奶山羊β-乳球蛋白基因及其调控区的克隆和序列分析

CLONING AND SEQUENCING OF β-LACTOGLOBULIN GENE OF DAIRY GOATS

从奶山羊组织提取基因组 DNA,根据已发表的山羊 β-乳球蛋白基因 (BLG)序列设计引物 ,用高保真长模板PCR法克隆得 6个奶山羊 BLG基因片段。这 6个基因片段的大小和部分限制性酶切图谱与已发表的山羊 BLG基因相同。部分序列测定结果显示 :6个奶山羊 BLG基因片段与山羊 BLG基因相应片段的同源性为 99%。进一步序列测定结果表明 :奶山羊 BLG基因 5′调控区与山羊、绵羊和牛的同源性分别为 99%、95%和 90 % ,3′调控区与这些动物的同源性分别为 99%、97%和 92 %。在乳腺特异表达调控元件集中的 -4 0 6bp区域内 ,奶山羊 BLG基因与山羊 BLG基因有 2个碱基的差异。所克隆的奶山羊 BLG基因调控区与山羊 BLG伪基因显著不同 ,其同源性仅为 83 % (上游调控区 )和88% (下游调控区 )。这些结果表明 :不同种动物的 BLG基因是高度保守的 ,高保真 PCR克隆的奶山羊

To construct mammary gland specific expression vectors, 6 β lactoglobulin (BLG) gene fragments of expected sizes were amplified from dairy goat genomic DNA using high fidelity long template PCR system. The 6 gene fragments were subcloned into pGEM T for restriction mapping and sequencing. Partial sequence analyses showed that all 6 gene fragments were 99% identical to goat BLG gene. Further sequencing showed that the 5′ end regulatory region of the dairy goat gene was 99%, 95% and 90% identical to that of goat, sheep and cattle BLG gene, while the 3′ end regulatory region was 99%, 97% and 92% identical to that of the above three genes, respectively. Over the mammary gland specific expression controlling region, the dairy goat gene differed from that of goat by only two bases. The dairy goat gene differed significantly from goat BLG pseudo gene with a homology of only 83% (upstream region) and 88% (downstream region), respectively. These results indicate that BLG gene was highly conserved among different animal species, and that the high fidelity PCR cloned regulatory regions were usable for constructing mammary specific expression vectors.