香菇漆酶高产菌株筛选及漆酶基因的表达研究

Screening of high‐yield laccase producing Lentinula edodes strains and transcriptional analysis of laccase genes

漆酶是一种含铜的多酚氧化酶,其作用底物范围广,在食品、能源和环保等领域具有重要的应用价值,因此筛选高产漆酶菌株对于其工业应用具有重要意义。本实验以78株香菇菌株为实验材料,利用愈创木酚平板法初筛,液体发酵复筛的方法筛选高产漆酶香菇菌株,并通过实时定量PCR分析香菇漆酶同工酶在不同菌株之间转录表达的差异性和特异性。结果表明,不同香菇菌株的生长速度、变色圈大小存在差异,以菌丝圈和变色圈大小及其比例为依据,从78株香菇菌株中筛选出19株潜在的漆酶高产菌株;液体发酵从这19株菌株中复筛出3株高产漆酶的菌株:香240、15和冕宁浅灰,其中香240在培养第3天酶活达到最高(3.583U/m L),香15和冕宁浅灰则在培养第7天酶活达到最高(分别为3.842U/m L和2.806U/m L);实时荧光定量PCR结果显示,10个漆酶同工酶基因在两株香菇菌株中的表达存在差异性和特异性,其中Lelac5和Lelac9只在菌株香240中检测到转录本;Lelac1–4在菌株15中表达量较高,而Lelac8、Lelac10和Lelac11在香240中的表达量较高。研究结果将为香菇漆酶的进一步研究提供基础。

Laccase is a type of multi-copper-containing polyphenol oxidases capable of oxidizing a wide range of substrate, and has important values in fields of food, energy, environmental protection and so on. Therefore screening of high-yield producing strains is important for industrial applications. The present work was carried out to screen high-yield producing strains from Lentinula edodes. PDA plates with guaiacol was used to preliminarily screen laccase producing accase accase strains from 78 L. edodes isolates according to growing characteristic of colony, and further screening was carried out by submerged fermentation. The specific or differential expression profiles of laccases in two different strains were analyzed by real-time quantitative PCR （qRT-PCR）. The results showed that changes of oxidized and hyphal zones varied among strains. Ninteen strains were selected based on the sizes of oxidized and hyphal zones. Further screening showed that three strains, Xiang 240, 15 and Mianningqianhui, displayed highest level of laccase activity at 3.583U/mL （on the 3rd day）, 3.842U/mL （on the 7th day） and 2.806U/mL （on the 7th day）, respectively. The results of qRT-PCR showed that differential and specific expression profiles of ten L. edodes laccase genes were observed in two strains, of which two genes were specifically expressed in Xiang 240, and four genes （Lelac.Z-4） displayed higher expression levels in strain 15, while three genes （Lelacg, Lelac.ZO and lelac.Z.Z） were higher in Xiang240. These results provide fundamental data for further study of laccase from L. edodes