过表达漆酶基因Lelcc1的香菇菌株构建及表型分析

Overexpression of a laccase gene Lelcc1 and phenotypic characterizations in Lentinula edodes

真菌漆酶(laccase)是一种多酚氧化酶,在真菌生长发育中具有重要作用。本研究采用根癌农杆菌介导转化的方法,以香菇Lentinulaedodes菌株W1为受体菌株,在Leactin基因启动子调控下过表达Lelcc1基因;对其中7个单拷贝插入的转化子进行qRT-PCR分析,7个Lelcc1基因表达量较出发菌株W1均上调了1.5–8倍。进一步分析了这7个转化子的遗传稳定性;挑取了3个稳定的转化子进行表型分析,主要包括不同培养基中的生长速度、代料栽培过程中菌棒转色程度、以及透射电镜观察菌丝细胞的超微结构。发现转化子和受体菌株W1的生长速度无显著差异,但代料栽培过程中转化子较W1转色更快,菌棒表面颜色更深;透射电镜观察菌丝细胞的超微结构发现在细胞壁厚度、细胞膜形态等方面,其中两个超量表达转化子与W1间存在显著差异。结果表明,香菇Lelcc1基因可能参与了转色过程中色素合成或积累,也可能与细胞壁形成有关。

Fungal laccase is an important polyphenol oxidase which plays important roles in fungal development.In the present study, the shiitake mushroom Lentinula edodes strain W1 was used as acceptor, and a laccase gene Lelcc1 was overexpressed under the control of actin promoter. The vector was transferred into L. edodes mycelia via Agrobacterium tumefaciens-mediated transformation method. Seven transformants were single-copy inserted,and qRT-PCR analysis showed that the expression of Lelcc1 in these seven transformants was 1.5–8 times higher than that in wild type strain W1. The genetic stability of these seven transformants were further analyzed,and three stable transformants were selected for phenotypic analysis including growth rate in different media,browning degree of substitution cultivation bags, and the ultrastructure of mycelial cells observed by transmission electron microscopy. The results indicated that the mycelium growth rate of three overexpression transformants growing in different medium was not significantly different from that of wild type strain. In sawdust-based cultivation,the transformants browned faster and the surface color was darker than W1. Significant differences were detected in the cell wall thickness and cell membrane morphology between the two overexpressed transformants and W1.These results indicated that Lelcc1 gene of L. edodes might be involved in the synthesis or accumulation of pigments during the process of brown film formation, and might also be related to the formation of cell wall.