Retrotransposons of rice as a tool for the functional analysis of genes.
2001
Hirochika, H. | Miyao, A. | Yamazaki, M. | Takeda, S. | Abe, K. | Hirochika, R. | Agrawal, G.K. | Watanabe, T. | Sugimoto, K. | Sasaki, T. | Murata, K. Tanaka, K. | Onosato, K. | Miyazaki, A. | Tamashita, Y. and Kojima, N.
Five endogenous active retrotransposons have been found in rice. Among them, the most active one, called Tos17, was characterized in detail. Tos17 is silent under normal conditions and becomes active only under tissue culture conditions. Five to 30 transposed Tos17 copies were found in each plant regenerated from culture. 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 32,000 regenerated rice lines carrying about 256,000 insertions was generated, and these lines are being used for forward and reverse genetic analyses. By using a transposon-tagging strategy, causative genes for viviparous, dwarf, semidwarf, brittle culm, pale green, and narrow leaf mutations, among others, have been cloned. For reverse genetic studies, two strategies are being employed. One is the polymerase chain reaction (PCR) screening of mutants of the gene of interest. We screened 12,000 lines and found mutants of 15 genes, including MAPK, MADS-box, and P450 genes, among the 47 genes analyzed. This suggests that at least 37,000 lines are required for saturation mutagenesis. Another important strategy is the random sequencing of mutated genes by isolating the sequences flanking transposed Tos17. The flanking sequences are amplified by TAIL (thermal asymmetric interlaced)- and suppression-PCR and directly sequenced. Until now, 7,376 independent flanking sequences from 2,134 lines have been determined and mutants of different classes of genes have been identified.
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