糙皮侧耳乳酸脱氢酶鉴定及其菌丝高温胁迫下表达特征分析

Identification of Lactate Dehydrogenase in Pleurotus ostreatus and Heat Stress Expression Analysis of Mycelium

乳酸脱氢酶(lactate dehydrogenase,LDH)可催化丙酮酸与乳酸(lactic acid,LA)之间的可逆转化,为探索乳酸脱氢酶基因在糙皮侧耳(Pleurotus ostreatus)菌丝体高温胁迫响应中的作用,在糙皮侧耳(P. ostreatus)菌株CCMSSC00389基因组中鉴定获得了8个乳酸脱氢酶编码基因。生物信息学分析结果显示,3个基因编码D型乳酸脱氢酶,5个基因编码L型乳酸脱氢酶。氨基酸序列比对、系统发育、三维结构和亚细胞定位分析显示,3个D型乳酸脱氢酶的同源性较高,可能属于细胞色素C依赖的D型乳酸脱氢酶,D-LDH2和D-LDH3位于线粒体中;5个L型乳酸脱氢酶的氨基酸序列同源性较低,并按电子受体和亚细胞定位的不同分为3个类型。通过检测36℃、40℃高温胁迫下乳酸脱氢酶基因的表达量变化,明确D-ldh3、L-ldh5和L-ldh7表达特征相同,L-ldh6和L-ldh8的表达特征相同,D-ldh1、D-ldh2和L-ldh4则表现出不同的基因表达趋势,表明乳酸脱氢酶基因对不同高温胁迫温度的响应方式存在差异。高温胁迫条件下,乳酸脱氢酶总酶活性降低,胞内乳酸含量升高;外源添加乳酸脱氢酶抑制剂和乳酸都能显著抑制菌丝生长,表明乳酸脱氢酶能够调控热胁迫下糙皮侧耳的菌丝生长。推测糙皮侧耳乳酸脱氢酶通过降低基因转录水平和酶活性,影响了胞内乳酸脱氢向丙酮酸的转化,导致胞内乳酸的过量积累造成细胞损伤,最终抑制了菌丝生长速率。

Lactate dehydrogenase(LDH)catalyzes the reversible conversion between pyruvate and lactic acid.In order to explore the role of LDH genes in heat stress response of Pleurotus ostreatus,eight coding genes of LDH were identified in the genome of P.ostreatus strain CCMSSC00389.The bioinformatics analyses showed that three genes encoded D-lactate dehydrogenase and five gene encoded L-lactate dehydrogenase.The results from the amino acid sequence alignment,phylogenetic analysis,three-dimensional structure and subcellular localization showed that three D-lactate dehydrogenases sharing a high homolog,which belonged to cytochrome C-dependent D-lactate dehydrogenase,and D-LDH2 and D-LDH3 were located in mitochondria.The five L-lactate dehydrogenases having a low homolog were divided into three different types according to electron acceptor and subcellular localization.The gene transcription changes of lactate dehydrogenase at 36℃and 40℃indicated the similar expression characteristics shared by D-ldh3,L-ldh5 and L-ldh7,as well as by L-ldh6 and L-ldh8;while the distinctive expression characteristics within D-ldh1,D-ldh2 and L-ldh4 was observed,which revealed varied response ways of the LDH genes to different stress temperatures.The total lactate dehydrogenase enzyme activity decreased and the intracellular lactic acid content increased under the heat stress.The inhibiting effects of lactate dehydrogenase inhibitor and exogenous lactic acid on the mycelia growth of P.ostreatus suggested that LDH regulated the mycelia growth under heat stress.It is speculated that P.ostreatus lactate dehydrogenase affects the conversion of intracellular LDH to pyruvate by reducing gene transcription level and enzyme activity,resulting in excessive accumulation of intracellular lactic acid and cell damage,and finally inhibits the growth rate of hyphae.