| Rotifers are useful model organisms for aging research, owing to their small body size (0.1-1mm), short lifespan (5-15days) and the relative easy in which aging and senescence phenotypes can be measured. Resveratrol (3,5,40-trihydroxy-trans-stilbene) is a polyphenolic phytoallexin found in a variety of plant products and has been reported to have beneficial effects on lifespan in many organisms. Dietary restriction (DR) is usually defined as a moderate (normally20-40%) reduction of available nutrients without causing malnutrition. In the laboratory, DR is one of the most commonly used life-history alterations among evolutionarily distinct eukaryotes, from single-cell to multicellular organisms. It has been reported that resveratrol may extend the lifespan through a CR-like mechanism. However, resveratrol’s anti-aging mechanisms is still controversial. In this paper, we study the effect of resveratrol and DR on the rotifer reproduction and lifespan, to study whether they are through with a same way to play the anti-aging effects. In this paper, We cloned the full cDNA encoding BcMnSOD, BcCuZnSOD, BcCAT, Bchsp40, Bchsp60, Bchsp70and Bchsp90in Brachionus calyciflorus through rapid-amplification of cDNA ends (RACE) technology. And to study the effects of resveratrol and DR on the mRNA expression of BcMnSOD, BcCuZnSOD, BcCAT, Bchsp40, Bchsp60, Bchsp70and Bchsp90. Main results of this dissertation included the following five parts:1. Gene clone and its structural and functional studiesThe degenerate primers were designed with reference to DNA nucleotide sequences of the MnSOD, CuZnSOD, CAT, hsp40, hsp60, hsp70and hsp90from other species. Obtain the full cDNA encoding BcMnSOD, BcCuZnSOD, BcCAT, Bchsp40, Bchsp60, Bchsp70and Bchsp90after confirmed the PCR amplified fragment. 2. Rotifer aging characterizationNewly hatched rotifers were～180μm long and grew to～300μm in length after-12h, when they matured into adults. Each rotifer usually carried1egg at1-day-old, and three or more (4-6) eggs at2-3-days old. After the fourth day, their reproduction began to decrease. Reproduction peaked at the age of2days, and was recorded as being approximately three offspring per female per day. Each female produced an average of15.5offspring in her lifetime. Under these conditions, rotifer reproduction was exclusively parthenogenetic. The first death occurred at72h, and numbers gradually increased with increasing age of the remaining rotifers. Under normal experimental conditions, the mean rotifer lifespan was102h. On the fifth day, the rotifers generally did not reproduce, and their morphology changed significantly; for example, the spines on the posterior lengthened in some individuals. Swimming speed also changed significantly with the aging process. Newly hatched rotifers swam at4.3-body lengths/s (～0.78mm/s). On the second day, the swimming speed peaked (1.89mm/s,～6.3-body lengths/s) and then began to slowly decline.3. The effects of aging process on gene expression in rotiferExamine the mRNA expression levels of antioxidant enzyme gene and four hsps during the aging process using highly sensitive real-time qPCR. The mRNA expression level of BcMnSOD decreased with aging (P<0.05), whereas that of BcCuZnSOD increased significantly (P<0.05). The expression of BcCAT did not change significantly (P>0.05). The expression level of Bchsp70mRNA significantly decreased with age (P<0.05), whereas a small but significant (P<0.05) increase in the mRNA levels of Bchsp40was only observed in4day-old individuals. The expression of Bchsp60mRNA significantly decreased (P<0.05) in1,2and3day-old individuals and significant decreases (P<0.05) in the mRNA levels of Bchsp90were observed in2,3and4day-old individuals.4. The effects of resveratrol and DR on reproduction, lifespan and oxidative stress tolerance in rotiferThe results showed that:resveratrol did not prolong the lifespan of B. calyciflorus. At low resveratrol concentrations (1-20μM), rotifer reproduction was slightly increased, and reached statistical significance in the R5group (Duncan’s test, P<0.05). Relatively high concentrations of resveratrol (50μM) significantly interfered with rotifer reproduction. Rotifers were able to carry eggs in medium containing50μM resveratrol, however, few eggs hatched. Reproduction recovered within24h when the rotifers were placed into normal medium. When adult rotifers which had been grown in normal medium and had produced at least one offspring were placed in medium containing a high concentration of resveratrol (50μM), reproduction stopped within24h. Fecundity of the rotifers was significantly reduced in both the RO-50and R50-0groups. In contrast to resveratrol, DR. extended the lifespan of rotifers. The average lifespan in the DR group was120.4±4.3h, which was significantly longer than that of the control group (95.0±3.7h)(log-rank test,P<0.0001). However, The average number of offspring produced per rotifer in the CR group was5.9±2.4, significantly less (Student’s t-test,P<0.0001) than that in the control group (13.6±5.5h). Resveratrol and DR have different effects on reproduction and lifespan suggest that resveratrol did not imitate the DR mechanism to lengthen the lifespan. In addition, resveratrol treatment did not increase rotifer swimming speed of4-days-old rotifers, whereas moderate DR (12h) increased the swimming speed (P<0.05). In the groups starved for24h and48h, there was no significant change in swimming speed. After being exposed to oxidative stress for12h, the survival rate of the DR pretreated group was0.78±0.02, significantly higher (P<0.05) than of the control group (0.60±0.25). A similarly significant result was recorded for the group exposed for24h: the survival rates of the DR pretreated and control groups were0.55±0.02and0.38±0.02, respectively (P<0.05). This result indicates that DR pretreatment can increase the rotifer survival rate under paraquat-induced oxidative stress. However, resveratrol pretreatment did not significantly improve survival under oxidative stress.5. The effects of resveratrol and DR on gene mRNA expression levelQuantitative real-time PCR showed that resveratrol significantly increased (P<0.05) the mRNA expression of the gene encoding BcMnSOD, whereas the expression levels of the genes encoding BcCuZnSOD and BcCAT were inhibited. The mRNA expression of BcMnSOD was enhanced after24-h DR, but was significantly lower (P<0.05) after48-h DR. Starvation also increased the mRNA expression of the genes encoding BcCuZnSOD and BcCAT, which peaked after12-h DR. This results suggest that BcCuZnSOD and BcCAT high. expression plays an important role in delaying the aging rotifer. DR also can improve rotifer Bchsp40, Bchsp60, Bchsp70and Bchsp90mRNA expression.6. The effects of Vitamin E and hydrogen peroxide (H2O2) on rotifer gene expressionReal-time quantitative PCR demonstrated that vitamin E (40μM) could instantly increase the expression of BcCuZnSOD, BcMnSOD and BcCAT (Fig.7). However, the mRNA expression levels returned to normal level if exposing beyond4hours. While, vitamin E (40μM) significantly decreased the expression of Bchsp40. However, the expression of Bchsp70 and Bchsp90was temporarily increased after exposure to vitamin E for1h, and after exposure for4h, the expression levels of Bchsp70and Bchsp90were significantly decreased compared to the control. The expression of Bchsp60was significantly increased after exposure to vitamin E, and the expression level was maximal after4h of exposure.Upon to H2O2(0.2mM) exposure, BcCuZnSOD, BcMnSOD and BcCAT genes mRNA expressions were up-regulated with different degree. After exposing0.1mM H2O2, the mRNA expression levels of BcCuZnSOD, BcMnSOD and BcCAT increased rapidly (P<0.05), however, the mRNA expression levels returned to normal levels (P>0.05) after exposing6hours. Following H2O2(0.1mM or0.2mM) exposure, the mRNA expression of the four hsps was up-regulated to different degrees. In both the0.1mM group and0.2mM group, the expression of the four hsps significantly increased (30-90fold) immediately after exposure to H2O2for1h, and even though expression decreased slightly after2h, the mRNA expression levels were still significantly higher (5-10fold) than the normal level (P<0.05). |
PDF Full Text Request