A rice gene activation/knockout mutant resource for high throughput functional genomics
Using transfer DNA (T-DNA) with functions of gene trap and gene knockout and activation tagging, a mutant population containing 55,000 lines was generated. Approximately 81% of this population carries 1–2 T-DNA copies per line, and the retrotransposon Tos17 was mostly inactive in this population during tissue culture. A total of 11,992 flanking sequence tags (FSTs) have been obtained and assigned to the rice genome. T-DNA was preferentially (∼80%) integrated into genic regions. A total of 19,000 FSTs pooled from this and another T-DNA tagged population were analyzed and compared with 18,000 FSTs from a Tos17 tagged population. There was difference in preference for integrations into genic, coding, and flanking regions, as well as repetitive sequences and centromeric regions, between T-DNA and Tos17; however, T-DNA integration was more evenly distributed in the rice genome than Tos17. Our T-DNA contains an enhancer octamer next to the left border, expression of genes within genetics distances of 12.5 kb was enhanced. For example, the normal height of a severe dwarf mutant, with its gibberellin 2-oxidase (GA2ox) gene being activated by T-DNA, was restored upon GA treatment, indicating GA2ox was one of the key enzymes regulating the endogenous level of GA. Our T-DNA also contains a promoterless GUS gene next to the right border. GUS activity screening facilitated identification of genes responsive to various stresses and those regulated temporally and spatially in large scale with high frequency. Our mutant population offers a highly valuable resource for high throughput rice functional analyses using both forward and reverse genetic approaches.
KeywordsRice T-DNA Gene knockout Gene activation Gene trap Mutant Flanking sequence tag
We thank Drs Tuan-Hua David Ho, Anthony H.C. Huang, and Kenrick Deen for their critical review of this manuscript and Drs Hua-Mei Chang and Peng-Wen Chen and Mr Cheng-Tung Lo from Vita Genomics, and Ms Lin-Chih Yu for technical assistance. This work was supported by grants from Academia Sinica (AS91IB2PP, AS90-AB-IMB-01, AS91-AB-IMB-01, AS92-AB-IMB-03, and AS93-AB-IMB-02 to Su-May Yu and 92S510921 and 92S508932-G to Yue-Ie Hsing) and the National Science Council (NSC92-2317-B001-032 and NSC92-2317-B-001-036 to Su-May Yu) of the Republic of China.
- Ichikawa T, Nakazawa M, Kawashima M, Muto S, Gohda K, Suzuki K, Ishikawa A, Kobayashi H, Yoshizumi T, Tsumoto Y, Tsuhara Y, Iizumi H, Goto Y, Matsui M (2003) Sequence database of 1172 T-DNA insertion sites in Arabidopsis activation-tagging lines that showed phenotypes in T1 generation. Plant J 36:421–429PubMedCrossRefGoogle Scholar
- International Rice Genome Sequencing Project (IRGSP) (2005) The map-based sequence of the rice genome. Nature 436:793–800Google Scholar
- Liu ZL, Han FP, Tan M, Shan XH, Dong YZ, Wang XZ, Fedak G, Hao S, Liu B (2004) Activation of a rice endogenous retrotransposon Tos17 in tissue culture is accompanied by cytosine demethylation and causes heritable alteration in methylation pattern of flanking genomic regions. Theor Appl Genet 109:200–209PubMedCrossRefGoogle Scholar
- Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y, Onosato K, Hirochika H (2003) Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 15:1771–1780PubMedCrossRefGoogle Scholar
- Sakamoto T, Miura K, Itoh H, Tatsumi T, Ueguchi-Tanaka M, Ishiyama K, Kobayashi M, Agrawal GK, Takeda S, Abe K, Miyao A, Hirochika H, Kitano H, Ashikari M, Matsuoka M (2004) An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol 134:1642–1653PubMedCrossRefGoogle Scholar
- Sallaud C, Gay C, Larmande P, Bes M, Piffanelli P, Piegu B, Droc G, Regad F, Bourgeois E, Meynard D, Perin C, Sabau X, Ghesquiere A, Glaszmann JC, Delseny M, Guiderdoni E (2004) High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. Plant J 39:450–464PubMedCrossRefGoogle Scholar
- Sunohara H, Kitano H (2003) New dwarf mutant controlled by a dominant gene, Twisted dwarf1. Rice Genet Newsl 20:20–21Google Scholar
- Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, Fankhauser C, Ferrandiz C, Kardailsky I, Malancharuvil EJ, Neff MM, Nguyen JT, Sato S, Wang ZY, Xia Y, Dixon RA, Harrison MJ, Lamb CJ, Yanofsky MF, Chory J (2000) Activation tagging in Arabidopsis. Plant Physiol 122:1003–1013PubMedCrossRefGoogle Scholar