摘要DNA甲基化是表观遗传学的研究热点之一。为了探索填饲是否能够引起朗德鹅(Anser anser)肝脏脂类代谢中DNA甲基化水平以及基因表达量的变化和DNA甲基化修饰作用和基因表达之间的关系。本研究运用焦磷酸测序技术分析鹅肝脏硬脂酞辅酶A去饱和酶5基因(stearovl-CoA desaturase 5, SCD5)、固醇调节元件结合蛋白2基因(sterol regulatory element-binding protein 2, SREBP2)和超长链脂肪酸延伸酶6基因(elongase of very long chain fatty acids 6, ELOVL6)的甲基化水平，再运用qRT-PCR技术进行定量验证。结果显示，SCD5、SREBP2和ELOVL6各2个片段的甲基化水平均在20%以上，均属于高甲基化状态；对照组3个基因各2个片段的甲基化水平均高于填饲组，填饲组与对照组的SCD5-1S和SREBP2-1S片段甲基化差异显著(P＜0.05)，SREBP2-2S片段甲基化差异极显著(P＜0.01)，SCD5-2S、ELOVL6-1S和ELOLV6-2S片段甲基化差异不显著(P＞0.05)；填饲组SCD5和ELOVL6的mRNA表达量均高于对照组，而SREBP2在填饲组的mRNA表达量显著低于对照组(P＜0.05)。综合各项指标，填饲引起鹅肝脏组织SCD5、SREBP2和ELOVL6甲基化水平降低，SCD5和ELOVL6 mRNA表达量上升，SREBP2的mRNA表达量降低，表明SCD5和ELOVL6的甲基化修饰对mRNA的表达起反向调控的作用。本实验结果为表观遗传学上DNA甲基化研究方法提供参考，为选育鹅肥肝高产高质系提供重要的分子遗传学依据，也为人类肥胖症和脂肪肝等代谢疾病的研究提供理论依据。
Abstract：DNA methylation is a hotspot in epigenetic research. This study was aimed to explore whether the force-feeding could change the levels of DNA methylation and gene expression related to lipid metabolism in liver of Landes(Anser anser), and to investigate the relationship between DNA methylation and gene expression. The levels of DNA methylation of stearovl-CoA desaturase 5 gene (SCD5), sterol regulatory element-binding protein 2 gene (SREBP2) and elongase of very long chain fatty acids 6 gene (ELOVL6) were analyzed with pyrosequencing method and quantified by qRT-PCR. The upstream promoter sequence of SCD5 (about 2 800 bp) was downloaded from University of California Santa Cruz (UCSC) website, there were 2 CpG islands, and the percentage of cytosine, G+C and CpG island were respectively reached of 22.7%, 50.06% and 3.81% after analyzing from the 406~1 220 bp CpG island (full-length fragment of 815 bp). The upstream promoter sequence of SREBP2 (about 2 248 bp) was also downloaded from UCSC website, there were 2 CpG islands, and the percentage of cytosine, G+C and CpG island were respectively reached of 24.87%, 56.61% and 5.29% after analyzing from the 1~965 bp CpG island (full-length fragment of 965 bp). The upstream promoter sequence of ELOVL6 (about 2 248 bp) was downloaded from UCSC website, there was 1 CpG island, and the percentage of cytosine, G+C and CpG island were respectively reached of 37.14%, 55.71% and 4.30% after analyzing from the 1 426~1 775 bp CpG island (full-length fragment of 350 bp). A total of 23 CpGs related DNA methylation were detected by using pyrosequencing method after amplified the promoter sequence of CpG island of SCD5, SREBP2 and ELOVL6 (each gene had two fragments of 37~55 bp). Results showed that the levels of DNA methylation of the 2 fragments of SCD5, SREBP2 and ELOVL6 in force-feeding group (FFG) and control group(CG) were respectivlely higher than 20%, belongs to high methylation status. The levels of DNA methylation of CG were greater than FFG. Significant difference were found on DNA methylation in the first fragment of SCD5 and the first fragment of SREBP2 between FFG and CG(P＜0.05), markedly significant difference (P＜0.01) in the second fragment of SREBP2, but no significant difference(P＞0.05) in the second fragment of SCD5 and the first fragment and the second fragment of ELOVL6 between FFG and CG. The mRNA expression of mRNA of SCD5 and ELOVL6 in FFG were greater than those of CG, and there relative gene expression of SREBP2 in FFG was lower than that of CG (P＜0.05). In conclusion, force-feeding decreased the expression of DNA methylation of SCD5, SREBP2 and ELOVL6, increased the mRNA expression of SCD5 and ELOVL6 in liver of goose, but decreased the mRNA expression of SREBP2, which indicated that the methylation of SCD5 and ELOVL6 played a negative regulation effect on the mRNA expression of SCD5 and ELOVL6. The result will provide a reference to the research method of DNA methylation on epigenetics and an important molecular genetics basis on breeding high quality and high yield goose fatty liver, it will also provide theoretical basis for researching the metabolic diseases such as obesity and fatty liver.
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