小麦热激转录因子基因TaHsfB2d的克隆和特性及其对耐热性的调控

摘要

植物热激转录因子(heat shock transcription factor,Hsf)是响应热胁迫的主要调节因子,通过调节热激蛋白基因表达进而增强植物耐热性.小麦Hsf家族至少含有56个成员,其中B族11个,含B2亚族5个.本研究采用同源克隆技术,从37°C热处理的二叶一心小麦幼叶中克隆获得TaHsfB2d(序列号为AK331994)cDNA序列,序列长1191 bp,编码396个氨基酸.蛋白序列包括DNA结合结构域DBD和核定位信号序列NLS.同源分析表明,TaHsfB2d蛋白与大麦未知蛋白的相似性最高,为92％.荧光定量分析表明,TaHsfB2d在小麦多个组织器官中组成型表达,其中在成熟植株根系中表达量较高.37°C热胁迫、外源水杨酸(SA)和H2O2处理均能不同程度上调TaHsfB2d的表达,热激能显著增强SA和H2O2对TaHsfB2d表达的诱导.H2O2合成抑制剂DPI和羟自由基清除剂DMTU联合处理显著抑制热激对TaHsfB2d表达的上调作用、完全抑制SA对TaHsfB2d表达的上调.通过在洋葱内表皮瞬时表达TaHsfB2d并观察GFP荧光发现,正常条件下,TaHsfB2d蛋白定位于细胞核.酵母中耐热性鉴定表明,正常条件下,转TaHsfB2d的酵母细胞与转空载体对照酵母细胞的长势没有明显差异,热激处理同时降低,但前者的长势相对更强,TaHsfB2d的导入不影响细胞的生长发育.推测TaHsfB2d通过水杨酸途径介导植株耐热性调控过程,该过程依赖于H2O2存在.%Heat shock transcription factors (Hsfs) are key components of heat shock signal transduction pathways involved in the activation of Hsp genes in response to heat stress in plants. There are at least 56 members in wheat Hsf family. Eleven of them belong to class B, among which 5 members belong to subclass B2. In this study, TaHsfB2d was isolated from wheat (Triticum aestivum L.) young leaves treated by heat shock at 37 ℃for 1.5 h using homologous cloning methods. Sequence analysis showed that the coding sequence (CDS) of TaHsfB2d was 1191 bp encoding a protein of 396 amino acids. The amino acid sequence analysis demonstrated that TaHsfB2d contained a DNA-binding domain (DBD) and nuclear localization signal (NLS). TaHsfB2d protein sequence shared 90％, 85％, and 80％ identities with the proteins from predicted protein of Hordeum vulgare, HsfB2c of Hordeum vulgare and Brachypodium distachyon, respectively. The qRT-PCR results showed that TaHsfB2d was expressed in multiple tissues and organs of wheat, and the relative expression level of TaHsfB2d was higher in roots at anthesis stage. TaHsfB2d was up-regulated by 37℃heat shock (HS), salicylic acid (SA) and H2O2 in leaves. Furthermore, HS significantly enhanced the expression of TaHsfB2d pretreated with SA or H2O2, the up-regulation expression of TaHsfB2d by HS was significantly inhibited by the combined treatment of 150 μmol L–1 DPI and 20 mmol L–1 DMTU, and the up-regulation expression by SA was completely inhibited. Through transient reporter assay with onion (Allium cepa L.) epidermal cells, we found that TaHsfB2d localized in the nuclei. Yeast overexpressing TaHsfB2d showed stronger growth potential than the control cells overexpressing pYES2 after HS at 50℃for 45 min, and overexpression of TaHsfB2d had no effect on the growth and development of yeast cells. The results re-vealed that TaHsfB2d perhaps plays a key role in regulating the response to HS through SA signal pathway in plants, which was dependent on existence of H2O2. These results will provide theoretical basis for analysing biological functions and regulating mechanism of TaHsfB2d further.