Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter
Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter
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DOI：
10.1002/yea.2946
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发表时间：
2013-03
期刊：
影响因子：
2.6
通讯作者：
Cheng Zhang;B. Zhuge;X. Zhan;H. Fang;H. Zong;J. Zhuge
中科院分区：
文献类型：
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作者：
Cheng Zhang;B. Zhuge;X. Zhan;H. Fang;H. Zong;J. Zhuge
使用简并引物克隆了产甘油假丝酵母 (Candida gearinogenes) WL2002-5 的 3950 bp 基因组片段，该片段包含编码甘油醛-3-磷酸脱氢酶的 CgGAP 基因，该基因与使用简并引物的其他酵母中的 GAP 基因同源。序列分析揭示了一个 1164 bp 的开放阅读框，编码由 387 个推导氨基酸组成的推定肽，分子量为 36 kDa。 CgGAP 蛋白由 N 端 NAD+ 结合结构域和中央催化结构域组成。在 CgGAP 基因的上游区域发现了 6 个应激反应元件。研究了 CgGAP 对糖酵解的影响。功能分析表明，用 CgGAP 转化的酿酒酵母在高渗透培养基中培养时恢复到野生型表型，表明它是一种功能性 GAP 蛋白。使用绿色荧光蛋白 (gfp) 基因作为报告基因在酿酒酵母中进行的启动子研究表明，GAP 启动子 (PCgGAP) 在葡萄糖上生长的酿酒酵母细胞中组成型表达。版权所有 © 2013 约翰·威利父子有限公司
A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD+‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (PCgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.