| Plant flowering is one of the most important biological phenomena.Deepening the understanding of its mechanism is of great significance for improving the plant rapid breeding technology,improving the variety cultivation strategy,optimizing planting pattern,and elevating the efficiency and capacity of agricultural production.The development regulation of plant flowering starts from fertilization,and ends at the next fertilization.The transition from vegetative to reproductive growth is the central part of plant flowering,which needs first to sense external stimulation such as light and temperature.After the complex gene regulation and signal transduction,flowering occurs.This is a hot field of developmental genetics research,with a lot of well-known reports,but still has many knowledge deficiencies and controversial arguments that need to be resolved.The major vernalization-related genes in wheat are VRN1,VRN2 and VRN3.But more genes,even indispensable ones,are needed for the effective regulation of reproductive development by vernalization.Inspired by the fact that the key vernalization gene in Brachypodium distachyon is RVR1,the expression and functional analysis of wheat RVR1 was performed.Meanwhile,a transcriptome analysis of wheat leaves collected before,during,and after vernalization was carried out to globally survey the involved genes during vernalization and their expression changes.In consideration of the significant inhibition of flowering by VRN2,the broader participation of CCT gene in plant flowering,and their complex regulation patterns,a genome-wide scanning of CCT genes and their sequences and expression analysis was conducted,and the functional analysis of some significant differentially expressed genes was also conducted.The results show as below:（1）TaRVR-A1,TaRVR-B1,and TaRVR1-D1 from the wheat sub-genome A,B,and D were cloned,which all contains 8 exons.Besides,a BAHplant3 domain and TFIISM domain also appears in their deduced protein sequences.Phylogenetic analysis of RVR1 proteins showed that there are only one or two RVR1 homologous copies appearing in a single plant sub-genome.The expression analysis found that this gene is slightly inhibited by vernalization.Ectopic expression of this gene in Arabidopsis results in delayed flowering and partially restores the defective phenotype of bdt1（RVR1 homologous gene）mutant.It is speculated that although the RVR1 gene is related to flowering,its role in plant development may be more extensive.（2）Transcriptome analysis of a strong winter wheat cultivar Shiluan 02-1 before,during and after vernalization showed that the most amount of differentially expressed genes（DEGs）including transcription factors appeared before vernalization and after six weeks of vernalization.A large number of DEGs were found to be related to substance synthesis and metabolism,light response and substance transportation,based on the functional enrichment of GO and KEGG.These genes were divided into 10 clusters by the time series expression cluster analysis.It was also shown that genes in a specific cluster often share similar functions,and the members of the same transcription factor family tend to cluster in the same or a few gene clusters.These results indicate that the gene expression changes obviously mainly at the beginning and the late stages of vernalization.Most of them may regulate the growth rate in response to the rapid change of temperature to resist low temperature stresses.（3）127 CCT genes were screened from wheat genome by HMM search against the CCT domain.These genes were divided into 4 sub-families as well as 40 gene clusters.A phylogenetic analysis also divided them into eight groups.The further analysis of the transcriptome sequencing data mentioned in Results（2）suggested 49 vernalization induced CCT genes,and 31 vernalization inhibited CCT genes.Real-time PCR further demonstrated 5 significant upregulated CCT genes（TaCMF11,TaCO18,TaPRR95,TaCMF6,and TaC016）during vernalization,which are down regulated immediately after vernalization.While TaCO1 and TaCO15 were the most significantly downregulated genes during vernalization which maintain their low expression after vernalization.Although TaZIM4 is inhibited by vernalization,it is not significant.A further VIGS functional verification showed that TaCO15,TaCO18,and TaZIM4 could promote flowering,which indicates the diversity of flowering regulation genes and the complexity of their mechanisms.This study found that vernalization can cause expression changes for a large number of genes,most of which are related to low temperature.The further analysis of relationships between these vernalization related genes and flowering regulation showed that TaCO15,TaCO18,and TaZIM4 promote flowering,while TaRVR1 inhibits flowering.Combined with their different expression changes during vernalization,their expression pattern is not completely related to flowering regulation.These findings further enrich the genes related to flowering development,and improve the interaction network between flowering genes.Meanwhile,this study provides some reference clues on the improvement of wheat rapid breeding technology,and optimization of agricultural production measures;and plenty of data support is also provided for the further research of plant flowering and development. |
