| Bud differentiation indicates one plant is undergoing the transformation from vegetative growth to reproductive growth. It is not only a shape establishing process but also an extremely complex biological process. The process of bud differentiation is usually involved by a large number of genes and to form a complex regulatory network. Flowering time depends on the time of bud differentiation is early or later. Floral transition is an irreversible process and could be infected by many factors, in which, temperature as one of the exogenous regulatory factors plays important role in the bud differentiation process. In this study, as the target materials, the bud differentiation processes of Agapanthus praecox ssp. orientalis under various cultivation conditions were observed clearly with the anatomical structures. Many techniques such as transcriptomics (RNA-seq) were applied to analyze the gene expression profiles of buds which from different cultivation conditions and differentiation stages. On this basis, the key genes affect the flower transformation process of Agapanthus praecox ssp. orientalis were obtained. Furthermore, genetic resources were also provided to regulate the blooming of Agapanthus praecox ssp. orientalis by the molecular method in this work. The main conclusions are as follows:1. The bud differentiation process of Agapanthus praecox ssp. orientalis was divided into6stages by observing the anatomical structures:vegetative period, induction period, late induction, early morphological differentiation, morphological differentiation and flower bud differentiation period. Comparing to the Agapanthus praecox ssp. orientalis plants which were cultivated in a greenhouse, it was found that the time of buds started to differentiate was brought forward significantly, the differentiation rate was accelerated and the florescence took place about50to66days ahead of schedule when the buds were cultivated with temperature-controlled method in their low-temperature vegetative growth period. There was no significant distinction within the differentiation processes of buds from the exogenous hormone treatment group and the control group, and the florescence of GA group occurred10days in advance. The floret number, inflorescence diameter, plant height and organ development degree of Agapanthus praecox ssp. orientalis from different cultivation conditions varied clearly. The floret number and inflorescence diameter from the thermostat treatment group was34.7%and11.2%less than that from the controlled group. Meanwhile, the floret number and plant height from PP333treatment group was increased32%and decreased29.4%than that from the controlled group, respectively.2. In this work, a mass of Unigene information was obtained from the previous transcriptome library and the RNA-seq technique was applied to analyze the expression profiles of floral gene from the varying stages of bud differentiation. The results showed that, there were many differential expressed genes in the buds from the varying differentiation stages. The differential expressed genes in buds of vegetative and other differentiation stages ranged from4310to7105with1957differential co-expressed genes. The specific gene expression in induction and inflorescence buds was2655and1895, respectively. The functions of differentially expressed genes GO mainly enriched in plasma membrane, cell wall, cytoskeleton, binding, transcription regulator activity, catalytic activity, reproduction, biological regulation and regulation of metabolic process. The specific expressed differential genes from induction and inflorescence buds showed significant distinction in the following functions:transcription, protein modification, cell cycle, gene expression and epigenetic regulation, flower development and response to biotic stimulus. PageMan biological functional annotation analysis showed that1957co-expressed genes had strong influence on the RNA transcription process, chromatin structure and epigenetic regulation. On the contrary, it had less effect on the metabolism of substance and energy, secondary metabolism and energy transport. The endogenous hormones such as IAA, GA and ABA and genes from more than20transcription factor families were involved in the flower transformation process of Agapanthus praecox ssp. orientalis. The pathways enriched significantly with differential expressed genes including signal transduction of plant hormone, DNA replication and the metabolism of starch and sucrose. Totally34important flowering genes related to the blossom way of Arabidopsis thaliana were identified in this work and most of them showed an up-regulated expression in the entire bud differentiation period of Agapanthus praecox ssp. orientalis.3. The buds of Agapanthus praecox ssp. orientalis with temperature-controlled treatment and advanced florescence were analyzed by RNA-seq method. Comparing to the simultaneous samples from a greenhouse, the differential expressed genes ranged from2778to4173. The expressed genes of ethylene and cytokinin signals showed up-regulated and the IAA signal was relative weak. The pathways enriched significantly with differential expressed genes including metabolism, biosynthesis of secondary metabolites, mutual transformation of pentose and glucuronic acid, metabolism of starch and sucrose, DNA replication and protein processing in endoplasmic reticulum. Some bud samples with temperature-controlled treatment were studied, and the Mapman analysis indicated that the specific expressed genes were up-regulated in the biological processes of lipid degradation, sucrose degradation, photorespiration, modification, the metabolism of cell wall and amino acids, and were down-regulated in glycolysis, Calvin cycle and FA synthesis. Massive genes involved in the photoperiod and gibberellin signal transduction pathways during the temperature controlling process, which may be closely associated with the bud differentiation of Agapanthus praecox ssp. orientalis.4. The homologous genes of Agapanthus praecox ssp. orientalis CO, FT and VIN3were obtained by using the RACE technique and named ApCOL, ApFT and ApVIN3. The results of real-time fluorescence quantitative analysis suggested that these3gene expressions in the tissues and organs of different developing stages were spatial different. The expressions of ApCOL and ApFT in leaves were higher than those in the shoot tip. The expression of ApFT in the induction period buds higher than that in the vegetative and bud differentiation period. The expression amount of ApVIN3in the shoot tip was regulated with temperature, and it increased with the lasting of low temperature. These three genes may play an important role in the flowering induction process of Agapanthus praecox, and their functions and regulatory mechanisms should be investigated in depth. |
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