湖北白猪IGF-1基因的克隆及序列分析

目的]以湖北白猪肝脏作为试验材料,克隆出湖北白猪胰岛素生长因子（IGF-1）基因,并对其序列进行分析。方法]采用Trizol法从湖北白猪的肝脏中提取总RNA,将其作为RT反应的模板,用P2（5＇-CAGGTAACTCGTGCAGAGCAAAGGA-3＇）引物合成IGF-1基因cDNA第一链,以其产物为模板,再以P1（5＇-CCCATCTCCCTGGATTTCTTTTTG-3＇）和P2为上下游引物扩增到大小约为607bp的产物,并将其克隆至pCRII载体上,经蓝白斑筛选、酶切、测序。结果]经筛选、酶切、序列分析,表明该片段为IGF-1基因的cDNA克隆,它由607个核苷酸组成,其中1～145位是5＇端非翻译区,从第146位ATG起始密码子开始至第538位TAG终止密码子,539～607位是3＇端非翻译区,包含一完整的ORF,该ORF共有393核苷酸,编码一个推断由130个氨基酸组成的多肽。与GenBank中的序列比较,该序列与Muller等报道的猪IGF-1基因编码序列完全同源。结论]成功克隆了湖北白猪IGF-1基因,并对其进行序列分析,证实了IGF-1基因的高度保守性,为采用转基因技术对湖北白猪进行育种奠定了基础和提供了技术依据。

Cloning and Sequence Analysis on IGF-1 Gene of Hubei White Swine

Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 (IGF-1) gene from liver of Hubei white swine. Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5’-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3’-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine.