Selection And Express Analysis Of The Genes Related To Floral Development Of Chrysanthemum In The Photoperiods Pathway

Chrysanthemum (Chrysanthemum morifolium) is traditional Chinese famous flowers,also is the one of the four world cut flowers, which production and sales are in the forefront ofthe world’s flowers.In this study, using cDNA-AFLP technique, the apical buds of Chrysanthemummorifolium (Ramat.) Kitam.‘jinba’, induced by different photoperiod treatments, were takenas the material to analysis gene expression profiling, separate and identify differentiallyexpressed genes, obtained6TDFs which were possible identified to be associated with theflowering of Chrysanthemum by cloning, sequecing and BLAST analysis. This laid afoundation for understanding the molecular mechanism of floral differentiation, and providednew information that can be used in molecular manipulation to breed different flowering timecultivars of chrysanthemum in the future. These results are as follows:1. The cDNA-AFLP reaction system suitable for chrysanthemum was established.The results showed that high purity and integrated RNA was extracted by TRNz olmethod; Double-stranded cDNA was synthesized with the Oligo(dT)18primer combined withBeyoRT M-MuLV reverse transcriptase after modification and optimization.1μg cDNAwas double digested by FastDigest EcoRⅠand MseⅠfor5min; The desirable result wasobtained with the ligation products diluted to10times as templates for an30-cyclepre-amplification PCR; Selected-amplification was carried out, using64pairs of AFLPprimer, with the pre-amplification products10times-diluted, adopting Touchdown procedures;The amplified products were determined by by6%PAGE electrophoresis using the improvedrapid silver-stained method and45pairs of AFLP primer with high polymorphic bands werescreened, and the legible cDNA-AFLP gel mapping was obtained. The cDNA-AFLP reactionsystem established and optimized in this experiment is suitable for the functional geneanalysis of chrysanthemum.2. Isolated the flowering related genes using the cDNA-AFLP reaction system established inthis study.Total of2917TDFs were screened by64primer combinations, and835TDFs wereidentified differentially expression, including584TDFs up regulation and251TDFs downregulation.50TDFs were successfully cloned and sequence analyzed. The results indicated that36TDFs were observed had homology in NCBI database, which of31TDFs displayedhomology to genes with known functions,5no had a match. Functional analysis indicatedthat31TDFs mainly participated in7kinds of processes of signal transduction, development,transcription regulation, protein degradation and synthesis, metabolism, stress responses etc.3. Semi quantitative RT-PCR analysis with selected transcripts of6flowering related genesSemi quantitative RT-PCR analysis with selected transcripts of6flowering related genesindicated that the6flowering related genes all differentially expressed or up regulated underthe short-day conditions, while they all did not expressed or down regulated under thelong-day conditions. These results was similar to those of cDNA-AFLP expression patterns inchrysanthemum.In this studies several flowering related gene pigments were selected by cDNA-AFLPtechniques. These novel genes could be used in future to functional analysis and strategies ofmolecular breeding.