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- 2019
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
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Abstract:
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789 bp，编码262个氨基酸，二倍体、四倍体泥鳅间有5 bp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2 341 bp和2 331 bp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 1st 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.
