Insertional mutagenesis in rice using the endogenous retrotransposon
2003
Hirochika, H.
Insertional mutagenesis is a powerful method for a systematic functional analysis of a large number of genes. In rice, five endogenous active retrotransposons have been found and the most active, Tos17, was characterized in detail. Tos17 is silent under normal conditions and becomes active only under tissue culture conditions. Tos17 was shown to transpose preferentially into low-copy-number, gene-rich regions, indicating that Tos17 can be used as an efficient insertional mutagen. A collection of 50,000 regenerated rice lines carrying about 500,000 insertions was produced and these lines are being used for forward and reverse genetic analyses. By using a transposon-tagging strategy, causative genes for more than 20 mutations have been cloned. For reverse genetic studies, two strategies are employed. One is polymerase chain reaction (PCR) screening of mutants of genes of interest. DNA pools derived from 40,000 lines have been produced for screening. By screening 31,000 lines for mutants of 53 genes, mutants of 17 genes have been isolated. Another important strategy is the cataloguing of mutants by sequencing the genomic sequences flanking Tos17 insertions. Sequences flanking transposed Tos17 copies are amplified by TAIL (thermal asymmetric interlaced PCR)- and suppression-PCR and directly sequenced. As of June 2002, 14, 300 independent flanking sequences from 3,700 lines have been determined and mutants of different classes of genes have been identified.
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