Manganese Peroxidase Gene Expression: Regulation by Mn

白腐担子菌黄孢原毛平革菌的木质素降解系统能够降解植物细胞壁聚合物、木质素和一系列芳香族污染物。这个非特异性氧化系统的主要成分是两种胞外含血红素的过氧化物酶，即锰过氧化物酶(MNP)和木质素过氧化物酶(LIP)。LDS在次生代谢期表达，次生代谢期由限制营养氮启动。最近，我们发现锰离子和热休克也在基因转录水平上调控着MNP的表达，本课题的主要目的是从分子水平上阐明锰对MNP基因转录的调控机制。将利用突变体的分离和分析以及启动子拷贝的滴定来确定调控类型。利用启动子-报告基因构建、缺失分析和合成启动子元件，对黄孢原毛霉MNP基因的启动子区域进行分析，以确定与转录的锰和热休克调控有关的序列。DNaseI足迹和凝胶迁移率改变分析将用于检测可能的DNA结合转录因子，这些因子将被分离出来。在转录后事件中对锰的可能需求将被研究。

The lignin degradative system (LDS) of the white rot basidiomycetous fungus Phanerochaete chrysosporium is capable of degrading the plant cell wall polymer, lignin, and a range of aromatic pollutants. The major components of this nonspecific oxidative system are two extracellular heme-containing peroxidases, manganese peroxidase (MnP) and lignin peroxidase (LiP). The LDS is expressed during secondary metabolic growth, the onset of which is triggered by limiting nutrient nitrogen. Recently, we discovered that MnP expression is also regulated at the level of gene transcription by Mn ion and heat shock and the primary goal of the proposed project is to elucidate on the molecular level the mechanisms of Mn regulation of mnp gene transcription. Isolation and analysis of mutants and titration with promoter copies will be utilized to determine the type of regulation. The promoter regions of P. chrysosporium mnp genes will be analyzed to determine the sequences responsible for Mn and heat shock regulation of transcription, using promoter-reporter constructs, deletion analysis and synthetic promoter elements. DNaseI footprinting and gel mobility shift assays will be used to assay putative DNA- binding transcription factors, and the factors will be isolated. A possible requirement for Mn in post-transcriptional events will be examined.