Piscataway, New Jersey
Rutgers University
A set of transgenic maize lines for localized mutagenesis based on the Ac-Ds transposon system --Dooner, HK The goals of our NSF Plant Genome project are: (a) to demonstrate that the transposon Ac (Activator) can be used as a gene identification and isolation tool, as well as a mutagen, in the complex maize genome, and (b) to develop the necessary tools to facilitate that use.

In the first phase of the project we confirmed that Ac inserts exclusively in or close to genes and is, therefore, an excellent gene-searching engine in the highly repetitive maize genome (Cowperthwaite et al., The Plant Cell 14: in press, 2002). A collection of 1225 independent germinal Ac transposants from the mutable allele wx-m7(Ac) was generated and deposited in the Maize Stock Center. All transposed Acs were mapped relative to the wx donor locus. In parallel, PCR methods were adapted for the isolation of DNA adjacent to the insertion (tac sites). Over 40 tac sites were isolated, sequenced, and deposited in GenBank, after confirming that they corresponded to bona fide germinal transpositions. All detected either one or two bands in genomic Southern blots and 54% had homology to sequences in the databases. Expression of most of these putative genes was confirmed by Northern blots. tac sites from several Ac-tagged mutants were also isolated, but most Ac insertions did not cause obvious phenotypes, as could have been anticipated from the highly duplicate nature of the maize genome (Gaut, B.S., Gen. Research 11,:55, 2001). Besides, Ac insertions differed significantly in the subtlety of their mutant phenotypes. At one end was an insertion in a chloroplast ribosomal protein gene, which resulted in a lethal embryo. At the other, was an insertion in a sesquiterpenoid cyclase gene involved in an indirect defense response against insects, which required special assays for detection (Shen, B. et al., Proc. Natl. Acad. Sci. USA 97:14807-14812, 2000). In addition, we began transformation experiments to eventually produce a comprehensive set of transgenic maize lines that will facilitate the isolation of genes from any location in the genome. A highly embryogenic bz wx inbred line was developed and transformed with Ac* and Ds* constructs that had been modified to facilitate the PCR isolation of the transposon-adjacent sequence.

The main goal of the second phase of this project is to create a comprehensive set of transgenic lines that will serve as starting points for the production of future insertion libraries. First, we will demonstrate the germinal transposition of an engineered Ac* or Ds* element and the ready isolation of the DNA adjacent to the transposon. This will be accomplished in transgenic plants transformed with either uniquely marked Ac* or Ds* elements. An Agrobacterium-based method is necessary for the former, whereas a biolistics method is sufficient for the latter. Upon deciding on a particular combination of transposon and transformation method, we will proceed to create a set of 124 transgenic lines with a uniquely marked Ac* or Ds* element at regularly spaced locations in the genome. In this set, most genes in the maize genome will be within 7 cM of a launching platform and will be, therefore, realistic targets in a localized transposon mutagenesis experiment. These lines will be deposited in the Maize Stock Center and will serve as starting materials for the generation of future insertion libraries by interested scientists.

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