Determination and Difference Analysis of DNA Methylation Content Both in Blood and Muscle Tissue of Pigs

In this experiment, DNA samples from parental lines Large White, Landrace, and Meishan pigs, and their hybrids Large White × Landrace, Landrace × Large White, Large White × Meishan, and Meishan × Large White pigs were used for the determination of DNA methylation content in both blood and muscle tissue. The differences about DNA methylation content between parental lines and their hybrids were analyzed. These will offer theoretical support from molecular level for heterosis. High performance liquid chromatography （HPLC） was firstly used to detect DNA methylation content. The average DNA methylation content in 163 DNA samples of muscle tissue was 16.92%, whereas, the average DNA methylation content in 182 samples of blood was 6.49%, the difference between which was especially prominent （P 〈 0.01）. In blood, the methylation content was lower than 10%, with the highest level of 9.93% in Landrace x Large White and the lowest of 2.97% in Landrace. The difference between Large White and Landrace was highly significant （P 〈 0.01）, but not significant between Large White and Meishan; and the differences between reciprocal cross hybrids in both hybrid systems were significant （P 〈 0.05）. In muscle tissue, the differences in methylation content among three parents were not significant （P 〉0.05）; and the differences between reciprocal cross hybrids in both hybrid systems were not significant （P 〉 0.05）, but between different hybrid systems, the hybrids had a significant difference （P 〈0.05）. The average methylation content in muscle samples was higher than that in blood samples, and the methylation in different tissues was different.     

猪血液和肌肉组织DNA甲基化含量的测定

 摘要: 【目的】通过测定纯种亲本大白猪、长白猪、梅山猪及其正反交大白猪×长白猪、长白猪×大白猪、大白猪×梅山猪和梅山猪×大白猪等杂种猪血液和肌肉组织DNA甲基化含量，分别比较基于不同杂交组合和不同组织，亲本与子代之间在DNA甲基化含量上的差异,为揭示杂种优势的分子机制提供依据。【方法】采用高效液相色谱法测定血液和组织中甲基化含量。【结果】经过测定及计算，163份肌肉组织样平均甲基化含量为16.92%；182份血液样平均甲基化含量为6.49%，两者之间差异达到了极显著水平（p<0.01）。在相同组织不同杂交组合中杂种后代表现不同。同样地，在相同杂交组合不同组织之间杂种后代表现也不尽相同。【结论】杂种甲基化含量升高，可能是利用甲基化关闭了一些影响生长的不利基因的表达。杂种甲基化含量的变化表现为杂交组合和组织特异性。     

Isolation,Identification and Tissue Expression Profile Analysis of One Novel Differentially Expressed Sequence Tag in the Longissimus dorsi Muscle from Meishan,Meishan × Large White Hybrid and Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus dorsi tissue from Meishan, Meishan × Large White hybrid and Large White pigs with nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 3 000 reproducible bands were examined. One novel expressed sequence tag (EST4,GenBank accession number: AY553914) that was differentially expressed in Meishan,Meishan × Large White hybrid and Large White pigs was isolated from the Longissimus dorsi muscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealed that the 350 bp long EST (EST4) was not homologous to any of the known porcine genes.Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.     

Isolation, Identification of Differentially Expressed Sequence Tags in the Backfat Tissue from Meishan, large White and Meishan×Large White Cross Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the backfat tissue from Meishan,Large White and Meishan×Large White cross pigs.Nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers were used to perform the differential display PCR and nearly 3 000 reproducible bands were examined .Fifteen expressed sepuence tags that were differentially expressed were isolated and then isdentifide through semi-quantitative RT-PCR.BLAST analysis revealed that the fifteen expressed sequence tags (ESTs) were ntt homologous to any of the known porcine genes or ESTs. These novel ESTs were then submitted to GenBank.     

高粱DNA甲基化变异的分子杂交验证

以2个高粱(Sorghum bicolor L.)亲本纯系及其F1杂交种为材料,利用甲基化敏感扩增多态性(MSAP)方法,对DNA甲基化水平和模式的遗传和变异进行了研究.结果表明:亲本纯系的大部分胞嘧啶甲基化位点稳定遗传给其F1杂交种,但3.94%的位点表现出了变异,即未从亲本遗传给杂交种,并通过Southern杂交方法进一步验证了杂交种和纯系亲本间的DNA甲基化模式的遗传和变异.     

猪 MKRN3 基因启动子区CpG岛在胚胎心脏组织甲基化模式分析

以梅山猪-大白猪正反交为模型,以65日龄和100日龄的胚胎心脏组织为研究材料,对MKRN3基因启动子区CpG岛进行预测,根据预测的CpG岛设计引物,采用重亚硫酸盐测序法(BSP法),分析MKRN3基因启动子区CpG岛在胚胎心脏组织的甲基化程度。结果显示:对于65日龄胚胎,猪MKRN3基因启动子区CpG岛在大白×梅山和梅山×大白杂交后代的胚胎心脏组织中均表现高度甲基化(75.6%、76.4%);对于100日龄的胚胎,大白×梅山杂交后代的胚胎心脏组织表现高度甲基化(80%),而梅山×大白杂交后代的胚胎心脏组织呈现低甲基化(35.6%)。由此可见,猪MKRN3基因启动子区CpG岛的甲基化模式随着正反交、胚胎发育时期不同而呈现出一定变化,即在胚胎发育65日龄时,正反交子代均具有高度甲基化;而在胚胎发育至100日龄,正交子代呈现高度甲基化,反交子代呈现低甲基化,说明在胚胎发育晚期父本或母本等位基因可能会发生明显的去甲基化。     

RAPD标记遗传距离与猪杂种优势相关性的研究

【目的】探讨利用RAPD分子标记技术预测杂种优势的可能性。【方法】利用RAPD技术测定6个亲本群体间的遗传距离，对亲本群体间遗传距离与10个经济性状的杂种优势率进行相关性分析。【结果】亲本间遗传距离与日增重、料重比、屠宰率和平均背膘厚的杂种优势率呈正相关，与瘦肉率、瘦肥肉比、皮率、眼肌面积的杂种优势率呈负相关。并且遗传距离与料重比和眼肌面积杂种优势率的相关系数达到显著程度（P＜0.05），而与肥肉率、内脂率等性状杂种优势率的相关系数则非常小。【结论】亲本间的遗传距离与杂种优势的相关程度随不同性状而异。