二倍体和四倍体泥鳅IGFBP-1基因的表达及其与DNA甲基化的相关性

Expression of IGFBP-1 and its correlation with DNA methylation in diploid and tetraploid Misgurnus anguillicaudatus

为探讨胰岛素样生长因子结合蛋白-1(IGFBP-1)基因在二倍体和四倍体泥鳅之间的表达差异及其与DNA甲基化的相关性,克隆二倍体、四倍体泥鳅的CDS序列和启动子序列,并分别采用qRT-PCR和亚硫酸氢盐测序技术(BSP)分析IGFBP-1在二倍体、四倍体泥鳅中的表达水平及启动子区和第一外显子区的甲基化水平。结果显示,泥鳅的IGFBP-1基因CDS序列长789bp,编码262个氨基酸,二倍体、四倍体泥鳅间有5bp的差异,但氨基酸序列完全相同。在二倍体、四倍体中分别克隆得到2341bp和2331bp的启动子序列,倍性间的序列相似度达99%,启动子序列中包含典型元件TATA-box,以及SP1、CREB、C/EBP、POU2F2、GATA-2/3/4和SRY等转录因子结合位点。泥鳅的IGFBP-1基因主要在肝脏中表达,且四倍体泥鳅肝脏中IGFBP-1的表达水平显著高于二倍体(P<0.01)。四倍体泥鳅IGFBP-1基因启动子及第一外显子区的甲基化水平均比二倍体低,其中四倍体肝脏中的甲基化水平显著低于二倍体(P<0.01)。综合分析研究结果表明,在IGFBP-1基因主要表达的肝脏组织中,其mRNA表达水平与DNA甲基化水平呈负相关,启动子区较高的甲基化可能抑制了二倍体泥鳅IGFBP-1基因的表达。

To understand the expression difference of IGFBP-1 between diploid and tetraploid Misgurnus anguillicaudatus,and their relationship with DNA methythylation,we cloned the CDS and promoter sequence of the IGFBP-1 gene,and analyszed the expression level and DNA methylatyion rate in diploids and tetraploids using qRT-PCR method and bisulfite sequencing technology (BSP),respectively.The results showed that,the CDS of IGFBP-1 was 789 bp,encoding 262 amino acid,and 5 base-pair difference but no amino acid difference were found between diploids and tetraploids.We cloned 2 341 bp and 2 331 bp promoter sequences in diploids and tetraploids,respectively,with the sequence similarity of 99%.The core transcription element TATA-box,and transcription factor binding sites such as SP1,CREB,C/EBP,POU2F2,GATA-2/3/4 and SRY were observed in the promoter.The IGFBP-1 gene was mainly expressed in the liver,and the expression level was significantly higher in tetraploids than in diploids ( P <0.01).The methylation level of promoter and 1 st exon were lower in teterploids than in diploids,expecially in the liver ( P <0.01).In conclusion,in the liver,the major expression tissue of the IGFBP-1 gene,the mRNA expression level and DNA methylation level were negatively correlated,suggesting that the higher DNA methylation rate in promoter may suppressed the expression of IGFBP-1 in the diploid M.anguillicaudatus.