The Expression Pattern And Functional Characterization Of Flowering Related Genes In Chrysanthemum Lavandulifolium(Fisch. Ex Trautv.) Makino

The flowering period of Chrysanthemum is important horticultural and cultivated characters. It is highly desirable to effectively manipulate the key genes involved in flowering and breed the new variety of Chrysanthemum with the natural florescence suitable for industrial production, while maintaining normal growth and without affecting their resistance to diseases and pests. However, research at the molecular level is limited by a lack of sequence data. Due to the complex genetic background of Chrysanthemum, an analysis of a Chrysanthemum relative species-Chrysanthemum lavandulifolium(Fisch. ex Trautv.) transcriptome aimed to obtain transcript sequence data will be helpful to elucidate the molecular mechanism of flowering control in Chrysanthemum. In this study, the transcriptome of C. lavandlifolium under different developmental stages and stress treatments was sequenced by Illumina/solexa technology, and then bioinformatically analyzed. The genes spatio-temporal expression profiles between seeding stage and squaring period were performed by up-graded version of the DGE. We perform the function analysis of DFL and DenFUL in Arabidopsis by transgenic technology. The major results and conclusions are described as follows:1. RNA-seq generated4GB raw data, which was then de novo assembled into108,738Unigenes with a mean length of349bp. A total of58,093Unigenes were obtained non-redundant annotation(E-value<10-5). GO, COG and pathway bioinformatically analysis showed that this transcriptome library provide a valuable resource for studying the C.lavandulifolium flowering mechanism and others basic biology.2. Based on the annotations, we screened56important candidate genes that can be used in genetically modified Chrysanthemum flowering. Those genes respectively belong to photoperiod pathway, vernalization pathway, GA pathway, autonomous pathway and FRI-dependent pathway, which indicated these flowering genetic regulatory pathways coexistence in C.lavandulifolium. In addition, we identified 6,204Unigenes that encode transcription factors and belong to57transcription factor families, of which8transcription factor families were related to flowering.3. Using up-graded version of DGE technology, we identified gene groups that specifically expressed during C.lavandulifolium squaring period. Most genes promoting flowering in the photoperiod pathway, GA pathway, autonomous pathway and vernalization pathways were up-regulated during C.lavandulifolium squaring period. In addition, the basalmetabolic pathway, such as sugar and starch metabolic pathways, nitrogen metabolism and metabolic pathway genes, as well as MIKC, SBP, YABBY, ARF, Dof and MYB transcription factor families were involved in C.lavandulifolium flowering.4. Heterologous overexpression of DFL extremely promoted Arabidopsis wildtype early flowering, the florescence was advanced about14days; and fully complemented Ify mutant. Using LFY promoter to drive DFL expressed in Arabidopsis can promote flowering about7days. We also found heterologous expression DFL in Arabidopsis wildtype drived by LFY promoter caused co-suppression.5. DenFUL transgenic Arabidopsis plants were significant early flowering, which advanced about14days than wild-type under long-day conditions and more than40days under short day conditions. In addition, DenFUL transgenic Arabidopsis producing terminal flowers and lateral flowers, the rosette leaves much small than WT and the style of silique was thin and elongate; but did not affect the fruit cracking.6. Transgenic plants vivo detection, transient expression using agrobacterium-mediated in onion epidermal cells and transient expression using PEG-mediated in Arabidopsis proplast were employed to investigate the subcellular location of DFL and DenFUL, respectively. All the analysis reached to the same patterns that DFL and DenFUL were localized in nuclear as Arabidopsis LFY and API did.7. Analysis on endogenous genes expression patterns in transgenic Arabidopsis, we found DFL widely activated the expression of B-and C-class floral organ identity genes, inhibited TFL1expression. Overexpression of DenFUL conferred the expression of endogenesis LfYand FT increased while TFL1and FLC decreased.Comprehensive results in this study, we gain the main conclusions:the flowering of C. lavandulifolium is not only controlled by photoperiod pathway, vernalization pathway, GA pathway, autonomous pathway and FRI-dependent pathway, but also MIKC, SBP, YABBY, AFR transcription factor family and basal metabolism such as sucrose and starch metabolism play important roles. These genes were excellent candidate genes to modify Chrysanthemum flowering. DFL has LFY function and might plays key roles in C.lavandulifolium transformation flowering. DenFUL might play key roles in flower development and also involved in fruit and leaves development. These studies preliminarily reveal molecular mechanism of flowering in C.lavandulifolium, laying foundations on genetic modification of Chrysanthemum flowering.