| The process of flowering, which is co-regulated by environmental and endogenous signals, is crucial to the whole lifecycle of higher plants, and has long been a hotspot of phytobiology and phytophysiology studies. Previous studies divided the flowering process into three progressive stages, termed flowering determination, flower evocation, and floral organogenesis and development. A number of genes that take part in the three stages have been cloned, including genes involved in the ABCDE model, and so on. LFY stands out as a major regulator in integrating the floral inductive signals at the first two stages as well as in controlling the floral homotic genes at stage three. Moreover, carbohydrates such as sucrose, besides being the substrates of energy metabolism, are found to be able to function as signal molecules. More importantly, sucrose as a signal has been reported to promote or delay flowering by affecting the expression of LFY. Oncidium Milliongolds is a member of the genus Oncidium, Unique flower shapes and color combinations make them ideal materials for chemical and molecular genetic analysis of flower development. To date, a few flowering associated genes have been cloned form different culcuvars of Oncicium.The cDNA amplified restriction fragment length polymorphism (cDNA-AFLP) technique has been proved to be a robust high-throughput, genomic-wide analysis tool for gene discovery. In this study, we found that reproducible cDNA-AFLP fingerprints could be generated by extracting the total RNA by TRIzol, digesting the ds-cDNA with EcoRⅠand MseⅠfor 3 h and using the 15-times diluted re-amplification products as selective amplification templates. Thus, a stable cDNA-AFLP analysis system was established in Oncidiun Milliongolds. 114 primer pairs were selected by primer screening for further studies.Using the cDNA-AFLP analysis system established above, we profiled transcripts differently expressed during flower buds development in Oncidium Milliongolds. Approximately a total of 15960 transcription-derived fragments (TDFs) were generated with 114 primer sets. Among them 1248 were sub-cloned and sequenced, through which 993 readable sequences were produced. BLASTX/N analysis of the 993 sequences showed that more than 80% of them had identity genes with known functions. Majority of the later group had putative functions involved in signal transduction (4.4%), energy (1.4%), metabolism (16.7%), gene expression regulation (7.3%), and transportation (1.5%), based on their homology with known genes or proteins in the NCBI databases.Full-length mRNA of SUCROSE SYNTHASE 1 (SUS1) and LFY were cloned using RT-PCR. Sequence analysis reveals that the SUS1 and LFY sequences of Oncidium Milliongolds showed higest homology to their homologous genes in Orchid plants such as Phalaenopsis and Mokara, and showed higher homology to the corresponding genes in monocotyledons than those in dicotyledons.Analysis of the expression patterns of SUS1 and LFY, along with that of HYTOCHTOME AND FLOWERING TIME PROTEIN 1 (PFT1), were characterized. The results showed that the expression levels of SUS1 and LFY shared a similar variation tendency, while the expression level of PFT1 displayed a different profile. To confirm the function of SUS1 in flower buds, carbohydrate contents and sucrose synthase (SS) activities were determined. The results revealed that the variation of sucrose content and sucrose synthase (SS) activity is in accordance with that of the expression level of SUS1. All these results illustrated that cDAN-AFLP is a reliable technique for studying differently expressed genes during the Oncidium flower development. Genes differently expressed were identified and their expression patterns were analyzed. The present study gives rise to an assumption that SUS1 may affect flower development by regulating the expression level of LFY via changing the sucrose content in the flower buds. |
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