Effect Of Light-dark Cycles On The Senescence Of Daphnia Pulex

The light-dark cycle,as the main zeitgeber for circadian rhythms,plays an important role in entraining.At the same time,circadian rhythms have a greater impact on the life activities and health of living organisms.The circadian rhythms that are developed for aging research has attracted more and more researchers' attention.In this paper,Daphnia pulex,an aquatic crustacean,was taken as the object of this research.The life table data,the oxides level and the activation of the antioxidant system in D.pulex under different light-dark cycles were detected.The characteristics of life span,the fluorescence images of oxidation and antioxidant balance in vivo,the quantitative detection results of ROS level,and antioxidant enzyme's activities were obtained.The effects of different light-dark cycles on growth,aging related pathway and base repair were studied by selecting the Erk pathway and base repair genes as two main directions.Ras1,Ras2,Raf,Mek,Msh2,and XRCC5 genes of D.pulex were cloned and obtained.The sequences of these genes and the characteristics of their corresponding amino acids were analyzed,and their amino acid structure as well as evolution characteristics were discussed.The results of gene expression under different light-dark cycles showed the response of D.pulex to environment for four generations.These findings can provide basic biological data for further study on the circadian regulation and rhythmic adaptation of D.pulex.The main results in this paper are as follows: 1.The effect of light-dark cycles on the life table and the balance of oxidation and anti-oxygen of D.pulexFour different light-dark cycle groups(L/D16:8,L/D8:16,LL,and DD)were set up in this paper.The neonates were divided into four groups and cultured under the above four conditions respectively.According to the recorded data,the body length,molting,reproduction and life span of D.pulex individuals in different light-dark cycles were plotted.In addition,D.pulex were collected every 4 hours for qualitative and quantitative detection of ROS,gene expression analysis of antioxidant enzymes and activity detection of antioxidant enzymes.The results showed that the average life span of D.pulex in L/D16:8,L/D8:16,LL,and DD groups was 43 days,48 days,36.5 days,and 40.5 days in turn,among which the life span of D.pulex in L/D8:16 was the longest,the life span of D.pule in LL group was the shortest,and there was a positive correlation between molting,reproduction times,reproduction amount and life span.The circadian changes of oxidation and antioxidant balance in D.pulex are the complex results of physiological process and biochemical reaction.The content of ROS was negatively correlated with the mRNA level of antioxidant enzyme genes.At the same time,the food supply may be a factor that indicates that D.pulex produces specific biological rhythm when there is no light-dark cycles.The fluorescence detection of ROS showed that ROS mostly concentrated in thoracic limbs,brood chamber,and rectum.2.Effect of light-dark cycles on ERK pathway gene of D.pulex in four generationsIn order to study the gene sequence of Erk pathway of D.pulex and its expression in successive generations,Ras1,Ras2,Raf,and Mek genes were cloned.At the same time,D.pulex were cultured in four conditions of L/D16:8,L/D8:16,LL,and DD until the 5th and 25 th day for sampling,RNA extraction and gene expression analysis.The results showed that Ras1 and Ras2 proteins had the same Ras domain responsible for binding or catalyzing the hydrolysis of GTP,which were located at 1-166 and 11-177 of amino acid sequences,respectively.They were very conservative,and even their amino acid numbers were similar among species.Ras1 protein in invertebrates is homologous with H-Ras of vertebrates,and Ras2 protein in invertebrates is homologous with R-Ras of vertebrates.In addition,Ras1 and Ras2 proteins evolve in their respective directions.Raf protein contains many domains such as serine / threonine kinase,mitogen activated protein kinase catalytic domain,and protein kinase domain.These structures act as important signal molecules in signal transduction pathways to further transmit signals to downstream proteins.Raf protein is less conserved among species than other genes in the pathway.Mek,as a downstream protein of Raf,belongs to Pkc-like superfamily.In addition,the catalytic domain Pkc_Mek of bispecific protein kinase and mitogen activated protein(MAP)/ extracellular signal regulated kinase(ERK)was also found in the domain of Mek protein.This protein in D.pulex is also more conservative,which is more similar to that of insect's in evolution.The results of Erk pathway gene expression in D.pulex for four consecutive generations under different circadian rhythms showed that Erk pathway had the characteristics of signal amplification step by step in different light-dark cycles,that is,Raf / Mek / Erk genes were significantly up-regulated grually,but the trend of signal amplification was significantly activated in light,the efficiency decreased in dark,and the efficiency of Erk activation cascade was age-related decreasing.3.Effect of light dark cycle on genes related to base repair in D.pulexIn order to study the gene sequence of Msh2 and XRCC5 of D.pulex and its expression in successive generations,the Msh2 and XRCC5 genes were cloned.At the same time,D.pulex were cultured in four conditions of L/D16:8,L/D8:16,LL,and DD until the 5th and 25 th day for sampling,RNA extraction and gene expression analysis.The results showed that the Msh2 gene belongs to the MutS superfamily,which contains a series of domains related to DNA mismatch repair,such as MutS_I and MutS_II.The results of multiple sequence alignment showed that the conservation of Msh2 protein among vertebrates was higher than that of invertebrates,with more identical sequences,and the number of amino acids of Msh2 protein in species was similar.In evolution,the relationship between Msh2 protein and the same kind of animals is close,and the homology is high.This is the first report of Msh2 gene in crustaceans.In addition,according to the analysis in this paper,the XRCC5 gene may be part of the gene or the cDNA sequence through variable splicing,although the homology with the reported Daphnia magna is 90.98%.The results of expression analysis showed that light promoted the expression of Msh2 gene related to the aging of D.pulex in a single generation.In successive generations,the increase of light duration increased the expression of Msh2 gene with aging,while the increase of dark duration decreased the expression of Msh2 gene with aging,and this up-regulation or down-regulation increased with the increase of generations.D.pulex showed the adaptation of increasing and decreasing light time in the environment for four consecutive generations.For the environment with long dark time,D.pulex would continuously down regulate Msh2 and XRCC5 genes.In addition,the expression of XRCC5 gene increased with aging.