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【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】 The objective of this study was to clone the PsNCS1 gene from wheat stripe rust, and analyze the expression of PsNCS1 at different developmental stages. 【Method】 The cDNA sequence of PsNCS1 was cloned by library screening and RT-PCR. Bioinformatics was used to predict the conserved domains and basic characteristics of the encoded protein. The phylogenetic tree was constructed by real-time fluorescence quantitative RT-PCR The expression level of PsNCS1 in different growth stages of germs. 【Result】 The full length cDNA of PsNCS1 was 1007bp (GenBank accession number GU134621). The open reading frame of PsNCS1 was 573bp and encoded 190 amino acids with a molecular mass of 22.17kDa and an isoelectric point of 4.96. The encoded protein contains four conserved domains of calcium-binding protein, EF-hand, and has an N-terminal myristoylation signature. The coding protein has the highest similarity with the basidiomycete NCS protein, and the closest relationship with NCS of wheat straw rust is 96%. The expression of PsNCS1 was higher than that of other developmental stages during the spore and germ tube stages. 【Conclusion】 PsNCS1 may be involved in the formation of stripe rust and extension of bud tube. The cloning and expression analysis of PsNCS1 laid the foundation for the further study of Ca2 + signaling mechanism and pathogenic mechanism of stripe rust cells.