摘要： 为明确玉米大斑病菌中是否存在漆酶以及影响酶活性的因素,以ABTS为底物采用分光光度计测定了420nm下不同Cu2+浓度和不同缓冲液pH值条件下的玉米大斑病菌胞内漆酶活性.根据子囊菌已知漆酶Cu2+结合位点的保守区域设计简并引物,以玉米大斑病菌cDNA为模板扩增漆酶基因的特异片段.结果表明,玉米大斑病菌中存在漆酶,且漆酶活性与Cu2+浓度和缓冲液pH值相关,pH值为2.6,Cu2+浓度为20 pmol⁄L时漆酶活性最高;通过PCR 技术克隆获得了一个929 bp的基因片段,生物信息学分析发现,该基因序列含有漆酶基因保守的Cu-oxidise superfamily,与其他子囊菌中漆酶的氨基酸序列具有60%以上的相似性.以上结果为进一步研究玉米大斑病菌的漆酶功能奠定了基础.

关键词: 玉米大斑病菌, 漆酶, ABTS, 克隆

Abstract: The object of this paper is to examine the existence of laccase in Setosphaeria turcica and influencing factors on enzyme activity.The laccase activity of S.turcica was tested by spectrophotometer under 420 nm with ABTS(2,2：azino-bis(3-ethyl-2,3·dihydro-1 3 benzthiazole-6-sulfonate))as the substrate,and the influence of Cu“concentration and reaction pH value on the enzyme activity were studied.A pair of degenerate primers was de- signed according to the conserved Cu2+domain of other fungal laccases and homologous fragment of laccase gene was cloned by polymerase chain reaction with cDNA as template.This study demonstrated that there was laccase in S. turcica and the laccase activity was related to copper concentration and pH.The results showed that optimum condi- tions were pH2.6 and 250 trmol/L CuS04 respectively.In our research,a 929 bp fragment was obtained from S.turcica.,named as StLacl,which shared over 60%identity with other fungus laccase and contained Cu-oxidise superfam- ily.This work is helpful to study and understand laccase function of S.turcica.

Key words: Setosphaeria turcica, Laccase, ABTS, Clone

中图分类号: 

• S432.1