奶山羊β-乳球蛋白基因5＇、3＇调控区的克隆和序列分析

目的：克隆奶山羊β-乳球蛋白（BLG）基因5＇、3＇调控区,并对其进行序列分析。方法：从徐淮奶山羊组织中提取基因组DNA,采用高保真长模板PCR法克隆得到奶山羊BLG基因4.2kb的5＇调控区和1.8kb的3′调控区;将纯化后的PCR产物克隆到pGEM-TEasy载体中,经酶切鉴定和序列测定验证克隆的正确性。结果：部分序列分析表明,奶山羊BLG基因5＇区与GenBank中登录的山羊和绵羊BLG基因5＇区的同源性分别为100%和95%,3＇区的同源性分别为99%和93%;克隆得到的奶山羊BLG基因调控区与山羊BLG伪基因显著不同,二者5＇区和3＇区的同源性分别为83%和88%。结论：克隆得到的奶山羊BLG基因调控区可用于构建乳腺特异性表达载体,用于外源基因在转基因动物乳腺中的高效表达。

Cloning and Sequencing of Goat β-Lactoglobulin Gene 5＇ and 3＇ Flanking Region

Objective：To clone and analyze the sequence of dairy goat β-lactoglobulin（BLG） gene 5＇ and 3＇ flanking region.Methods：The 4.2 kb 5＇ end regulatory region and 1.8 kb 3＇ end regulatory region of goat BLG gene fragments were amplified from goat genomic DNA by high fidelity long template PCR system.These two fragments were subcloned into pGEM-T Easy vector for restriction mapping and sequence analysis.Results：Partial sequence analysis showed that dairy BLG gene 5＇ end regulatory region were 100% and 95% identical to goat BLG gene and sheep BLG gene in GenBank,respectively,while the 3＇ end regulatory region was 99% and 93% respectively.The dairy goat gene differed significantly from goat BLG pseudogene with a homologyofonly 83%（upstreamregion） and 88%（downstreamregion）,respectively.Conclusion：The dairy goat BLG gene cloned can be uesd to construct mammary gland-specific expression vectors,and it offers a reliable guidance to the further study of highly experssing foreign gene in mammary gland of transgenic animal.