Ding Liu-jun, Pu Ming-yu, Wei Bo, Wang Xian-ping, Fan Ren-chun, Zhang Xiang-qi
1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; 2. University of Chinese Academy of Sciences, Beijing 100049, China.
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Yi Chuan. 2016 Dec 20;38(12):1090-1101. doi: 10.16288/j.yczz.16-133.
Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. &Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% β-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.
小麦条锈病由条形柄锈菌小麦专化型(Puccinia striiformis West. f. sp. tritici Eriks. &Henn.,Pst)引起,是一种严重的真菌病害。鉴定与条锈病抗性相关的新基因对于培育抗病小麦品种和研究抗病机制具有重要意义。三螺旋是植物特有的转录因子家族,参与生长发育、形态建成以及胁迫响应的调控。迄今为止,尚无关于三螺旋家族与小麦条锈病关系的研究报道。在本研究中,从乌拉尔图小麦(Triticum urartu Tum.,2n = 2x = 14,AA)中克隆了三螺旋家族GTγ亚家族的一个基因,命名为TuGTγ-3。测序结果表明,TuGTγ-3基因包含一个完整的开放阅读框(ORF),其编码序列长度为1329 bp,编码一个含有442个氨基酸的蛋白质。该蛋白质的预测分子量为50.31 kDa,理论等电点为6.12。生物信息学分析显示,TuGTγ-3蛋白具有一个单分型核定位信号(GLPMQKKMRYT),既没有跨膜结构域也没有信号肽。保守的三螺旋结构域、第四个α-螺旋和CC结构域分别位于Q115 - R187、F234 - Y241和K362 - K436区域。二级结构分析表明,TuGTγ-3蛋白由43.89%的α-螺旋、9.51%的延伸链、9.95%的β-转角和36.65%的无规卷曲结构组成。基于对国际小麦基因组测序联盟(IWGSC)普通小麦基因组数据库的BLAST搜索,TuGTγ-3位于5A染色体长臂上。利用洋葱内表皮细胞进行的瞬时表达实验表明,融合蛋白TuGTγ-3-GFP主要分布在细胞核中,在细胞质中分布较少。不同器官中的表达谱表明,TuGTγ-3在叶片中的表达水平远高于根或叶鞘,且在叶片中的表达受Pst小种CYR32侵染的极大上调。此外,大麦条纹花叶病毒(BSMV)介导的病毒诱导基因沉默(VIGS)实验表明,转录因子TuGTγ-3正向调控乌拉尔图小麦对条锈病的抗性。