The Establishment Of The Rice Promoter Trapping System

We had previously constructed two kinds of promoter trapping plasmids based on T-DNA (GUS) and Ac/Ds (GUS), respectively. The coding region of promoterless GUS reporter gene was inserted into the T-DNA region of pCAMBIA1300 to construct the T-DNA (GUS)-based plasmid pl3GUS. The plasmid pl3GUS was introduced into calli of Oryza sativa subsp. japonica cv. Zhonghua 11 and then 31 transgenic plants showing GUS activity were obtained by histochemical staning. Homozygotes in some of them were derived by screening with hygromycin.Construction of the Ac/Ds (GUS)-based plasmid pl3B is as follow: Both left and right border of Ds element were inserted into the T-DNA region of vector pCAMBIA1300, respectively. The promoterless GUS gene and promoterless Ac transposase gene located between the two borders of Ds element. The orientations of the GUS gene and Ac transponase gene are opposite and the 5' end of GUS gene is closed to the left border of Ds element. The promoter sequence of Ubiquitins was linked outside of the right border of Ds, while the promoterless Bar gene was linked outside of the left border of Ds. The orientations of the Ubiquitons' promoter, promoterless Ac and Bar gene are identical. When the plasmid pl3B is integrated into rice genome after being transformed, transponase activity of Ac will be driven by Ubiquitins' promoter to make Ds element transpose from the initial insertion site. Meanwhile Ac element is also carried to the new site and separated from the Ubiquitons' promoter, and the Ds element becomes stable at the new site. Then the affair of transposition can be screened by PPT conveniently and quickly because Bar gene express was driven by the Ubiquitons' promoter.My work was further focused on constructing of another kind of promoter trapping plasmid containing the report gene GFP and analyzing of GUS-positive transgenic plants:Firstly, the coding region of promoterless GFP reporter gene was inserted into the T-DNA region of vector pCAMBIA1300 in turn to construct promoter trapping plasmid pl3EGFP. The plasmid pl3EGFP was introduced into calli of Oryza sativa subsp. japonica cv. Zhonghua 11 and then 363 transgenic plants were obtained. Sreening by flurescent co-focal microscope and hygromycin, GFP activity was observed in 5 homozygous transgenic plants.Secondary, the plasmid p13B was introduced into calli of Oryza sativa subsp. japonica cv. Zhonghua 11 by Agrobacterium tumefaciens-mediated transforming. Eighteen independent transgenic lines were obtained and propagated. T2 generationsof 18 independent transgenic lines were screening by herbicide (Basta) and the derived herbicide-resistant plants were analyzed by PCR. Ds element transposed in a hereditable manner was found in 37 plants, in which 5 plants showed GUS activity.Thirdly, the copy number of 8 homozygous transgenic plants showing GUS activity was estimated by real time quantitative PCR and the flanking sequences were analyzed by Inverse PCR.