The Study Of DNA Methylation Level Of Loquat Leaf And The Correlation Between Chemical Composition And DNA Methylation

Objective:To study the methylation level of Eriobotrya japonica which from different strais and fields with the methods of high performance liquid chromatography(HPLC) and methylation sensitive amplified polymorphism(MSAP),then explore the relationship between methylation level and chemical component.Methods:1. Collect43samples in Fujian.2. Using the improved CTAB method to extract loquat leaf genomic DNA,and then testing DNA concentration and purity by0.8%agarose gel electrophoresis and uv specetrophotometer.3. Using the70%perchlorate solution to hydrolytis total genomic DNA and being separated segments of the bases,then take the reversed-phase high performance liquid chromatography (HPLC) method to test the content of cytosine and5-methyl cytosine, and calculate the rate of methylation.4. Adopt the method of orthogonal design respectively the the main factors of pre-amplification and selective-amplification is optimized, then the6%denatured polyacrylamide gel electrophoresis to check the result.According to the different position of bands to analysis methylation types and rate.5. Reversed phase high performance liquid chromatography is used to detecte the content of ursolic acid and oleanolic acid in loquat leaves.6. Using statistical software SPSS18.0, Origin8.0to analysis.Results:1. The UV specetrophotometer test the DNA which extracted by the improved CTAB method showed that OD260/280between1.98to2.15and OD260/230between1.79to2.25means that there are without protein,polysaccharides,polyphenols left,and0.8%agarose gel electrophoresis presented a small neat bands,is pure enough to next analysis.2. HPLC method can separate the cytosine and5-methyl cytosine well, according to the formula of methylation rate=5mC/(5mC+C)%, methylation rate calculation, the results showed that the methylation rate from6.93%to54.11%in the43samples.3. Is obtained by orthogonal test the best dosage of pre-amplification reaction system which contain3uL linked product, E00and H00105ng, dNTPs tendsity is0.25mmol/L, Taq DNA polymerase2U, and the best selective-amplification reaction system is Taq DNA polymerase1.5U, dNTPs0.25mmol/L,primers37.5pmol, mg2+2mmol/L, and pre-amplification product30times dilution. By verifying the reaction conditions can get more clear strip, can be used for the analysis of loquat leaf methylation.4. The analysis results of MSAP by NTsys-pc version2.10e software show that the coefficient of similarity between0.6125to0.9625, the total methylation ratio is between15.31to26.92.5. The determination of ursolic acid and oleanolic acid concent in loquat leaf by HPLC shows that content differences between different varieties, the total average of oleanolic acid and ursolic acid content in the Baili were the highest, ursolic acid content in the Wugongbai is the lowest, and ursolic acid content in the Jiefangzhong is the lowest.Conclusion:1. DNA methylation affected by strains and geographic factors. This experiment was analysis six varieties and four origins of loquat leaf methylation, is a sign of methylation between varieties and producing field have significant difference.The leaves come from Huating have the highest methylation rate,and the ones from Changji are lowest.2. The methylation types of Zaozhong6is given priority to full methylation, however Jiefangzhong is given priority to half methylation.3. The results of correlation analysis between content of ursolic acid, oleanolic acid and methylation rate showed that ursolic acid, oleanolic acid and half-methylation rate, full-methylation rate with a certain negative correlation. But cannot think have correlation with total methylation rate.4. The results of HPLC and MSAP of detecting DNA methylation level are similar.