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本研究从糙皮侧耳中克隆了乙醛脱氢酶基因PoALDH1(Gen Bank登录号为KT026035)和其全长c DNA序列。长为2 016bp的PoALDH1序列编码478个氨基酸,分子量约为52k Da,氨基酸序列中含有乙醛脱氢酶保守的谷氨酸活性位点和半胱氨酸残基活性位点。PoALDH1基因在大肠杆菌中表达后显示,重组菌株的乙醛脱氢酶比活力为0.58U/mg,且重组菌株的乙醛耐受性显著高于对照菌株。实时荧光定量PCR(quantitative real-time PCR)分析PoALDH1基因在糙皮侧耳不同发育时期的表达结果显示,PoALDH1基因在原基期的表达量约为双核菌丝期的5倍。PoALDH1基因在原基发育起始阶段经光照及温差刺激后均上调表达。当糙皮侧耳菌丝暴露在不同浓度的氯化钠及甘露醇的条件下时,菌丝生长均受到抑制且PoALDH1基因的表达量均高于对照。研究结果将为进一步从分子水平揭示糙皮侧耳的抗逆机制以及食用菌的发育奠定一定基础。
In this study, we cloned the aldehyde dehydrogenase gene PoALDH1 (GenBank accession number KT026035) and its full-length cDNA sequence from Pleurotus ostreatus. The 2 016 bp PoALDH1 sequence encodes 478 amino acids with a molecular weight of about 52 kDa. The amino acid sequence contains the glutamic acid active site and the cysteine residue active site conserved by acetaldehyde dehydrogenase. After the expression of PoALDH1 gene in E. coli, the specific activity of acetaldehyde dehydrogenase of the recombinant strain was 0.58U / mg, and the acetaldehyde tolerance of the recombinant strain was significantly higher than that of the control strain. Quantitative real-time PCR analysis of PoALDH1 gene expression in different developmental stages of Pleurotus ostreatus showed that the expression level of PoALDH1 in primordial stage was about 5 times higher than that of the double-mycelium stage. The PoALDH1 gene was up-regulated after light and temperature difference in the initial stage of primordium development. When the Pleurotus ostreatus exposed to different concentrations of sodium chloride and mannitol conditions, mycelial growth was inhibited and PoALDH1 gene expression were higher than the control. The results will lay a solid foundation for further revealing the anti-retrogradation mechanism of Pleurotus ostreatus at the molecular level and the development of edible fungus.