Analysis Of Gene Expression Profile And DNA Methylation Characteristics Of Apostichopus Japonicus(Selenka) During Aestivation

Sea cucumber Apostichopus japonicus(Selenka) is of high nutritional and medicinal value, which has the most economic output as a single aquaculture species in China. When sea water temperature rises to some level in summer, A. japonicus will enter into aestivation. The annual period of aestivation usually lasts more than 4 months with obvious torpor characters: complete cessation of feeding and locomotion, loss of body weight, strong metabolism depression and reorganizing of energy. Aestivation undoubtedly shortens the growing cycle of A. japonicus and brings economic losses, hence aestivation research has important scientific significance and industry guidance significance.So far, most studies of sea cucumber aestivation have focused on morphology, physiology and immunology. However, studies of molecular regulatory mechanisms that control aestivation are just beginning. This study explored the DNA methylation characteristics and gene expression profile of A. japonicus during aestivation, which may facilitate the understanding of the molecular regulation mechanisms of aestivation in sea cucumber.In this paper, qRT-PCR, RACE, F-MSAP and high-throughput sequencing techniques were used to analyze the gene expression profile and DNA methylation characteristics of A. japonicus during aestivation. First, we optimized the optimal reference gene combination for different tissues of A. japonicus during aestivation; then we constructed a global gene expression pro?le of intestine and respiratory tree tissues of A. japonicus using RNA-seq to identify transcriptional responses associated with transitions between different states during aestivation; then we cloned the two key genes Dnmt1 and Mbd2/3 of the DNA methylation system in A. japonicus and analyzed the expression levels during aestivation; at last, we analyzed DNA methylation levels of various tissues of A. japonicus during key aestivation states. Main results were as follows:1. The expression stability analysis of eight candidate reference genes in sea cucumber showed the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. The optimal reference gene combination for intestine was 40 S ribosomal protein S18(RPS18), β-tubulin(TUBB) and NADH dehydrogenase(NADH); for respiratory tree, it was β-actin(ACTB) TUBB, and succinate dehydrogenase cytochrome B small subunit(SDHC); and for muscle it was α-tubulin(TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13(NDUFA13).2. We have then analyzed the global gene expression pro?le of intestine and respiratory tree tissue of A. japonicus during aestivation by constructing and screening three libraries representing key stages of aestivation: non-aestivation(NA), deep-aestivation(DA), and arousal from aestivation(AA) using RNA-seq. A set of respiratory tree speci?c DEGs was identi?ed the ?rst time and common DEGs that were responsive to aestivation in both respiratory tree and intestine were then identi?ed involving with metabolism, immune defense and energy and cell cycle regulation. Most key DEGs in both sea cucumber tissues during aestivation were associated with metabolic depression, especially energy-expensive processes, detoxi?cation and tissue protection, and metabolism and energetic reorganization, which may be excellent candidates for further investigating molecular mechanism of aestivation in sea cucumber.3. Two key genes Dnmt1 and Mbd2/3, which comprise the DNA methylation system in A. japonicus were cloned and analyzed. The deduced amino acid sequences and characteristic motifs of sea cucumber Dnmt1 and Mbd2/3 showed high homology to those of their mammalian counterparts. Quantitative real-time RT-PCR analysis showed Dnmt1 expression in the intestine was up-regulated during deep-aestivation(P<0.05), while Mbd2/3 was over-expressed in both the intestine and respiratory tree during the same period(P<0.01). The results of this study suggest that the above genes may be involved in DNA methylation of sea cucumber during aestivation, and the major sites are the intestine and respiratory tree.4. DNA methylation levels of A. japonicus were further analyzed by the ?uorescence-labeled methylation-sensitive ampli?ed polymorphism(F-MSAP) technique over three stages of the aestivation cycle. The incidence of DNA methylation was different across tissue types in the non-aestivation stage ranging from 28% to 56%. Our results show that hypermethylation accompanied deep-aestivation in intestine and respiratory tree tissues of A. japonicus, and that full-methylation but not hemi-methylation levels exhibited signi?cant increases in the deep-aestivation stage. No significant differences were observed in DNA methylation levels of muscle and body wall tissues over the three aestivation states. Hence we speculate that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation and the main DNA modi?cation sites were focused on intestine and respiratory tree tissues, which were most affected organs during aestivation.