Abstract: Wheat leaf rust is an important disease in wheat production, and the utilization of resistant varieties is a safely, efficient and economical method to control the disease. It is clear that the role of leaf rustrelated genes in leaf rust resistance is of great significance for the effective control. The Ca2+ CaM/CML messenger system plays an important role in disease resistance, and different CaM subtypes may regulate different resistance pathways. Previous studies showed CaM/CML sequences significantly differentially expressed in diseaseresistant and susceptible transcription. Based on this, this study cloned the open reading frame (ORF) of this differentially expressed sequence in wheat nearisogene line TcLr19. Through Blastx and sevral kinds of software, the structural properties were preliminarily determined. The gene encoded a novel CML isoform with 458 bp in length and 147 amino acids and contained no transmembrane region, no signal peptide, located in other organelles rather than mitochondria, and had an EFhand_8 and multiple EFh conservative domains. The amino acid sequence of this gene shared 99% identify with CML25/26 from Aegilops tauschii. The gene was tentatively designated as TaCML25/26. SWISSMODEL was used to construct a threedimensional model of CML to initially determine its structural characteristics. The differential expression of CML gene under leaf rust infection was detected by realtime fluorescence quantitative PCR (qRTPCR). The gene was expressed in different levels in roots, stems and leaves. It was induced by Puccinia triticina, and the peak time of expression was significantly different in wheat leaf rust resistance nearisogenic line TcLr19 and the susceptible mutant Mu19. And the peak time was 96 h earlier in TcLr19 than that in susceptible mutant. The exogenous phytohormone salicylic acid and abscisic acid induced TaCML25/26 upregulated expression in TcLr19. The results laid a foundation for the future analysis of the potential biological function of wheat calmodulin gene in plant wheat leaf rust resistance.

Key words: wheat leaf rust, calmodulin, gene cloning, expression analysis, resistance

摘要： 小麦叶锈病是小麦生产中的重要病害，抗病品种的利用是防治该病安全、高效、经济的方法。明确抗叶锈病相关基因的作用对于有效防治叶锈病具有重要意义，而Ca2+CaM/CML信使系统在抗病过程中起着重要作用，并且不同的CaM亚型可能调控不同的抗病途径。前期研究发现在抗病和感病转录中存在显著差异表达的CaM/CML序列，在此基础上，在小麦近等基因系TcLr19中克隆该基因的开放读码框（open reading frame，ORF）。通过用Blastx及多种软件进行序列分析，初步确定其结构特性。该基因包含一个完整458 bp的开放阅读框，编码147个氨基酸；编码的蛋白不含跨膜区、无信号肽、定位在线粒体以外的其他细胞器中，具有一个EFhand_8以及多个EFh保守结构域。该基因与粗山羊草CML25/26 基因的亲缘关系最近，相似性高达99%，确定其为一个新的小麦CML亚型，命名为TaCML25/26。用SWISSMODEL构建CML的三维模型。利用实时荧光定量PCR（qRTPCR）分析该基因在TcLr19和其感病突变体Mu19中表达的特性。该基因不仅在根、茎、叶等组织中均有不同程度的表达，而且在叶片中表达受叶锈菌诱导，表达高峰出现时间在小麦近等基因系TcLr19和感病突变体Mu19中显著不同，在抗病的TcLr19中比在感病突变体中高峰早出现96 h；外源植物激素水杨酸、脱落酸在TcLr19中诱导TaCML25/26上调表达。为今后解析小麦类钙调素基因在植物抗叶锈病中的生物学功能奠定基础。

关键词: 小麦叶锈病, 钙调素, 基因克隆, 表达分析, 抗病性