Journal of Photosciences

Random Insertional Mutagenesis with Subtracted cDNA Fragments in Arabidopsis thaliana

Volume 7(2000), 103-108 page

저자: Euna Cho, Young Myung Kwon, Ilha Lee

   7-3-103.pdf (529.1K)

Keywords: Random insertional mutagenesis  cDNA subtraction  antisense  posttranscriptional gene silencing


We have evaluated a new mutagenesis strategy called random insertional mutagenesis with subtracted cDNA fragments.
The cDNAs from long day Arabidopsis plants were subtracted by cDNAs from short day plants using PCR based cDNA subtraction.
The subtracted cDNAs were inserted between 35S promoter and 3 -NOS terminator regardless of orientation.
When the cDNA library was used for the random insertion into Arabidopsis genome by Agrobacterium-mediated transformation, approximately 15% of transformants showed abnormal development in leaf, floral organ, shoot apex. When 20 mutants were further analyzed, 12 mutants showed single cDNA fragment insertion and 8 mutants showed more than 2 transgene insertions.
Only two mutants among 12 mutants that have single cDNA insert showed consistent phenotype at T2 generation, suggesting the genetic instability of the mutants.