Chen Liang, Zhang ZengYan, Liang HongXia, Liu HongXia, Du LiPu, Xu Huijun, Xin Zhiyong
National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
J Exp Bot. 2008;59(15):4195-204. doi: 10.1093/jxb/ern259. Epub 2008 Oct 26.
Wheat sharp eyespot, primarily caused by a soil-borne fungus Rhizoctonia cerealis, has become one of the most serious diseases of wheat in China. In this study, an ethylene response factor (ERF) gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the T. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERF1 is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmai12, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down from the T0 to T4 generations. Quantitative reverse transcription-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the expression levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-dependent signal pathway, such as a chitinase gene and a beta-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ethylene-dependent pathway.
小麦纹枯病主要由土传真菌禾谷丝核菌引起，已成为中国小麦最严重的病害之一。在本研究中，对来自小麦近缘种中间偃麦草的一个乙烯响应因子（ERF）基因TiERF1进行了进一步鉴定，培育了表达TiERF1的转基因小麦株系，并研究了转基因小麦株系对禾谷丝核菌的抗性。Southern杂交分析表明，中间偃麦草基因组中存在至少两个拷贝的TiERF1基因。酵母单杂交试验表明，TiERF1的激活结构域对于激活含有GCC-box顺式元件的报告基因转录至关重要。通过基因枪轰击将TiERF1基因导入中国小麦品种扬麦12。PCR和Southern杂交分析结果表明，TiERF1已成功整合到转基因小麦基因组中，并可从T0代遗传至T4代。定量逆转录PCR分析表明，TiERF1在稳定转基因植株中能够过量表达，其中主要在乙烯依赖信号途径中的小麦病程相关（PR）基因如几丁质酶基因和β-1,3-葡聚糖酶基因的表达水平显著提高。病害试验表明，与野生型和TiERF1沉默植株相比，TiERF1的过量表达使转基因小麦株系对纹枯病的抗性增强。这些结果表明，TiERF1的过量表达通过激活主要在乙烯依赖途径中的PR基因增强了转基因小麦株系对纹枯病的抗性。
