贵州矮马IGF-I启动子-529 CpG甲基化及个别核苷酸变异与贵州矮马矮小表型有关

Methylation of -529 CpG Site and Base Variation in IGF-I Promoter Associated with the Petite Phenotype of Guizhou Pony

胰岛素样生长因子(insulin-like growth factors,IGFs)和生长(激)素(growth hormone,GH)是动物机体主要的促生长因子,它们的表达水平直接决定成熟个体的高度。贵州矮马的体高明显低于伊犁马等大型马,但其原因尚不清楚。本研究从贵州矮马基因组DNA中克隆了GH和IGF-I基因5'-侧翼序列,分别为239 bp和817 bp,包括部分启动子结构;进而采用生物信息学、比较基因组学方法对比分析了贵州矮马与伊犁马两个基因5'-侧翼序列/启动子的转录因子结合位点及潜在甲基化位点(Cp G岛)分布。亚硫酸氢盐PCR测序法(bisulfite sequencing PCR,BSP)显示,两个马群的GH基因5'-侧翼区域(239 bp)内的6个Cp G位点均发生了甲基化,甲基化频率无明显差异。然而,在IGF-I基因5'-侧翼区的148 bp片段内含有4个Cp G位点中,贵州矮马的-529 bp处Cp G位点的甲基化程度明显高于伊犁马(P<0.01),且该甲基化位点处于基本启动子邻近3'端;此外,两个马群IGF-I基因5'-侧翼区的-561 bp处检测到T、C碱基改变,导致贵州矮马的顺式调控元件/转录因子结合位点较伊犁马少1个,有可能影响IGF-I基因的转录效率。血清IGF-I浓度测定揭示,贵州矮马血清IGF-I含量极显著低于伊犁马(P<0.01)。Spearman相关性结果显示,贵州矮马及伊犁马的IGF-Ⅰ基因甲基化频率与血清IGF-I浓度呈中度负相关(r=-0.468),提示IGF-Ⅰ基因甲基化抑制其编码蛋白质的表达。结果证明,IGF-I启动子高甲基化及某些核苷酸(碱基)序列变异可能是贵州矮马个体矮小的部分原因。

Insulin-like growth factors（ IGFs） and growth hormone（ GH） are the major growth-promoting factors in determining the size of animals. To explore the reason why Guizhou pony is shorter in height than Ili horse,the 5＇-flanking sequences at 239-bp and 817-bp of the GH and IGF-I genes were isolatedfrom the genomic DNA of Guizhou pony. The biding sites of transcription factors and the potential methylation sites（ Cp G islands） were analyzed by bioinformatics and compared to the sequences from Ili horse. Bisulfite sequencing PCR（ BSP） method revealed that six Cp G sites upstream of GH gene were heavy methylated in both species. Therefore,we excluded these sites from being considered as phenotype determining components. For the four Cp G sites upstream IGF-I,the methylation at the position- 529 in Guizhou pony was significantly higher than that in Ili horse（ P 0. 01）. A transition variation from T to C at the position- 561 was detected in Guizhou pony,resulted in the loss of a transcription factor binding site. ELISA assays showed that the levels of serum IGF-I in Guizhou pony were lower than that in Ili horse（ P 0. 01）. Spearman correlation showed a negative correlation between the serum IGF-I and methylation level of the IGF-I gene（ r =- 0. 468）. We concluded that hypermethylation and variation of certain nucleotide（ s） / base（ s） in the IGF-I promoter might contribute to the shorter and smaller phenotype of Guizhou pony.