小麦条锈菌PsNCS1基因的克隆及转录表达特征  

Cloning and transcriptional profiling of PsNCS1 from Puccinia striiformis f.sp.tritici

作　　者：郭军[1] 张洪[1] 丁可[1] 代西维[1] 陈玥颖[1] 段迎辉[1] 黄丽丽[1] 康振生[1] 

机构地区：[1]西北农林科技大学植物保护学院,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《微生物学报》2010年第7期962-968,共7页Acta Microbiologica Sinica

基　　金：公益性行业(农业)科技专项(200903035-2);国家"973项目"(2006CB100203);现代农业产业技术体系建设专项资金资助;高等学校学科创新引智计划资助项目(B07049);教育部科学技术研究重点项目(107104);西北农林科技大学青年学术骨干支持计划~~

摘　　要：【目的】克隆小麦条锈菌神经钙离子感应蛋白基因PsNCS1,分析其在病菌不同发育时期的表达水平。【方法】利用文库筛选和RT-PCR技术克隆PsNCS1的cDNA序列,采用生物信息学技术预测分析该基因编码蛋白的保守结构域及基本特性,构建系统发育树;运用实时荧光定量RT-PCR技术分析PsNCS1在病菌不同生长发育时期的表达水平。【结果】PsNCS1全长cDNA为1007bp(GenBank登录号GU134621),开放阅读框为573bp,编码190个氨基酸,分子量为22.17kDa,等电点为4.96。编码蛋白含有4个钙结合蛋白的保守结构域EF-hand,并且具有N末端豆蔻酰化特征。编码蛋白与担子菌门真菌NCS蛋白相似性最高,其中与小麦秆锈菌的NCS亲缘关系最近,序列相似性达96%。PsNCS1在夏孢子和芽管时期表达量较高,均超过其他发育阶段基因表达量的2倍。【结论】PsNCS1可能参与了条锈菌夏孢子的形成和芽管的延伸。PsNCS1的克隆与表达分析为进一步研究条锈菌细胞钙信号传导机理和致病机制奠定了基础。[Objective]We cloned PsNCS1 encoding neuronal calcium sensor from Pst and analyzed its transcriptional profile.[Methods]A full-length cDNA of PsNCS1 was cloned by using RT-PCR in combination with cDNA library screening,the sequence was analyzed with different bioinformatic tools and the gene expression pattern was characterized via real-time RT-PCR.[Results] PsNCS1 （Genbank accession no.GU134621） encoded 190 amino acids,with a molecular weight of 22.17 KDa and a pI of 4.96.PsNCS1 contained four conserved EF-hand domains and was Nterminally myristoylated.Phylogenetic analysis indicated that PsNCS1 was highly similar to the NCS from Basidiomycetes and the highest similarity was with that from Puccinia graminis （96%）.Real-time RT-PCR analysis indicated the amount of PsNCS1 transcripts of urediospores and of germinated urediospores were doubled or more comparing with those of fungal bodies at other different developmental stages.[Conclusion]PsNCS1 might be involved in the process of urediospore formation and germ tube elongation.The present results may provide basic data for further analysis of the role of PsCNS1 in pathogenesis process and calcium signaling.

关 键 词：小麦条锈菌 神经钙离子感应蛋白 基因克隆 实时定量RT-PCR 

分 类 号：S435.121[农业科学—农业昆虫与害虫防治]