玉米弯孢叶斑病菌NADPH氧化酶生物信息学分析和ATMT敲除载体的构建

从JGI数据库玉米弯孢叶斑病菌(Curvularia lunata)基因组中获得2个编码NADPH氧化酶基因ClNox1和ClNox2。采用生物信息学方法对2个基因编码的蛋白质进行性质、结构和功能等方面的预测。结果表明,ClNox1和ClNox2的分子量分别为6.35和6.41 KD,等电点PI为8.87和8.93,不稳定指数为44.50和39.45;ClNox1是亲水性膜蛋白,包含6个明显的跨膜结构域;ClNox2也是膜蛋白,包含8个明显的跨膜结构域。在亚细胞水平上,ClNox1定位于线粒体上,而ClNox2定位于细胞质膜上;在氨基酸序列方面,ClNox1和ClNox2都含有多个苏氨酸、丝氨酸和酪氨酸激酶磷酸化位点;在功能方面,ClNox1和ClNox2都包含NADPH氧化酶特征结构域,并且与Cochliobolus heterostrophus的NADPH氧化酶基因具有较高的同源性。根据ClNox1和ClNox2序列设计特异性引物扩增目的片段,与标记基因NPTII和HphGFP分别连入骨架载体pPZP100,构建完成ClNox1和ClNox2基因的敲除载体pPZP100NPTIIClNox1和pPZP100HphGFPClNox2,为根癌农杆菌介导的遗传转化提供基础材料。

Bioinformatic Analysis and Deletion Vector Construction of Curvularia lunata NADPH Oxidase

Two NADPH oxidase genes ClNox1 and ClNox2 within Curvularia lunata genomic sequence were found. Bioinformatic analysis showed that the molecular weight of the protein encoded by ClNox1 and ClNox2 was 6.35 and 6.41 kD respectively, and the theoretical isoelectric point pI was 8.87 and 8.93. ClNox1 was a hydrophilic membrane protein, containing six distinct transmembrane helix topologies, and ClNox2 also was a membrane protein, containing eight distinct transmembrane helix topologies. At subcellular level, ClNox1 protein located in the mitochondria, ClNox2 protein located in the cytoplasmic membrane. More than one of the serine, threonine and tyrosine kinase phosphorylation sites in ClNox1 and ClNox2 amino acids sequence were found. In the function, ClNox1 and ClNox2 protein contained a typical conserved NADPH oxidases function domain, and was highly of homology to NADPH oxidases of Cochliobolus heterostrophus. Vector construction was based on the skeleton of pPZP100, HphGFP, NPTII and flanking sequences of ClNox1 and ClNox2 of C.lunata were linked into pPZP100 by restriction enzymes digested and ligase connection. Deletion vector pPZP100NPTIIClNox1 and pPZP100HphGFPClNox2 of C.lunata NADPH oxidases(ClNox1 and ClNox2) had been constructed and the basis of Agrobacterium tumefaciens-mediated transformation(ATMT).