T‐DNA insertional mutagenesis for functional genomics in rice

Abstract

SummaryWe have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless b-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature¯owers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6±2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ-or tissue-speci®c or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Molecular characterization, expression pattern, and function analysis of the OsBC1L family in rice

“…The GUS expression pattern mimicks the target gene in the promoter trap lines (Jeon et al 2000;Jeong et al 2002), and the GUS expression pattern in the OsBC1L3 promoter trap line is similar with the results from RT-PCR and microarray analysis (mainly in culms, roots, and sheaths; Supplementary Fig. 1).…”

“…Completion of the rice genome sequence has enabled gene identification and functional analysis on a large scale. In order to elucidate the gene function in rice genome, a global effort to generate insertional mutants, mainly by T-DNA and transposable elements insertion, has been carried out and a large number of mutants has been collected in the last few years (Hirochika et al 2004;Jeon et al 2000;Martienssen 1998;Osborne and Baker 1995;Wu et al 2003). Further, additional research tools of functional genomics in rice, such as full-length cDNA collection and whole genome expression profiling, have been established and have enhanced the pace for elucidating gene function throughout the genome .…”

Impaired Function of the Tonoplast-Localized Sucrose Transporter in Rice, OsSUT2, Limits the Transport of Vacuolar Reserve Sucrose and Affects Plant Growth

“…To produce transgenic rice (cv Hwayoung) plants expressing CaMV35S:: OsSUT2-GFP and pOsSUT2::GUS constructs, Agrobacterium tumefaciens LBA4404 strains harboring each of these vectors were grown on Agrobacterium broth medium supplemented with 25 mg L 21 kanamycin for 3 d at 28°C, and transgenic calli were obtained via the Agrobacterium-mediated cocultivation method as described previously (Jeon et al, 2000). Transgenic rice plants were regenerated from the transformed calli on selection medium containing 50 mg L 21 hygromycin and 250 mg L 21 cefotaxime.…”

RNA interference for wheat functional gene analysis

“…In Arabidopsis and rice, large gene knockout collections have been assembled from chemical mutagenesis and T-DNA or transposon insertional mutagenesis (Jeon et al 2000;Alonso et al 2003;Sallaud et al 2004). In wheat, a powerful reverse genetics approach was recently implemented through the combination of EMS-mediated mutagenesis and TILLING technology (Slade et al 2005).…”