Poster: Emerging Technologies

Abs # 881: Gateway Binary Vectors (pGWBs) -Application and Improved pGWBs-

Presenter:	Nakagawa, Tsuyoshi , tnakagaw@life.shimane-u.ac.jp
Authors	Nakagawa, Tsuyoshi  (A)   Kurose, Takayuki  (A)   Jinbo, Tetsurou  (B)   Kimura, Tetsuya  (B)
Affiliations:	(A): Res Inst Mol Genet, Shimane University (B): Fac Biores, Mie University

Previously, we reported binary vectors applicable for Gateway Cloning Technology. They included reporter/tag (GUS, GFP, 6His, FLAG, 3HA, 4Myc, 10Myc and GST)-fusion ones. In this paper, we report the application of these vectors. We made GUS entry clone and introduced it into all kinds of 35S driven pGWBs to obtain fusion genes, GUS-GFP, GFP-GUS, GUS-6His, 6His-GUS, and so on. All of them were introduced into tobacco BY-2 cells, and expression of fusion proteins were analyzed by immunoblotting using anti-GUS antibody and anti-tag antibodies. In all constructs except for GUS-3HA, we confirmed expression of tag-added GUS proteins. We also detected GUS activity in each constructs. In this time, we made new pGWB seriese which have Nos-pro::HPT::Nos-Ter as a hygromycin resistant marker. Because previous pGWBs had 35S-pro::HPT::Nos-Ter (reverse orientatio to reduce the effect of 35S promoter against the expression of cloned gene), we replaced it to the Nos promoter version to remove the effect of 35S promoter completely. These new pGWBs should have an advantage in the experiments using native promoter.