The Optimization And Application Of Msap In DNA Methylation Analysis In Fenneropenaeus Chinensis

In order to explore the molecular mechanisms that affecting the growth ofFenneropenaeus chinensis from the perspective of epigenetics and provide atheoretical basis for the breeding of new Fenneropenaeus chinensis. The three keyPCR procedures, including digestion, pre-selective amplification and selectiveamplification in MSAP (Methylation Sensitive Amplification Polymorphism) systemwere optimized in allusion to Fenneropenaeus chinensis firstly. Then MSAP wereapplied to analyze methylation patterns of genome DNA in muscle, gill and blood ofwild and cultivated shrimp Fenneropenaeus chinensis.“Huanghai No.1” is a selectedstrain with faster growth rate and better performance in diseases resistance. The third,MSAP were applied to analyze the patterns of Fenneropenaeus chinensis which underthe ammonia stress and pH stress.The part one is the establish and optimize of MSAP in allusion to F. chinensis,The pivotal steps of MSAP technique were optimized based on its mechanism and aprevious study. Such as DNA digestion, pre-selective amplification and selectiveamplification. The digestion time, the concertration of buffer, DNA template andPAGE are optimized specifically. The result of experiment appear that DNA qualityand quantity is fundamental and the reaction time of isoschizomers is crucial for thesuccess of MSAP. Insufficient digestion could reduce the amount of products.Over-digestion may lead to deficiency and nonspecific reaction and prolong reactioncycle. Wherein, dilution factor becomes especially important. Insufficient dilutionmay lead to entrainment phenomenon while over-dilution could result in failure indetecting polymorphisic bands.8h digestion produced the most desired products.10-fold dilution of ligation products and2.2μL PCR buffer are adopted inpre-selective amplification.10-fold dilute pre-selective amplification produce astemplate are more uniform and clear, thus it was used in further MASP analysis.The part two apply MSAP in the first part to investigate the differences of cytosine methylation of genomic DNA from muscle, gill and blood between the wildstock and “Huanghai No.1” F. chinensis. and last explored the possible molecularmechanisms affecting growth traits.30pairs of selective-amplification primers werechosen in the experiment and the results of PAGE (Polyacrylamide GelElectrophoresis) presented that the methylation ratios in muscle, gill and blood ofwild stock were23.1%,22.3%and19.7%, while those were21.4%,19.6%, and18.9%in cultivated shrimps, respectively. Which are much lower than some of plantsand animals possiblily due to the different amount of genome NDA. Methylationlevels of CCGG site were specific in different tissues of the same stock and the sametissue of different stocks, and the genome DNA methylation level is always higher inorgans than in blood. The same result also happened in DNA methylationpolymorphism, DNA polymorphic methylation of gill and blood between wild stockand “Huanghai No.1” varied to some extent, while those of muscle kept in a balanceddegree. Three classes of band chains among nine categorized classes based on twomethylation type were analyzed as a meaning of DNA metylation polymorphism inthree tissues between two stocks of F.chinensis, which could be used as a basis forfurther studies on the mechanism of methylation-mediated DNA methyltransferases inF.chinensis. Furthermore, polymorphic methylation was associated withdemethylation and methylation of CCGG loci and further studies need to be carriedout to understand the biological meaning of variation in methylation of CCGG loci.The part three apply MSAP technology to analye the genomic DNAmethylation patterns of F. chinensis,which under the different ammonium chlorideconcentrations and pH gradient acute stress. In the ammonium stress, the methylationlevels of F. chinensis are among23.66%—24.17%. when F. chinensis were under lowconcentrations of ammonium chloride (≤8mg/L) treatment, DNA methylation levelsare relatively stable compared with the control group, there is a slight decreasehemimethylated levels changed significantly rise. But when ammonium chlorideconcentrations were higher than8mg/L, DNA methylation levels were rised. Aboutthe pH part, F. chinensis methylation levels are among21.73%—22.11%, which had arelatively narrow range of pH value. When the pH is among8.0-8.8, the livingconditions is similar to the control group, but the mortality is significantly higherunder other pH. The DNA methylation levels are slight changed under the ammoniastress with a relatively stable levels. The decrease of methylation levels are0.34%and0.38%when pH are7.2and7.6, which is very obvious than0.21%and0.28%which are the methylation levels under pH are8.4and8.8. Hemi-methylation levelsresponse to the increase of the pH with a decrease trend.