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  

Bioinformatic Analysis and Promoter Identification of clt-1 Gene in Curvularia Lunata

作　　者：倪璇[1,2] 高金欣[1,2] 余传金[1,2] 刘铜[1,2] 李雅乾[1,2] 陈捷[1,2] 

机构地区：[1]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200240 [2]上海交通大学国家微生物代谢重点实验室,上海200240

出　　处：《中国生物工程杂志》2017年第3期37-42,共6页China Biotechnology

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

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。The clt-1 gene within Curvularia lunata genomic sequence was screened through Agrobacteriummediated transformation（ ATMT） method. Bioinformatic analysis showed that the molecular weight of the protein encoded by clt-1 was 81. 984 8k Da,the oretical isoelectric point p I was 8. 62,and the instability index was 55. 08. CLT-1 was a hydrophilic protein,containing one transmembrane helix topologies. At subcellular level,CLT-1 protein located in the nucleus. More than one of the serine,threonine and tyrosine kinase phosphorylation sites in CLT-1 amino acids sequence were found. In the aspect of its function,CLT-1 protein contained a typical conserved BTB function domain,which may regulate the expression of some functional protein. The expression vector pC1300th-Pclt1-GFP was constructed for detecting its promoter functional activity by the fusion of clt-1 promoter fragment to the reporter GFP gene. Transformation of plasmid pC1300th-Pclt1-GFP was conducted by ATMT method of the spores of C. lunata wild-type CX-3. The putative transformants were obtained from selective regeneration medium and confirmed by PCR detection and green fluorescence analysis. The results indicated that GFP gene was integrated into the genome of CX-3 strain,and the clt-1 promoter could drive the GFP gene expression. Strong green fluorescence activity was detectable in the mycilia and spores of transformants under the confocal laser scanning microscope.

关 键 词：玉米弯孢叶斑病菌 生物信息学 启动子活性 clt-1 GFP 

分 类 号：Q811.4[生物学—生物工程]