The Overexpression Vectors Construction Of Pt Flc Transcripts And The Optimization Of Genetic Transformation Systerm Of PtFLC Transcripts

Citrus has a long juvenile phase, at least 8 years, it seriously hindered the progress of breeding, so it is very useful to shorten the juvenile phase. As the only one plant in Citrus which is deciduous in winter, Poncirus Trifoliata(L.) Raf needs to be exposed in winter cold for a period of time(vernalization) before flower formation. FLC is a key gene which is important in the vernalization pathway; its transcription factor inhibits the expression of floral pathway integrator genes. Winter annual plants germinate in the autumn and flowering is repressed before winter, during the cold period, FLC expression is reduced and in the spring the plants have the competence to flower. A FLC homolog gene, Pt FLC was cloned in Poncirus Trifoliata(L.) Raf, and it was alternatively spliced in different phase in plant development, then 5 transcripts were separated. Compared with annual plants, it is a different way to regulate.To verify the function and regulation of Pt FLC in Poncirus Trifoliata(L.) Raf and to create materials for further research, this study constructed expression vector35S::Pt FLC-GUS and 35S::Pt FLC-EGFP. The main results are as follows:1.Successfully constructed the vectors 35S::Pt FLC-GUS and the vectors35S::EGFP and 35S::Pt FLC-EGFP. After enzyme digestion and sequencing, these vectors can be utilized for the next steps of the experiment.2.Subcellular localization of the Pt FLC transcripts in onion epidermal cells via Agrobacterium-mediated shows that Pt FLCv1-EGFP is located in cell nucleus and suggests that Pt FLCv1 might be a transcription factor. Pt FLCv3 is widely distributed in cells, compared with its expression pattern, Pt FLCv3 may have a wider range of functions.3.The genetic transformation of 35S::Pt FLC-GUS into precocious trifoliate orange via Agrobacterium-mediated is performed(hygromycin selection). Now we can get the resistance buds. For the plant cell, hygromycin toxicity is very large,further optimization and testing is needed in hygromycin selection system.4.The genetic transformation of 35S::Pt FLC-EGFP into precocious trifoliate orange via Agrobacterium-mediated is performed(kanamycin selection). At first, we confirmed the GFP fluorescence detection standards, then we got homozygote buds of35S::EGFP and some chimaeriferus buds of 35S::Pt FLC-EGFP with GFP fluorescence detection. The total regeneration efficiency of transformation into precocious trifoliate orange is 29.9%. In the process of the epicotyl genetic transformation via Agrobacterium tumefaciens-mediated, chimeras was emerging, as GFP fluorescence detection showing. How to reduce or avoid the formation of chimeras in the process of the genetic transformation via Agrobacterium tumerfaciens-mediated is one of the most important contents in genetic transformation research.5.Callus of Citrus and tobacco have a huge advantages in validation of gene function research, so the genetic transformation of 35S::Pt FLC-EGFP into tobacco and callus of Citrus ‘Marsh’ via Agrobacterium-mediated is performed. Now we get the resistant callus and buds of tobacco, resistant callus of Citrus ‘Marsh’.