Analysis Of Gene Activity During Flowering Transition In Precocious And Normal Trifoliate Orange [Poncirus Trifoliata(L.) Raf.] And Characterization Of PtFCA Correlated With Flowering Time

Citrus is a woody perennial tree crop grown around the world for the production of fresh fruit and juice amongst other products. During the lifecycle of citrus, two transitions of citrus flowering are important. Firstly, the transition from vegetative to reproductive growth is characterized by the emergence of flowering shoots. The internal cues and environmental factors, such as age, seasonal changes in temperature and day-length, activate a series of signaling processes targeting shoot and axillary meristems, which culminate in the emergence of flowers. Secondly, it is the transition from juvenile to adult period. Whereas the reproductive phase of most woody fruit trees is preceded by a prolonged period of juvenility during which the above-ground part of the plant produces only vegetative shoots. The long juvenile stage is the biggest handicapt in fruit trees breeding. Most of what we know about the molecular genetics of flowering time regulation comes from studies in the model plants. However, little is known about the regulation of flowering transition in perennial species especially fruit trees compared with model plants. Precocious trifoliate orange and trifoliate orange had nearly the same morphology characteristics but had significant flowering habit. They are optimal materials for the studying on flowering-regulation mechanism of perennial woody plant. Here comparative transcriptome profiling of spring shoots was performed on them. In addition, we characterized the function of trifoliate orange FCA which is a key gene for flowering. The main results are as follows:1. A total of36,523unigenes were observed in precocious trifoliate orange and trifoliate orange by MPSS. Of these,6,859and5,055genes were specifically observed in precocious trifoliate orange and trifoliate orange libraries, respectively. There were2,735genes that were expressed differently in the two species. To validate the expression profiles obtained by MPSS, real-time PCR was performed on30differentially expressed genes selected for high or low expression levels, real-time PCR revealed that28of the30genes had the same expression tendency as the MPSS data. MPSS was able to successfully detect the expression of genes.2.569putative TFs belonging to60TF families were identified.There were552TFs in trifoliate orange library and564TFs in precocious trifoliate orange library. The MADS family was the most prevalent, followed by the AP2/EREBP and WRKY families.Meanwhile, MADS、SEP�'�MYB families which were related to flowering were identified.3. Gene Ontology (GO) categories and KEGG pathway were assigned to the significant2735genes. The three most overrepresented GO terms were binding, catalytic, and transcription regulators. A total of200different metabolic pathways were found in this study.4. In this research, we explored110putative components for the genetic flowering pathways in citrus by identifying homologs of Arabidopsis flowering-time genes. The expression of selected flowering-time genes corresponding to each flowering pathway were compared in precocious trifoliate orange library and trifoliate orange library, most flowering-promoting genes had up-regulated expression in the precocious trifoliate orange library, while most repressors of flowering genes had down-regulated expression.5. Isolated the FCA gene ortholog (PtFCA) from early flowering trifoliate orange and trifoliate orange. Sequence analysis that PtFCA contained two RNA recognition motifs (RRMs), a WW domain and a proline-rich region immediately downstream of the RRM2.6. PtFCA contained three alternatively spliced transcripts. PtFCA1encoding the entire FCA protein, PtFCA2and PtFCA3encoded truncated proteins with the RRM2and WW domain absence. The alternative splicing at the third intron of PtFCA displayed conservation and divergence between precocious trifoliate orange and model plants. The nuclear localization of PtFCA-GFP suggests that PtFCA1containing NLS might be a transcription factor. Nevertheless, PtFCA2and PtFCA3are widely distributed in cells.7. Gene expression analysis via real time PCR showed that PtFCAs may be involved in flowering and root development. Functional complementation experiment among three alternatively spliced transcripts indicated that PtFCA1partially rescued the late flowering phenotype of the fca-1mutant. PtFCA2delayed the flowering time in the fca-1background, but PtFCA3made no influence on flowering time. Moreover, the three transcripts also exhibited different abilities of regulating root development in the fca-1background. 8. Yeast-two hybrid assay and bimolecular fluorescence complementation (BiFC) showed PtFCA interacting with PtFY was conserved in plant. PtFCA can interact with several genes through its WW domian, and may play several roles and respond to seasonal signals in trifoliate orange development.9. Gene expression analysis via real time PCR showed that ABA and ambient temperature treatment led to change the level of PtFCA and interaction proteins.