Construct The Pyba Plant Binary Vectors And Functional Analyse The Alcm Promoter Of Aspergillus Nidulans In Arabidopsis

Binary vectors are the basic tools of Agrobacterium-mediated plant transformation protocols. In order to facilitate gene operation and application in plant transformation, and can easily excise the selectable markers after obtaining transgenic plants, a set of versatile plant binary vectors pYBA series are constructed, which meet the demands for producing bio-safety transgenic plants. The fusion PCR method was used in vector construction for eliminating all the nonessential sequences. The pYBA is high copy vector in Escherichia coli, and the backbone is minimized3.69kb. The pYBA100vector is5.37kb, including a NptⅡ plant selectable marker gene cassette; the pYBA200vector is5.60kb, including a Hyg plant selectable marker gene cassette; the pYBA300vector is5.12kb, including a Bar plant selectable marker gene cassette.They all contain22unique multiple cloning sites which is convenient for genetic engineering operation. Both sides of the selectable marker cassette of T-DNA have the LoxP-FRT sites, which can conveniently excise the plant selectable marker cassette via Cre or FLP recombinase after obtaining the transgenic plants. The pYBA vectors can replicate in Escherichia coli and Agrobacterium, and has been successful transformed to Arabidopsis thaliana. They are consistent with the requirements of Agrobacterium mediated plant transformation. In vitro assay of pYBA100demonstrates the plant selectable marker cassette of the pYBA vectors can be excised by Cre recombinase.Ethanol-induced AlcR/alcA expression system is widely used in research. In order to obtain the new ethanol-induced system with independent patent. in this study we cloned alcM(S-M) full-length promoter from Aspergillus nidulans genome, constructed pBBB-AlcR-SM:GUS expression vector. It is transferred to Arabidopsis by Agrobacterium-mediated inflorescence dip method. The GUS staining shown the whole plant was blue after ethanol-induced. while the uninduced seedling only shown the blue in roots. When SM promoter in Arabidopsis induced by ethanol. the downstream gene is strongly expressed.