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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。
Two NADPH oxidase genes ClNox1 and ClNox2 were obtained from the genome of JGI database Curvularia lunata. Bioinformatics methods were used to predict the nature, structure and function of the two proteins encoded by the gene. The results showed that the molecular weights of ClNox1 and ClNox2 were 6.35 and 6.41 KD, respectively. The isoelectric point PI was 8.87 and 8.93, and the instability index was 44.50 and 39.45. ClNox1 was a hydrophilic membrane protein and contained 6 distinct transmembrane domains. ClNox2 is also a membrane protein that contains eight distinct transmembrane domains. At the subcellular level, ClNox1 is located on the mitochondria and ClNox2 is localized on the plasma membrane. In terms of amino acid sequence, both ClNox1 and ClNox2 contain multiple sites of threonine, serine and tyrosine kinase phosphorylation; in terms of function , ClNox1 and ClNox2 both contain the NADPH oxidase signature domain and have high homology with the NADPH oxidase gene of Cochliobolus heterostrophus. According to the sequence of ClNox1 and ClNox2, specific primers were designed to amplify the target fragment, and inserted into the backbone vector pPZP100 with the marker genes NPTII and HphGFP respectively to construct the knockout vectors pPZP100NPTIIClNox1 and pPZP100HphGFPClNox2 which are ClNox1 and ClNox2 genes, which are mediated by Agrobacterium tumefaciens Conversion to provide basic materials.