Isolation And Functional Analysis Of Vernalization Related Gene TaVRN2 And A NF-Y Family Gene TaNF-YB3 From Triticum Aestivum L.

The transition from vegetative growth to reproductive growth is one of major developmental transitions in the life cycle of plants. Wheat (Triticum aestivum L.), an important crop plant, requires a long photoperiod and vernalization to flower. To study the molecular mechanism of flowerring time in this specie, we isolated TaVRN2 gene regulating flowering time in wheat, photoperiod gene CO (WCO1 and TaHd1), and NF-Y (HAP) family genes. The relations between those genes were studyed by sequence analysis and protein interaction. Moreover, we examined the expression pattern and biological function of TaVRN2 and TaNF-YB3. The results are as follows:1. Isolation and characterization of TaVRN2The TaVRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2). We isolated ZCCT-A1, ZCCT-B1, ZCCT-D1, ZCCT-A2 and ZCCT-D2 genes from hexaploid wheat, and their ORFs were 621 bp, 642 bp, 639 bp, 639 bp and 639 bp in length respectively. The deduced amino acid sequences are 206 aa, 213 aa, 212 aa, 212 aa and 212 aa respectively. ZCCT1 and ZCCT2 proteins had mutations at arginine amino acids at positions R39C, R/K16C of the CCT domain, respectively. These positions were conserved among CCT-domain and core region of HAP2. The clustering analysis revealed that the TaVRN2 protein was closer to HvZCCT-Ha, HvZCCT-Hb, and HvCO9 of barley than to CO-like proteins of Arabidopsis. As we known, there is no homolog of TaVRN2 in Arabidopsis.2. Isolation and characterization of wheat CO and NF-Y family genesThree different clones WCO1-2, WCO1-13 and WCO1-16 of WCO1 and one clone TaHd1-3 were isolated. In addition, NF-YA 1, 3, 5, 7 and 9 genes, NF-YB3, 4, 8 and 11, NF-YC6, 7 and 14 were also isolated. Based on the CCT-domain of these related proteins and core region of NF-YA proteins, sequence analysis revealed that both CCT-domain and the core region were conserved. NF-YA proteins also contained a DNA binding region, a short linker region and a region involved in heterodimer formation. However, NF-YB and NF-YC were not so conserved than NF-YA.3. Relations of TaVRN2, CO and NF-Y proteinsThe Yeast Two-Hybrid System was used to study the interactions between obtained the wheat CCT-domain proteins with NF-YB and NF-YC proteins. However, further investigation was needed to prove the results.4. Expression analysis of TaVRN2The expression pattern of TaVRN2 was studied by Real time RT-RCR. The results revealed that TaVRN2 transcripts were detectable mainly in early vegetative tissues, and showed low or no signals in reproductive tissues. Moreover, it seemed that ZCCT1 exhibited a higher expression level than ZCCT2 under same conditions. TaVRN2 transcript levels cycle in both long and short day conditions, indicating that the circadian expression patterns of TaVRN2 is regulated by day lengths and circandian clock. The results also showed that both ZCCT1 and ZCCT2 had the highest peak in the end of light, and generally ZCCT1 was peaked 2 h earlier compared to ZCCT2.5. Functional analysis of TaVRN2We analyzed not only the expression pattern of TaVRN2 but also the function of TaVRN2. Late flowering occurred in overexpressed Ubi::TaVRN2 plants of Brachypodium distachyon under long day condition. Variation of flowering times was observed in different transformants and most likely caused by differences in TaVRN2 expression levels. However, by given 4 weeks or 6 weeks vernalization, transformed plants flowered as early as wild type. Real-time RT-PCR was carried out to investigate the expression level of VRN1, VRN3 and VRT2-like genes in wild type Brachypodium distachyon, weak and strong transgenic plants of Ubi::TaVRN2. The transcript levels of VRN1 and VRN3 were lower in plants overexpressing TaVRN2 than in wild type plants under long day photoperiod. In contrast, VRT2-like showed a higher expression level. It suggested that the late flowering phenotype was correlated with expression levels of these genes to some extent.6. Sequence analysis of TaNF-YB3The full-length cDNA sequence of TaNF-YB3 is 809 bp in length and contained an ORF of 597 bp encoding 199 amino acids. Using the genomic DNA of ditelocentric wheat lines, the ORF sequence was mapped to chromosome 3D. Sequence analysis showed that TaNF-YB3 has high similarities of 85% and 66% to OsHAP3I and AtNF-YB3, respectively, and presented low similarities of 50% and 47% to yeast and human, respectively. A phylogenetic analysis based on the core sequence showed that the NF-YB proteins were located in several different clades. Among them, TaNF-YB3 belonged to a non-LEC1-like clade, which also includes OsHAP3F and OsHAP3I, both of which showed similarities even in the non-conserved regions.7. Expression analysis of TaNF-YB3 geneExpression patterns of TaNF-YB3 were analyzed by Real time RT-PCR. TaNF-YB3 transcripts accumulated mostly in the leaves, less in the stems, and were hardly detectable in the roots. In addition, its expression was weakly detected in young spikes, pistils, and young embryos. However, TaNF-YB3, which belongs to the no-L1L clade, was also strongly expressed in the stamen, as high as that in the stem. It was also regulated by salt, mannitol, abscissic acid, wounding, and cold. Moreover, TaNF-YB3 was down-regulated by short days and vernalization, and sensitive to the day length. It was mainly induced by light and exhibited a similar diurnal rhythmic expression pattern with the CCT-domain family gene TaVRN2 (TaZCCT1 and TaZCCT2), but not with CO (WCO1 and TaHd1). Overall, results suggested that TaNF-YB3, aside from having a role in regulating day length and vernalization responses, may integrate signals from other environmental stresses to perform its functions in winter wheat adaptability and development.8. Arabidopsis plants overexpressing TaNF-YB3 have no distinct difference with wild type in flowering time35S::TaNF-YB3 was transformed into Arabidopsis, but showed no significant difference in flowering time. The phenotypes of transformants could be divided into mild phenotype and severe phenotype. Comparing with wild type plants, the transgenic plants with mild phenotype showed curled or lobed leaves, and the severe phenotype showed line map leaves or transparent needle leaves. In addition, some leaves appeared etiolated phenomenon. As transgenetic plants were dicots and different from wheat, further study was required.