【作者】 赵翔宇；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 植物学， 2005， 博士

【摘要】 小麦(Triticum aestivum L.)是一种重要的粮食作物。为了理解小麦花发育的分子调控机制,本研究分离了调节小麦开花时间的TaGI1(Triticum aestivum GIGANTEA 1)基因和调控小麦花发育的TaMADS1(Triticum aestivum MADS box gene 1)基因,并对它们的表达模式与生物学功能进行了深入研究。主要结果如下: 1、TaGI1 基因的序列特征TaGI1 基因cDNA 的全长为4012 bp,其中包括长度为3522 bp 的开放阅读框,由此推测TaGI1 蛋白包含1174 个氨基酸残基,并且含有核定位保守序列。瞬时表达实验表明pBI121-TaGI1-GFP 融合蛋白定位于细胞核内。氨基酸序列比较分析与系统进化树分析表明TaGI1 蛋白与拟南芥GI 蛋白以及GI 在其它植物中的同系物高度同源,并且与单子叶植物的GI 蛋白亲缘关系较近,而与双子叶植物的GI 蛋白亲缘关系则相对较远。2、TaGI1 基因的表达分析为了研究TaGI1 基因的时空表达模式,我们利用Northern 杂交对其进行了分析。结果显示,除了胚乳之外,TaGI1 基因的转录物在根、茎、叶、穗原基、小穗和胚等组织器官中都能被检测到。在短日照和长日照条件下,虽然TaGI1 的转录水平不同,但是都呈现周期性变化。这说明TaGI1 基因的表达呈现昼夜节律性,而且这种周期性表达模式受到日照长度和昼夜节奏钟的调节。尽管在连续光照或连续黑暗条件下,TaGI1 基因转录物在小麦叶中有一定水平的积累,但是并没有检测到TaGI1 基因表达水平的周期性变化。此外,RT-PCR 结果还表明,种子萌发后TaGI1 基因和TaHd1-1 基因(拟南芥中CO 基因的同源基因)在幼叶中的周期性表达是对光周期快速响应的结果。由于植物感受光信号的部位在叶片,所以我们利用原位杂交技术分析了TaGI1 基因转录产物的细胞定位。结果显示,在苗端分生组织和叶原基中均检测到杂交信号,而且信号强度在0 小时和10 小时两个时间点很相似,这表明TaGI1 基因在苗端的表达不呈现周期性。有趣的是,TaGI1 mRNA 在叶内定位于近轴端表皮细胞中,而且这些细胞恰恰靠近维管束。同时,当光照10 小时的时候,TaGI1 基因在这些细胞中的表达信号要比在光照0 小时的信号强,这进一步证明了TaGI1 基因在叶片中具有节律性表达。以上结果表明,TaGI1 基因响应光周期的节奏性表达是发生在叶中的特异细胞内,而不是发生在苗端。此外,长日照条件下TaHd1-1 基因在叶片的维管束中被检测到,尤其在小的更多还原

【Abstract】 Wheat (Triticum aestivum L.) is an important crop and requires long day and short night to flower. To study the molecular mechanism of flower development in this species, we isolated TaGI1 (Triticum aestivum GIGANTEA 1) gene regulating flowering time in wheat and TaMADS1 (Triticum aestivum MADS box gene 1) gene involved in the flower development of wheat. The results are as follows: 1. Isolation and characterization of TaGI1 The nearly full-length cDNA of TaGI1 is 4012 bp in length and it has an open reading frame of 3522 bp. The deduced amino acid sequence of TaGI1 contains 1174 amino acid residues. The conserved region for nuclear localization in TaGI1 was identified. The pBI121-TaGI1-GFP fusion protein is clearly targeted to the nucleus in a transient transfection assay. The clustering analysis reveals that the TaGI1 protein is closer to HvGI of barley and OsGI of rice in monocots than to BrGI of cabbage and GI of Arabidopsis in dicots. Thus, these data suggest that TaGI1 is a GI homolog. 2. Expression analysis of TaGI1 The expression pattern of TaGI1 was studied by RNA blot hybridization. The results revealed that TaGI1 transcripts were detectable in both vegetative and reproductive tissues, with the exception of endosperm. TaGI1 transcript levels cycle in both long and short day conditions, indicating that the circadian expression patterns of TaGI1 is regulated by daylengths and circandian clock. The results also show that the rhythmic cycling of TaGI1 transcript levels was not observed although the transcripts were accumulated in the leaves in continuous light or continuous dark for 6 days. In addition, the results of RT-PCR indicate that the rapid rhythmic expression of TaGI1 and TaHd1-1 (CO ortholog in Arabidopsis) in the leaves of seedling after germination occurs in response to photoperiods. In order to explore subcellular localization of TaGI1 transcrips, in situ hybridization was performed. The results show that hybridization signals were detected in the vegetative shoots which contained shoot apical meristems and leaf primordia at 10 h and 0 h, and the levels of signals were quite similar at both time points, suggesting the TaGI1 expression does not cycle in the shoots. Interestingly, TaGI1 mRNA in leaves was localized in the cells of adaxial epidermis and those cells were just above the vascular bundles. Signals in these cells were much stronger at 10 h than at 0 h. Thus, these results indicated that the TaGI1 rhythmic expression occurred in the specific cells of leaves rather than shoot apices in response to photoperiod conditions. In addition, it is interesting that hybridization signals of TaHd1-1 were detected only in the vascular bundles, in particular, in the small vascular bundles. Further observation indicates that the signals are mainly accumulated in the xylems of vascular tissues. Thus, the results indicate that the tissues of TaGI1 expression are close to those of TaHd1-1 mRNA accumulation in leaves. 