| The optimal timing of transition from vegetative to reproductive growth is crucial for successful sexual reproduction in flowering plants, and is also the central process in the life cycle of the individual plant. Citrus is one of the most important economic fruit trees and has planted world wide. Because of the fact that most of the citrus cultivars have a long juvenile period, coupled with high heterozygosity, makes conventional citrus breeding very difficult. Therefore, to reveal the mechanism of citrus flower development at the molecular level has important theoretical and practical significance for shortening the juvenile phase and improving the efficiency of breeding as well the important agronomic traits of citrus. MADS-box genes and miRNAs play a critical role in reproductive development. In the present study, according to the research hotspot of the model plant flower development, we investigated the function of several key regulatory factors in FLC pathway of MADS-box family and the expression profiling of flowering related miRNAs from precocious trifoliate orange, expecting to further enrich the flowering regulatory network in woody plants, and provide a basis for citrus genetic engineering breeding. The main results are as follows:1. The function and mechanism of PtFLC transcripts. On the basis of the discovery that the PtFLC gene of precocious trifoliate orange involved in alterative splicing, we first isolated the transcripts and full-length DNA of PtFLC gene, and then analyzed the structure and sequence feature in all transcripts. The sequence of PtFLC gene contained a quite long intron between the first two exons. Different coding sequences of the transcript induced different transcriptional activity. Among these transcripts, PtFLCV3and PtFLCV5have stronger transcriptional activity. The flowering statistics of transgenic Arabidopsis for each transcript showed that they have different ability in the inhibition of flowering with related to their transcriptional activity. We also performed yeast two-hybrid screening by using the transcript bait vector, and found different transcript screened different interacting proteins. Furthermore, each yeast bait strain were co-transformed with the screened prey, the results indicated that the interaction between the proteins and each transcript had some universality and specificity, for example, the PtNAD(P) can interact with all transcripts.We also isolated the promoter sequence of PtFLC by genome walking, and predicted various cis-elements in this sequence. From yeast one-hybrid assay the clones that bind to PtFLC promoter directly were obtained, some of them contained the coding region of dormancy breaking related protein. This suggested that PtFLC gene might also play a role in seed germination and reproductive development. To further investigate the expression pattern of PtFLC, the PtFLC::GUS vector was constructed and introduced into Arabidopsis. GUS activity was found in the early stage of seed germination in transgenic Arabidopsis, and then it was presented throughout the whole developmental stages of plant. The expression of PtFLC promoter was observed in almost all tissues on the ground, and it also play a role in the process of reproduction. Furthermore, the activity of this promoter could be inhibited by vernalization, but restored after removing the vernalization.2. Functional analysis of APETALA1-like homologue gene(PtAPl) from precocious trifoliate orange. APETALA1is a sign of flowering transition. Here the cDNA of PtAPl from precocious trifoliate orange was isolated by homology cloning. The sequence alignment and phylogenetic analysis indicated that PtAPl protein falls in the euAPl clade. Expression analysis showed the presence of PtAPl transcription in almost all reproductive tissues, but only faint expression in vegetative tissues, which different from the API in Arabidopsis. Moreover, PtAPl expression was also fluctuated according to the alteration of shoot self-pruning and flowering ability. Ectopic expression of PtAPl resulted early flowering and changed architecture in transgenic Arabidopsis. In addition, the35S::PtAPl transgenic plants partially complemented the apl-1mutant. This showed that PtAPl has functional conservation in flowering time regulation.To further understand the regulatory function of PtAPl, the1000bp upstream promoter of PtAPl was cloned. A number of putative cis-regulatory elements were predicted by the sequence analysis. The GUS activity of PtAPl::GUS transgenic Arabidopsis indicated that the PtAPl promoter had strong activity and showed developmental phase and tissue specificity. Furthermore, we obtained some proteins that bind to PtAPl promoter such as early light-inducible protein (ELIP), gibberellin-regulated protein and so on, by performing yeast one-hybrid screening.3. Expression analysis and functional characterization of PtAGL2’4. The full-length of PtAGL24was isolated from precocious trifoliate orange by RACE method. Alignment of the cDNA with the genomic sequence revealed that it contained eight exons and seven introns. And the deduced amino acid sequence of PtAGL24had the highest similarity (79%identity) to PtrMADS9of Populus trichocarpa over the entire coding region. Phylogenetic analysis showed that PtAGL24was clustered into the clade of AGL24homologues from other species, but seperated with AGL24from Arabidopsis. The transcript of PtAGL24can be detected in most organs of precocious trifoliate orange, with the higher expression in mature flowers. The results of subcellular localization indicated that PtAGL24protein was mainly localized in the nucleus.In order to unravel the biological function of PtAGL24, ectopic expression in Arabidopsis was performed. The transgenic Arabidopsis of35S::PtAGL24were placed into two classes Based on the phenotypic variation. In class I transgenic lines, PtAGL24expression was evidently high, moreover, these plants exhibited early flowering and the morphologic alteration in leaves and reproductive tissues. While the morphology of reproductive tissues in class II transgenic lines was similar to wild-type. PtAGL24can interacted with endogenous AGL24and other AGL24partners in Arabidopsis, this suggested that the inappropriate protein-protein interactions between exogenous and endogenous proteins might result in abnormal morphology directly or indirectly. The expression of PtAGL24promoter could be detected once the radicle break through the seed capsule, and higher GUS activity was shown in seedlings. This promoter can also respond to low temperature induction.4. Comparative profiling of miRNAs related to development in precocious trifoliate orange and its wild-type. Small RNA library construction and Solexa sequencing were performed at different developmental stages on both precocious trifoliate orange (MT) and its wild-type (WT), resulting in the obtainment of141known miRNAs and75novel miRNAs in four libraries. The expression profile analysis revealed that down-regulation of conserved miRNAs appeared to be more important during adult development stage. Some miRNAs that showed different expression patterns between the MT and WT were also found. For75novel identified miRNAs, most of them were only obtained in the MT or the WT. For example,17miRNAs were only detected in two MT libraries, and23miRNAs were only detected in two WT libraries. Among these novel miRNAs,51miRNAs had targets predicted. A total317potential target genes were predicted, and they involved in a broad range of functions such as transcriptional regulation, developmental process, defense response and so on. |
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