GC含量丰富的人Ⅰ型原胶原蛋白基因片段克隆及其序列分析

鉴于做转基因牛羊乳腺生物反应器的需要,根据GenBank中所公布的人Ⅰ型原胶原蛋白基因序列设计引物,利用健康人的胎盘中提取的基因组DNA为模板,通过优化的长距离PCR,成功地克隆出GC含量丰富的人Ⅰ型原胶原基因组DNA片段,序列测定与分析的结果显示中国人Ⅰ型原胶原蛋白基因组DNA片段与GenBank中所公布出来的序列所对应的部分有99.5%的同源性,其中外显子部分与GenBank中的对应部分完全一致;而在内含子中,本研究克隆到的基因与GenBank中公布出来的序列有不少差异,尤其表现在内含子1和2中,其中内含子2要比GenBank中公布的基因内含子2多出14个碱基,这14个碱基具体有什么作用尚需要进一步阐明,内含子间的差异凸显了东西方人种之间基因序列的多态性.

Cloning and Sequencing of Human COL1A1 Gene Fragment with High GC Content

According to the human COL1A1 sequence reported in GenBank,primers were designed,and human COL1A1 gene fragment with high GC content was amplified by optimized long-distance PCR with healthy human placenta genomic DNA as template DNA.The cloned fragment was inserted into vector and sequenced.Sequencing result showed that Chinese COL 1A1 fragment was 99.5% identical to the counterpart of the gene sequence reported in GenBank(Access NO.AF 017178).Their exons were completely identical;in introns,there were many differences between Chinese COL1A1 fragment and the sequence in GenBank.The discrepancy was especially obvious in intron1 and intron2.Nucleotides in intron2 of Chinese COL1A1 were more than in inton2 reported in GenBank by 14 bases.The role of these nucleotides should be unraveled further.Differences in introns also demonstrated the sequence polymophrism between Chinese and Caucasian.