叶锈菌及信号分子诱导小麦TcLr35中β-1,3-葡聚糖酶基因的表达分析

Expression and Analysis of β-1, 3-Glucanase Gene in Wheat TcLr35 Induced by Wheat Leaf Rust Pathogen and Signal Molecule

【目的】β-1,3-葡聚糖酶是一种病程相关(pathogenesis-related,PR)蛋白,了解小麦TcLr35中β-1,3-葡聚糖酶基因在叶锈菌(Puccinia triticina)与TcLr35小麦互作体系中的表达模式,明确其与TcLr35小麦抗叶锈病相关性,进而探讨PR蛋白介导的小麦成株抗叶锈病的分子机理。【方法】在前期研究基础上,利用生物信息学方法分析已克隆β-1,3-葡聚糖酶类病程相关蛋白基因TaLr35PR2结构特征,利用半定量RT-PCR方法结合genetools和SPSS软件检测叶锈菌及信号分子诱导后不同时间点该基因表达量,并明确其在小麦根、茎和叶各生长器官及小麦不同生长时期的基因表达模式。【结果】小麦TaLr35PR2含有信号肽,编码定位于液泡的碱性蛋白,蛋白序列与其他物种PR2类蛋白序列具有较高同源性,与普通小麦病程相关蛋白2亲缘关系最近,与水稻病程相关蛋白亲缘关系最远。小麦成株期接种叶锈菌后不同时间点,TaLr35PR2在亲和反应(Thatcher)中表达量变化不大,趋于平缓;而在非亲和反应(TcLr35)中的表达有显著差异,总体上,该基因在非亲和组合中表达量明显高于亲和组合中表达量。检测TaLr35PR2在成株小麦TcLr35各器官中基因表达,该基因在叶片中表达量随接菌时间点延长明显增加,并在接菌后48 h达到表达高峰;而在根中接菌后各时间点均未检测到基因表达;在茎中接菌后48 h开始有少量表达,但表达量变化不大,趋于平缓,总体表达丰度为叶>茎>根。在小麦生长发育的不同时期,除1叶期外,该基因在TcLr35和Thatcher中均有表达,Mock(未接菌处理)反应中,TaLr35PR2在小麦不同生长期的表达呈上升趋势;接种叶锈菌后,TaLr35PR2在亲和反应和非亲和反应小麦各生长时期的表达量均高于Mock反应,且在成株期表达稳定。信号分子水杨酸(salicylic acid,SA)、脱落酸(abscisic acid,ABA)均能诱导该基因的表达,信号分子预处理后接种叶锈菌,基因表达量显著上调。【结论】TaLr35PR2的表达受叶锈菌诱导,TaLr35PR2可能参与SA、ABA介导的信号通路,并参与小麦TcLr35成株抗叶锈病防御反应。

【Objective】β-1, 3-glucanase, a kind of pathogenesis-related protein, plays an important role in signal transduction in plants. The objective of this study is to understand the expression patterns of β-1, 3-glucanase gene in the interaction between Puccinia triticina and wheat TcLr35, identify the relativity between the aim gene and resistance to leaf rust pathogen attack, and explore the molecular mechanism of PRs mediated resistance in adult wheat to P. triticina.【Method】Based on the previous studies, the structure characteristics of β-1, 3-glucanase gene（TaLr35PR2） were analyzed using bioinformatics methods. In the meanwhile, the gene expression profiles of TaLr35PR2 induced by P. triticina and chemical molecule at different time points were analyzed by semi-quantitative RT-PCR method combining genetools and SPSS software. In addition, the similar analysis was also conducted on the different tissues of wheat including roots, stems and leaves, and different growth stages of wheat.【Result】TaLr35PR2 coded an alkaline protein positioned in the vacuole contained a signal peptide. The protein sequence of TaLr35PR2 had high homology with other plants, and had the closest relationship with pathogenesis-related protein 2 gene in Triticum aestivum, but farthest relationship with the PR protein in rice. The expression profile analysis of TaLr35PR2 at the adult stage in wheat at different time points after P. triticina inoculation indicated that the transcription level changed little in the compatible interaction（Thatcher）, but changed much in the incompatible interaction（TcLr35）. The transcription level was much earlier and higher in the incompatible interaction than that in the compatible interaction at different points. Different tissues of the adult wheat TcLr35 detection results showed that the gene expression in leaves increased significantly with the extension of inoculation time, the expression level reached a peak at 48 hpi, but the gene was not expressed in roots at different time points, and there was a little expression level at 48 hpi in stems and changed a little at the later period, so its expression abundance was leafstemroot. In different periods of wheat growth and development, the TaLr35PR2 expressed at certain levels without inoculation both in TcLr35 and Thatcher except at one-leaf stage, and the expression levels were much higher both in the incompatible and compatible interactions than that in the Mock reaction, then expressed stably at the adult stage. In addition, the expression level of the TaLr35PR2 increased significantly after pre-treatments with chemical molecule such as salicylic acid（SA） and abscisic acid（ABA）.【Conclusion】The expression of TaLr35PR2 was induced by P. triticina. TaLr35PR2 may be involved in the defense response against rust pathogen and participate in SA, ABA mediated signaling pathway and take part in defense reaction to P. triticina of wheat TcLr35 adult stage.