3. Functional analysis of TaGI1 We not only analysised the expression pattern of TaGI1 but also explored the function of TaGI1. Early flowering occurred in overexpressed 35S::TaGI1 plants under both long and short day conditions. Variation of flowering times was observed in different transformants and most likely caused by differences in TaGI1 expression levels. The flowering time of gi-2 plants expressing TaGI1 gene is very similar to the wild type plants in long day photoperiod. These data demonstrate that expression of TaGI1 alters flowering time and complements gi mutant phenotype. Real-time PCR was carried out to investigate the expression level of CO in wild type Arabidopsis, 35S::TaGI1 transgenic plants, gi-2 mutants and 35S::TaGI1/ gi-2 cross progeny. The results show the mutation of GI resulted in lower levels of CO transcripts in gi-2 mutant. The transcript levels of CO are higher in plants overexpressing TaGI1 than in wild type plants under long day photoperiod. In contrast, in the gi-2 mutant plants, CO is expressed in the same phase as in wild type plants, but at lower amplitude. However, when 35S::TaGI1 was transferred into gi-2 mutant plants, the levels of CO transcripts were restored. 4. Sequence analysis of TaMADS1 The nearly full-length cDNA of TaMADS1 is 1197 bp and encodes one of the typical MIKC MADS proteins in plants. Sequence analysis shows that TaMADS1 shares higher homology with SEP3 and SEP-like proteins. Further, the phylogenetic tree was constructed based on the alignment of amino acid sequences of MADS box full-length proteins. The results indicate that TaMADS1 is closer to E function genes, in particular, SEP3 and SEP3 homologs. Thus, it is most likely that the TaMADS1 belongs to a SEP3 group. In addition, the results of Southern hybridization show that although a few bands were detected in wheatgenome, only one band shows a stronger signal. The result implies that there may be a single TaMADS1 gene in wheat genome. 5. Expression analysis of TaMADS1 gene Exression patterns of TaMADS1 were analyzed by Northern hybridization and in situ hybridization. Northern hybridization shows the transcripts of TaMADS1 were detected in carpels and stamens, but no signals were detectable in others tissues. Further, in situ hybridization results show that TaMADS1 transcripts begin to accumulate in the tissues of spikelet primordia after the formation of glume primordia, while the signals are weaker than those in floret primordial. In addition, TaMADS1 mRNA is accumulated in floret primordia and foral organ primordial. Thus, these data suggests that the activity of TaMADS1 could be involved in floret development. 6. Arabidopsis plants overexpressing TaMADS1 have the phenotypes of early flowering and abnormal floral organs In order to explore the function of TaMADS1, sense TaMADS1 was transferred to Arabidopsis. The phenotypes of transformants carrying sense TaMADS1 could be divided into mild phenotype and severe phenotype. Comparing with wild type plants, the transgenic plants with mild phenotype show reduced size and curled leaves, and these plants flower after the formation of three or four rosette leaves. Interestingly, the transgenic plants with severe phenotypes have two curled cotyledons and a solitary terminal flower. We did in situ hybridization to analyze the expression of LFY. The results show the strong signal is detected in the shoot apical meristem of the transgenic seedling at day 5 after germination, while the signal is very weak in the shoot apical meristem of the wild type seedling. The results indicate that the formation of flower primordia might occur in the embryos of transgenic plants with severe phenotype. Overexpression of TaMADS1 not only caused early flowering of transgenic plant but also altered the morphology of floral organs. Some sepals are converted into leaf-like structures, and the number of petals in most of transformants is reduced and the morphology of petals is altered. The number of stamens is also reduced, and they have short filaments and petaloid anthers that are sterile. Real-time quantitative PCR was performed to analyze the transcript levels of some genes更多还原