草菇锰过氧化物酶编码基因生物信息学分析及其转录水平和酶活性的测定

Bioinformatic,gene expression and enzyme activity analyses of manganese peroxidases in Volvariella volvacea

在草菇全基因组中发现了4个担子菌锰过氧化物酶MnP基因同源物(vv-mnp1、vv-mnp2、vv-mnp3和vv-mnp4)的基础上,对这4个MnP进行了生物信息学分析,并对其基因表达和酶活性进行测定。结果表明vv-mnp1与vv-mnp2基因结构相同,vv-mnp3与vv-mnp4基因结构相似。在4个草菇MnP基因启动子区域存在AP-2、cAMP、MRE、XRE和HSE等调控元件,预示着草菇中MnP基因的转录可能会受到氮源、碳源、重金属离子、芳香族化合物以及热击的影响。草菇MnP具有信号肽,不存在跨膜区域,表明其是分泌蛋白。草菇MnP中形成4个二硫键的8个半胱氨酸、潜在的锰离子和钙离子结合位点,在担子菌MnP中高度保守。系统进化树分析显示草菇MnP1和MnP2与香菇MnP1亲缘关系最近,而MnP3和MnP4与灰盖鬼伞过氧化物酶有较近的亲缘关系。RT-PCR检测到vv-mnp1、vv-mnp3与vv-mnp4能在含锰离子的培养基中表达,但运用锰离子氧化法没有检测到MnP活性,推测草菇中的MnP不具有将Mn2+氧化成Mn3+的能力。

We identified four putative manganese peroxidases （MnP） in the genome of the straw mushroom Volvariella volvacea, and further analyzed their bioinformatic traits. The mnp gene expression and enzyme activity analyses were also conducted in this study. The upstream region of mnp genes of V. volvacea contains putative AP-2, cAMP, MRE, XRE and HSE response elements, which are known to be involved in regulation of carbon, nitrogen, heavy metal ions and aromatic compounds. Each MnP protein has a signal peptide but without transmembrane helices, suggesting they are the secretory proteins. MnP protein sequences all contain the conserved sites related to Mn2＋ and Ca2＋ binding and disulfide bond formation. Phylogenetic analysis indicated that MnP1 and MnP2 of V. volvacea are close to the MnP1 ofLentinula edodes, but MnP3 and MnP4 are closely related to the peroxidase of Coprinopsis cinerea. The vv-mnpl, vv-mnp3 and vv-mnp4 genes were transcribed by the fungus during the mycelial growth by using RT-PCR. However, the V. volvacea MnP activity was not detected, indicating that V. volvacea MnPs lacked of the ability to oxidize Mn2＋ to Mn3＋.