醇母PHO81基因的克隆和表达  

Cloning and Expression of Yeast PHO81 Gene

作　　者：陈金莲[1] 吴健生[1] 李江琪 敖世洲[1] 

机构地区：[1]中国科学院上海生物化学研究所分子生物学国家重点实验室

出　　处：《生物化学与生物物理学报》1994年第1期59-65,共7页

摘　　要：从酵母染色体ＤＮＡ中获得１个约３．０ｋｂ的ＢａｍＨＩ的克隆片段和１个约５．０ｋｂ的ＰｓｔＩ片段，前者包含１７４５ｂｐ的ＰＨＯ８１基因编码序列及１２４４ｂｐ的上游序列，后者包含２２３６ｂｐ的编码序列及约２．８ｋｂ的下游序列，经拼接得到完整的ＰＨＯ８１基因。以ＵＲＡ３基因取代部分ＰＨＯ８１的编码序列，通过体内同源重组，获得ＰＨＯ８１基因缺陷的酵母细胞株。构建ＰＨＯ８１－ＬａｃＺ融合基因，以β－半乳糖苷酶的活力表示ＰＨＯ８１基因的表达水平，研究了它的表达作用。ＰＨＯ８１基因为阻遏型表达，受无机磷浓度的控制，高磷使基因表达阻遏，低磷去阻遏。ＰＨＯ８１对其自身的表达有正调控作用，它与ＰＨＯ５和ＰＨＯ１１基因的调控模式相似，但ＰＨＯ８１上游调控序列和ＰＨＯ５及ＰＨＯ１１的同源性很低。Through in situ hybridization, a 3.0 kb BamHI fragment and a 5.0 kb PstI fragment were obtained from the yeast gene library. The BamHI fragment contains 1745 bp N-terminal coding sequence and 1244 bp upstream sequence of PHO 81 gene, the PstI fragment contains 2236 bp C-terminal coding region and 2 .8kb downstream sequence and their overlapping region is about 450 bp. We constructed the complete PHO 81 gene by ligating these two fragments. The part of coding region of PHO 81 gene was replaced with URA 3 gene and used as donor to transform YPH499 to URA 3. A PHO81 mutant resulted from disruption of the chromosomal counterpart. The coding region of PHO81 gene was fused in frame with Lac Z and assayed by β-galactosidase aetivity. The results show that the PHO81 gene is under the same transcriptional control as PHO5 and PHO11 genes. The PHO81 gene is transcribed at high level in media containing low amounts of inorganic phosphate (Pi). Transcription of the PHO81 gene is repressed when cells are supplied with large amounts of Pi in the media. Sequence comparisons revealed that the upstream sequence of PHO81 gene shared littlehomology with PH05 or PHO 11 genes.

关 键 词：酵母 PHO81基因 基因表达 

分 类 号：Q939.506[生物学—微生物学]