The Application Of MGFP4 And Cre/loxP System In The Transgenic Rice

With the development of transgenic technology in plant, it has become a developing direction in plant genetic engineering in the world that to set up a practical and effective genetic transformation system with marker free and to cultivate maker-free transgenic crops. To optimize the maker-free system established by our laboratory, two T-DNAs contrformation systems, the present experiment studied the expression of GFP and GFP-Hyg fusion protein in the trangenic rice. At the same time, we also detected the deletion efficiency of Cre/loxP system, which made preparations for setting up a new maker-free system(Cre/loxP site-specific system) in transgenic rice.Four constructs of plant expression vetctor carrying mgfp4 gene were introduced into rice Minghui 86(Indica Sativa L.) mediated by Agrobacterium in the present experiment. We have obtained 522 independent clones' transgenic plants. PCR and southern blot analysis showed that exogenous genes were most integrated into rice genome at lower copies. GFP was proved to be expressed in the transgenic rice via fluorescence microscope and RT-PCR detectation, even though we could not get a good result of green fluorescence. Based on PCR analysis, we presumed that the ere gene might be expressed under the control of promoter wcs and it could carry out the deletion of exogenous genes.In addition, a new rapid and simple method of preparing PCR template with alkali-treated was established in this study, which was superior to CTAB. It had more practical significance in screening and identifying transgenic rice on a large scale.