Isolation Of TaGI1 And TaMADS1 Genes And Functional Analysis In Triticum Aestivum L.

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...