Poster: Genomic & proteomic resources

Abs # 871: Development of a novel versatile vector for gene discovery in Arabidopsis

Presenter:	He, Chengkun , ch226@cornell.edu
Authors	He, Chengkun  (A)   Lin, Zhihong  (A)   Li, Fengling  (A)   Wu, Ray  (A)
Affiliations:	(A): Department of Moleculr Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

We describe here the development of a novel versatile gene trap, Ac/Ds-mediated vector, pDsG7, which can be used to construct a non-redundant, indexed, near-saturation insertional mutant library in dicots and to trap expressed genes. The pDsG7 was introduced into Arabidopsis by Agrobacterium-mediated transformation to produce Ds-containing anchor lines that are inserted in different regions of the Arabidopsis genome. Next, after introducing the Ac gene, a number of transposants can be generated to saturate a 200- to 300-kb region adjacent to a specific anchor location with an insertion every 3-5 kb. Eventually, when each anchor line saturates the region within 200-300 kb, the entire genome can be saturated. To speed up the analysis, we have developed a one-step PCR approach to identify transposition events, to display Ds element reinsertion, empty donor site, and full donor site, and to analyze the stable transposition frequency and efficiency in the F2 generation. By generating many independent insertion lines from several anchor lines, we have obtained plants with diverse patterns of reporter gene expression and isolated an interesting mutant. This Ds insertional mutant flowers very late in long day length condition and has altered plant architecture. The advantage of this new system is that a genomic region around any specific anchor line can be used to generate many mutants to saturate the region and identify mutants that correspond to a specific gene of interest by reverse genetics. In summary, we believe that this novel strategy could be employed for construction of a non-redundant, indexed, near-saturation insertional mutant library for any dicot.