低温对罗非鱼基因组DNA甲基化的影响

Effect of low temperature on genomic DNA methylation in Nile tilapia (Oreochromis niloticus)

以经过连续多代抗寒选育获得的尼罗罗非鱼耐寒品系为实验材料,采用DNA甲基化敏感扩增多态性(methylation sensitive amplified polymorphism,MSAP)分析方法从全基因组水平上探讨了低温适应对罗非鱼DNA序列中CCGG位点的甲基化水平的影响。结果显示,选用的18对选择性扩增引物共检测到849个位点。耐寒品系检测到的位点数为411个,对照组为438个,其中发生甲基化的位点数分别为72和104个,总甲基化率分别为17.52%和23.74%;全甲基化位点分别为37个和65个,全甲基化率分别为9.00%和14.84%;半甲基化位点分别为35个和39个,半甲基化率分别为8.52%和8.90%。结果分析表明,尼罗罗非鱼耐寒品系的总甲基化水平、全甲基化水平和半甲基化水平较对照组均有一定程度的下降。与对照组相比,尼罗罗非鱼耐寒品系基因组DNA甲基化总体水平下降了6.22%,其中以全甲基化位点变异为主,其下降幅度比例明显,为5.84%。由此可见,经过连续多代的低温胁迫可导致尼罗罗非鱼DNA甲基化水平发生改变,发生了去甲基化反应,表现为基因组甲基化程度降低的特征,说明了DNA甲基化与罗非鱼抗寒性反应密切相关。

In this study,using the methylation sensitive amplified polymorphism（MSAP）method,we globally assessed the effect of low temperature adaptation on the level and variations of cytosine methylation alterations at CCGG sites of genomic DNA in Nile tilapia（Oreochromis niloticus）strains with good cold-resistance characters developed by the successive and directional selection breeding of multi-generations.Total of 849 fragments were identified by 18 pairs of se1ective primer combinations of which 411 and 438 were detected in cold-resistance tilapia strain and the control,respectively.Among these fragments,the methylation sites were 72 and 104,and the corresponding total-methylation levels were at 17.52% and 23.74%,respectively.The full methylation sites at the internal Cs were 37 and 65,and the corresponding full methylation levels were at 9.00% and 14.84%,respectively,and the hemi methylation sites at the external Cs were 35 and 39,and the corresponding hemi-methylation levels were at 8.52% and 8.90% in cold-resistance tilapia strain and the control,respectively.Further analysis of DNA methylation indicated that a statistically decrease in the overall level of total-methylation levels and both methylation types was detected in cold-resistance tilapia strain compared with the control（decrease at 6.22%,5.84% and 0.38%,respectively）,and the changes of DNA methylation pattern were recognized mainly in the full methylation sites.The level of decrease in DNA methylation suggested that the levels of genomic DNA methylation were changed during the successive cold stress of multi-generations in tilapia,and it also indicated that DNA methylation alteration in tilapia treated with cold stress was mainly through de-methylation that occurred in some CCGG sites.All these results implied that the change of DNA methylation was closely associated with cold tolerance of tilapia.The present findings are valuable to further explore the application potential of DNA methylation alteration in tilapia genetic improvement and provide a new method and theoretical basis for fish stress resistance breeding.