新月旋孢腔菌胞内漆酶的活性测定及漆酶基因ClLac1克隆

漆酶是一种含铜的多酚氧化酶，与植物病原菌致病性、黑色素合成及降解木质素等方面相关。为明确漆酶在新月旋孢腔菌的催化作用及其催化活性，以2,2′-连氮-双(3-乙基苯并噻唑-6-磺酸)（简称ABTS）为底物，利用分光光度计在420nm下测定胞内漆酶活力，结果表明酶活测定最佳反应条件为缓冲液pH2.8、Cu2+浓度500μmol/L和0.6mmol/L ABTS。根据漆酶Cu2+结合保守结构域设计了1条引物，对新月旋孢腔菌漆酶基因进行克隆，并通过RACE技术克隆了其全长cDNA序列。开放阅读框长1,803bp，

Intracellular laccase activity assays and cloning of laccase gene from Cochliobolus lunatus

Laccases are copper-containing phenol oxidases which are involved in plant pathogenesis,pigment synthesis,and delignification.In order to understand the catalysis and catalytic activity in Cochliobolus lunatus,the intracellular laccase activity of the fungus was tested by spectrophotometer under 420nm with 2,2’-azobis(3-thylbenzothiazole-6-sulfonaic acid)(ABTS) as the substrate and the absorbance results suggest that the optimal reaction conditions were pH 2.8,500μmol/L Cu2+ and 0.6μmol/L substrate concentration.The homologous fragments of ClLac1 gene was cloned by PCR amplification with degenerated primer sets designed around the copper-binding-sites.Its full length cDNA sequence was obtained by RACE method with 1,803bp encoding 600 amino acids and the deduced protein amino acids were highly identitical with the laccases of other fungi.It contained four conserved copper-binding sites,eight putative N-glycosylation sites and a signal peptide sequence was found in the N-terminal by bioinformatics analysis.The promoter region contained one MRE(metal responsive element),one STRE(stress responsive element),two CreA-binding sites,four nitrogen factor binding sites.