| Chrysanthemum morifolium is both a traditional famous flower in Chinese history and an important cut flower in the world. The ground-cover Chrysanthemum used in this study is a kind of cultivar owning great application value due to the characters of low height, dense flowers and excellent resistance. Currently, transgenic technology, which has become an important means of breeding, possesses huge potential in the directed improvement of plant traits. On the basis of a flowering-related gene CmFT cloned by our research team recently, this study constructed a plant expression vector named pBI121-CRE-CmFT, trying to obtain new cultivars of photoperiod insensitive to Chrysanthemum by further transformation experiments. However, The safety of transgenic plants acquires increasing concerns and doubts. With aim to solve this problem, this study constructed a plant safety expression vector named pBI121-CRE combined with Cre/loxP site-specific recombination system, managed to obtained a safe transgenic cultivar of Chrysanthemum by the deletion of selectable marker gene. The main results are as follows:1.A Cre/loxP induction deletion component B2939Gn was designed. In this component, CRE gene is regulated by the inducible promoter rd29A. The promoter can be activated in the stress condition and causes the expression of its downstream gene CRE. CRE enzyme is able to identify the two loxP sites in the same direction, which will cause the recombination reaction with all genes between the two sites deleted including the npt II.2.With the vector pBI121and pBI121-CmFT as basic skeleton, accompanied by the B2939Gn as core component, this study constructed two plant safety expression vectors pBI121-CRE and pBI121-CRE-CmFT, and then they were transformed to Agrobacterium tumefaciens respectively.3.Three positive transgenic seedlings were obtained through transformation to ’Wan Fen’ using the A. tumefaciens with pBI121-CRE. The further GUS and PCR experiments proved that the rd29A had been induced and the selectable marker gene npt II had been deleted after the stress-induction to the transgenic seedlings. Besides, the transformation experiment with the A. tumefaciens containing pBI121-CRE-CmFT is ongoing. |
