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T-DNA Insertional Mutagenesis for Activation Tagging in Rice1

Summary: T-DNA Insertional Mutagenesis for Activation
Tagging in Rice1
Dong-Hoon Jeong, Suyoung An, Hong-Gyu Kang, Sunok Moon, Jong-Jin Han, Sunhee Park,
Hyun Sook Lee, Kyungsook An, and Gynheung An*
Department of Life Science and National Research Laboratory of Plant Functional Genomics,
Pohang University of Science and Technology, Pohang 790784, Republic of Korea
We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice
(Oryza sativa) genes. The binary vector contains the promoterless -glucuronidase (GUS) reporter gene next to the right
border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located
next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS
assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed
with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present
in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected
pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly.
Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter
activity using the gus reporter, as well as for creating gain-of-function mutants.
Recent completion of the draft sequence for the rice
(Oryza sativa) genome has resulted in an explosion of
information on rice genes (Goff et al., 2002; Yu et al.,
2002). The challenge for the post-sequencing era is to


Source: An, Gynheung - Department of Life Science, Pohang University of Science and Technology


Collections: Biotechnology; Biology and Medicine