糙皮侧耳乙醛脱氢酶基因PoALDH1的克隆与非生物胁迫下的表达分析

摘要

本研究从糙皮侧耳中克隆了乙醛脱氢酶基因PoALDH1(GenBank登录号为KT026035)和其全长cDNA序列.长为2 016bp的PoALDH1序列编码478个氨基酸,分子量约为52kDa,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.%Aldehyde dehydrogenase gene PoALDH1 and its full-length cDNA sequence were cloned from Pleurotus ostreatus.The PoALDH1 is 2 016bp encoding a putative protein of 478 amino acids.The amino acid sequence contains conservative glutamic acid and cysteine residue active sites of aldehyde dehydrogenase family.The measurement of aldehyde dehydrogenase activity and aldehyde tolerance by PoALDH1 gene expressing in Escherichia coil showed that ALDH specific activity of the recombinant strain was 0.58U/mg and acetalde tolerance of recombinant strain was significantly higher than that of control strain.The analysis of PoALDH1 differential expression between dicaryotic mycelium stage and primordial stage through quantitative real-time PCR (qRT-PCR) showed that the expression level in primordial stage was about four times higher than that in dicaryotic mycelium stage.The analysis of PoALDH1 differential expression under the stress of light or low temperature during primordial formation showed that PoALDH1 was significantly up-regulated.When exposed to different concentrations of sodium chloride or mannitol,the mycelial growth was inhibited and the expression level of PoALDH1 was significantly higher than that of the control ones.The research results will lay the foundation for both the further exploration of the stress resistant mechanism of Pleurotus ostreatus and the development of edible fungi at molecular level.