The Method To Assess Genome DNA Methylation Of Apostichopus Japonicus By HPLC And MSAP Analysis In Different Tissues

DNA methylation can regulate the gene expression on the premise ofnot change the genetic structure, it is related to embryonic development,histological differentiation and senium. Correlational research in animal andplant genetic breeding about DNA methylation mainly focused on genomeDNA methylation status, DNA methylation and regulation of geneexpression, DNA methylation and molecular marker, etc.1. MSAP Analysis of Genome DNA Methylation in Different Tissues ofApostichopus JaponicusGenomes of respiratory tree, intestine, muscle, body wall of Seacucumber Apostichopus Japonicus were compared for the methylation leveladopting methylation-sensitive amplification polymorphism technique(MSAP). Genome DNA was digested by two pair of restrictionendonucleases MspI/EcoRI and HapII/EcoRI. The products of two steps ofPCR-amplified were separated by polyacrylamide gel electrophoresis(PAGE) to obtain the MSAP map.The results showed that electrophoretic bands concentrated in between 100bp and750bp. Appear in the MspI and EcoRI enzymes cut lane forwhole-methylation sites, appear in the HapII and EcoRI enzymes cut lane asthe half-methylation sites, at the same time appear in the two lanes, therewas no methylation. Statistical analysis by Spss17.0Statistics organizationsbetween the valid data, we learned that the methylation probability of thefour tissues from high to low were respiratory tree>bodywall>muscle>intestine, as35.77%,33.51%,32.72%and28.0%in sequence,among which the full methylation took up19.46%,18.39%,19.18%and15.97%orderly. The statistic results showed that the intestine had significantdifference with the other three tissues (P<0.05); and the difference ofmethylation between muscle, respiratory tree and body wall was not obvious(P>0.05), while there was significant difference between muscle andrespiration tree in hemi-methylation (P<0.05).The no significant differences between body wall and muscle maybewere caused by the attachment of the two tissues. And intestinal andrespiratory tree is a separate existence of the organization, the intenseinterference each other is not so significant difference with the otherorganization. DNA methylation in different periods, different organizationsof differentially expressed is required by the animal and plant growth anddevelopment. It was found that the genome methylation phenomenon andApostichopus Japonicus methylation level is different between different organizations, the probability of no-methylation in genome was greater thanmethylation, and the locus of whole-methylation was greater than thehalf-methylation. This may perform different functions with differentorganizations and methylation plays a certain role in gene regulationfunction. Compared with other aquatic animals, Apostichopus Japonicusmethylation level belongs to medium level.Four specific fragments were got through analysis of difference site inMSAP strips, and the functions of those fragments were unknown aftersequencing and blasting. It was supposed that those sequences were newfunctional genes or control sequence area of Apostichopus Japonicus.2. A Method to Assess Genome DNA Methylation of ApostichopusJaponicus by HPLCHydrolysis DNA into a single nucleotide, using high performanceliquid chromatography (HPLC) testing Apostichopus Japonicus genomeDNA methylation status, the detection wavelength of283nm, the columntemperature30℃, the flow rate of1ml/min. The experiment was carriedon the DNA sample pretreatment and chromatographic conditions two partsrespectively.Sample preparation including two kinds of method, it was acid solutionusing perchloric acid and enzyme solution by nuclease and phosphatase.Different hydrolysis temperature(85℃,90℃,95℃,100℃) and time(45min、 55min、60min) was set up respectively. The pH of all the samples was4.5or6.7, also the mobile phase. The pH of samples by enzyme solution andmobile phase was6.7or4.5.The kinds of mobile phases (Potassium dihydrogen phosphate,Ammonium acetate and Pentane sulfonic acid sodium) for chromatographicconditions were mainly optimized by different ratio of methanol (0%、2%、5%、7%). Optimum conditions were obtained by comparing with each other:50μl DNA (6μg DNA was contained) with70%perchloric acid30μl,incubation55min under95℃, pH3-5, mobile phases was Pentane sulfonicacid sodium, pH4.5; For the samples of enzymatic hydrolysis, the optimumchromatographic conditions were5%-methanol-7mmol/L Ammoniumacetate (pH6.7), the pH of samples had no obvious influence to the results.The HPLC results of the two hydrolysis methods showed that the enzymesolution had a higher probability of methylation than that of acid solution. Itwas mainly caused by the mild of the enzyme, less damage on the5-mC washappened, so the results were more accurate. But the high consumption ofenzymatic method made the acid solution economical and convenient.