摘要
利用原位杂交方法从野生型酵母菌染色体ＤＮＡ中克隆了ＰＨＯ８０基因，全长约４．２ｋｂ，包括１．１ｋｂ的上游序列和８７９ｂｐ的编码序列。以ＵＲＡ３基因为筛选标记，通过体内同源重组和营养互补筛选获得了ＰＨＯ８０基因缺失突变株。进一步研究了ＰＨＯ８０在细胞内的功能，它是酵母阻遏型酸性磷酸酯酶结构基因以及调控基因ＰＨＯ８１表达的负调控因子，但不影响ＰＨＯ４和ＰＨＯ８５的表达。还构建了ＰＨＯ８０－ＬａｃＺ融合基因，探索它在细胞内的表达规律。β－半乳糖苷酶活力测定结果表明，ＰＨＯ８０基因在细胞内呈低水平表达，并受自身产物和ＰＨＯ８５的抑制，推测它与ＰＨＯ５和ＰＨＯ８１基因的调控模式可能相似。
Through in situ hybridization,a 4.2 kb PstⅠ-BamH Ⅰfragment was obtained from S.cerevisiae gene library.The cloned fragment contained 1100 bp upstream sequence and 879 bp coding sequence of the PHO80 gene.Coding region of PHO80 gene was substituted with URA3 gene and used as donor to transform YPH499 to URA3.A pho80 mutant resulted from deletion of the chromosomal counterpart in PHO80 was oblained.In vivo functional study of the PHO80 gene indicated that PHO80 was a negative regulator in the Pi-repressible acid phosphatase system including the structural genes PHO5 and PHO11 and the regulatory gene PHO81,whereas the expression of PHO4 or PHO85 was independent of PHO80.The coding region of PHO80 was fused in frame with LacZ and β-galactosidase activities in various cells was deteimined.The results Showed that the PHO80 gene was expressed at a low level and suppressed by itself and PHO85.
