Structural Analysis And Functional Vermcation Of FT-like Gene In Precocious Trifoliate Orange

Citrus occupies an important role in the fruit production. It has a very long juvenile phase which can reach6-8years, this seriously hindered the progress of genetic breeding. Thus how to shorten the juvenile phase is gaining more attention. In1976, a mutational trifoliate orange-precocious trifoliate orange was found in YiChang, HuBei, which can blossom in2years after germination and then flowered twice or three times per year, this provides a good material for citrus breeding research. Here we got2different PtFT cDNA when cloning the key flowering gene FT in precocious trifoliate orange, named PtFTl and PtFT2. According to the comparison with other normal gene, we designed PtFT3and PtFT4. Therefore we constructed five expression vectors including4PtFT-like genes and a PtFT DNA. To analyze the relations the between function of PtFT-like genes and their structures in precocious trifoliate orange, ectopic expression analysis in model plant tobacco were performed. At the same time, in order to observe the influence of this gene in citrus and create materials for further research, we also transform the PtFT in citrus. The main results are as follows:1. The structure analysis of PtFT-like genes showed that the full length of PtFT DNA contained4exons and3introns, PtFTl is the mature cDNA sequence of PtFT, has534bp and include4exons. When we cloned the PtFT1, another sequence, PtFT1was also obtained, which only missing the first intron compared with PtFT DNA, while retained2introns compared with PtFTl. The transcription of PtFT2was terminated in advance at the second intron and formed PtFT3. We concluded that the main regions for the function of PtFT were in the former two exons. But there are some research suggested that the fourth exon of the FT gene may also play an important role, therefore we got the redundant sequence as PtFT4based on the PtFT3.2. Expression vectors of35S::PtFT DNA,35S::PtFTl and35S::PtFT4were constructed and transformed into tobacco. Transgenic lines were obtained and then positive detection from the level of DNA and transcriptional level respectively were also performed.3. The phenotypic analysis of transgenic lines. The analysis of the flowering time and leaf numbers of T2generation of transgenic tobaccos showed that35S::PtFT DNA,35S::PtFT1and35S::PtFT2can make transgenic tobaccos flower90-100days earlier compared to wild type. When the plants growing up to5-centimeters-tall and had4-5leaves, the transgenic tobaccos began to form the first blossom, while the wild type tobacco flowering needed130days and19leaves. We also found that these transgenic lines showed a phenotype of multiple-branches obviously. But the flowering time of35S::PtFT3and35S::PtFT4transgenic tobaccos was the same as wild type. The results indicate that the first intron of PtFT probably plays a role in transcriptional regulation. It do not affect the function of the FT gene, but the sequences result from terminate transcription and redundant sequences had lost the function. The integrity of exons in a gene was the prerequisite of its function.4. We got the35S::PtFT1transgenic trifoliate orange by the method of Agrobacterium-mediated. Positive identification of the transgenic plants through specific primers and RT-PCR respectively, proved that PtFT1had been integrated into the genome of trifoliate orange. But the transgenic plants were too small, the phenotype remains to be seen.5. The optimization of genetic transformation system of Valencia Orange. We analyzed the impact of different hormone and concentration to the genetic transformation system, the results indicated that the most appropriate medium for bud induction culture was MT+1.0mg/L6-BA, and the optimal culture medium for root induction was1/2MT+3.0mg/L IB A.