The Transformation And Function Analysis Of PtFLC Alternatively Spliced Transcripts

Trifoliate orange, as the only deciduous plant of Citrus, needs to be exposed in winter cold before flower formation. This phenomenon is closely linked to the expression of vernalization-related genes. FLOWERING LOCUS C(FLC) is an important flowering inhibiting factor in plant vernalization pathway. FLC were alternatively spliced in different phase of trifoliate orange development. Five FLC homolog transcripts were cloned from trifoliate orange, respectively named PtFLCv1～PtFLCv5.PtFLCv1and PtFLCv2were expressed only in the juvenile stage, while PtFLCv4and PtFLCv5were expressed in the adult stage. PtFLCv3was expressed in the whole growth process. In order to verify the function of different PtFLC transcripts in regulating the development of Citrus, this study constructed over-expression vector of PtFLCv1～PtFLCvs and over-express PtFLCv1～PtFLCv5in precocious trifoliate orange and pomelo. Furthermore, we preliminarily analyzed the expression of different transcripts and attempted to find out the possible diversity function among the five transcripts. The main results are as follows:1. Constructed overexpression vector of PtFLCv1～PtFLCv5and genetic transformation. Including35S::PtFLCv1-GFP-PtFLCv5-GFP and35S::PtFLCvl-FLAG-PtFLCv5-FLAG. Generate two precocious trifoliate orange transgenic lines over-expressing PtFLCv4and PtFLCv5. Over-expression plants were detected with PCR on the levels of DNA and expression detection on the levels of RNA.2. Analyzed the expression of PtFLC in transgenic plants. Using Real-time quantitative analysis, the PtFLCv4and PtFLCv5transgenic plants showed higher expression level of PtFLC, when compared with wild type controls. In three PtFLCv5positive plants, the expression level is more than5000folds than the one of the control, while PtFLCv4transgenic plants over-expressed20～300folds than the expression in wild-type.3. Examined the PtSOC1, PtFT expression in transgenic plants. SOC1and FT are downstream target genes and down-regulated by FLC. In this study, the results showed that all PtSOC1expression level in the transgenic plants was ten times as low as that in the control group, indicating PtSOCl was really strictly down-regulated by PtFLC. The down-regulation degree of PtFT in two different transcriptions was not consistent with each other, which might result from different exons constitution of PtFLC transcripts.4. Transformed the genes PtFLCv1～PtFLCv5into pomelo callus. After screening, we got four transcription types of resistant callus, including pCAMBIA1302、PtFLCv1、 PtFLCv2、PtFLCv5.