expression level of MnSOD gene was about 3.2 times higher than that of the control group at 1 h,and both decreased.The gene expression change of CuSOD was lower than the normal level at 6h,which was 0.63 times higher than that of the control group(P<0.05),while the gene expression change of MnSOD was also significantly lower than the normal level by 0.4 times at 6h(P<0.001). |
PDF Full Text Request