玉米弯孢叶斑病菌clt-1基因生物信息学分析和启动子的功能鉴定
发布时间：2018-08-08 21:37
【摘要】：利用农杆菌介导的基因转化(Agrobacterium-mediated transformation,ATMT)技术构建玉米弯孢叶斑病菌突变体库,并从中筛选到了一个毒素合成相关基因clt-1。对clt-1基因编码的蛋白质进行生物信息学分析,结果表明CLT-1的分子质量为81.984 8k Da,等电点p I为8.62,不稳定指数为55.08;CLT-1是亲水性蛋白,包含1个膜外区域。在亚细胞水平上,CLT-1定位于细胞核内;在氨基酸序列方面,CLT-1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,CLT-1包含BTB特征结构域,可能参与一些功能蛋白质活性的调控。利用GFP(green fluorescent protein)基因作为报告基因,构建了用于鉴定clt-1基因启动子活性的p C1300th-Pclt1-GFP真菌表达载体,采用ATMT方法转化弯孢菌萌发的分生孢子,通过PCR及GFP荧光检测clt-1基因启动子在弯孢菌中调控GFP基因表达的活性。结果表明,在共聚焦激光扫描显微镜下观察到菌丝和孢子发绿色荧光,说明弯孢菌clt-1基因启动子具有较强驱动外源gfp基因表达的活性。
[Abstract]:A mutant library of Curvularia corniculata was constructed by Agrobacterium-mediated transformation technique, from which a toxin synthesis-associated gene clt-1 was screened. The bioinformatics analysis of the protein encoded by clt-1 gene showed that the molecular weight of CLT-1 was 81.984 kDa, the isoelectric point I was 8.62, and the instability index was 55.08% CLT-1, which was a hydrophilic protein containing an extracellular region. At the subcellular level, CLT-1 is located in the nucleus; in amino acid sequence, the CLT-1 contains several threonine, serine and tyrosine kinase phosphorylation sites; and in the functional aspect, CLT-1 contains the characteristic domain of BTB. It may be involved in the regulation of the activity of some functional proteins. Using GFP (green fluorescent protein) gene as reporter gene, the expression vector of p C1300th-Pclt1-GFP fungi was constructed to identify the promoter activity of clt-1 gene. The conidia of Curvularia sprouting were transformed by ATMT method. PCR and GFP fluorescence were used to detect the activity of clt-1 promoter regulating GFP gene expression in Curvularia. The results showed that the green fluorescence of mycelium and spores was observed under confocal laser scanning microscope, which indicated that the promoter of clt-1 gene of Curvularia had strong activity to drive the expression of exogenous gfp gene.
【作者单位】： 上海交通大学农业与生物学院农业部都市农业(南方)重点开放实验室;上海交通大学国家微生物代谢重点实验室;
【基金】：国家自然科学基金(31471734) 国家玉米产业技术体系(CARS-02)资助项目
【分类号】：S435.13
[Abstract]:A mutant library of Curvularia corniculata was constructed by Agrobacterium-mediated transformation technique, from which a toxin synthesis-associated gene clt-1 was screened. The bioinformatics analysis of the protein encoded by clt-1 gene showed that the molecular weight of CLT-1 was 81.984 kDa, the isoelectric point I was 8.62, and the instability index was 55.08% CLT-1, which was a hydrophilic protein containing an extracellular region. At the subcellular level, CLT-1 is located in the nucleus; in amino acid sequence, the CLT-1 contains several threonine, serine and tyrosine kinase phosphorylation sites; and in the functional aspect, CLT-1 contains the characteristic domain of BTB. It may be involved in the regulation of the activity of some functional proteins. Using GFP (green fluorescent protein) gene as reporter gene, the expression vector of p C1300th-Pclt1-GFP fungi was constructed to identify the promoter activity of clt-1 gene. The conidia of Curvularia sprouting were transformed by ATMT method. PCR and GFP fluorescence were used to detect the activity of clt-1 promoter regulating GFP gene expression in Curvularia. The results showed that the green fluorescence of mycelium and spores was observed under confocal laser scanning microscope, which indicated that the promoter of clt-1 gene of Curvularia had strong activity to drive the expression of exogenous gfp gene.
【作者单位】： 上海交通大学农业与生物学院农业部都市农业(南方)重点开放实验室;上海交通大学国家微生物代谢重点实验室;
【基金】：国家自然科学基金(31471734) 国家玉米产业技术体系(CARS-02)资助项目
【分类号】：S435.13
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