Cloning and Bioinformatic Analysis of 6-Phogluconate Dehydrogenase Gene from Aspergillus oryzae
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摘要: 采用PCR技术从米曲霉CICC2012菌株基因组中克隆6-磷酸葡萄糖酸脱氢酶基因(gnd),并利用生物信息学手段对其氨基酸序列、进化树、理化性质、蛋白质结构等进行分析。序列测定和分析结果表明,gnd基因序列长为1 723 bp,包含1个1 551 bp的开放阅读框,编码516个氨基酸;gnd基因编码的6PGDH氨基酸序列与黄曲霉6PGDH基因的同源性为99%,存在的丝氨酸、苏氨酸和酪氨酸磷酸化位点分别有11、2和6个;6PGDH蛋白分子量为57.3 kD,等电点为5.63;gnd基因编码蛋白二级结构α-螺旋区域占44.57%,β-折叠区域占12.79%,无规则卷曲区域占42.64%;氨基酸残基11~195位点为NADP+结合区域。
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关键词:
- 米曲霉 /
- 6-磷酸葡萄糖酸脱氢酶 /
- 克隆 /
- 生物信息学
Abstract: 6-Phogluconate dehydrogenase (6PGDH, EC 1.1.1.44)is one of the key enzymes in the pentose phosphate pathway(PPP). In this study, the 6-phogluconate dehydrogenase gene (gnd) of Aspergillus oryzae CICC2012 was cloned by means of PCR. Subsequently, the bioinformatic methodology was applied to determine the amino acid sequence homology, phylogenetic trees, physical-chemical properties and protein structure of the gene. The results revealed the gene’s length to be 1 723 bp, which encompassed an open reading frame with 1 551 bp encoding 516 amino acids. The 6PGDH encoded by gnd showed a 99% homology with the 6PGDH gene of Aspergillus flavus, which had 11 serine phosphorylation sites, 2 threonine phosphorylation sites and 6 tyrosine phosphorylation sites. The 6PGDH had a molecular weight of 57.3 kD and an isoelectric point of 5.63. The secondary structure of the gnd protein was 44.57% alpha helix, 12.79% beta sheet and 42.64% random coil. The 11-195 amino acid residues appeared to be the NADP+ binding sites.-
Keywords:
- Aspergillus oryzae /
- 6-phosphate glucose dehydrogenase /
- cloning /
- bioinformatics
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