Molecular analysis of rice plants harboring an Ac/Ds transposable element‐mediated gene trapping system

Abstract

SummaryIn rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging ef®ciency. The 3¢ end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identi®ed at the 3¢ end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.

State IIDissociationElement Formation FollowingActivatorExcision in Maize

“…For instance, we are currently amplifying germinal transpositions of the Ds resident at the r1-sc:m3 locus, by exploiting the unique breakpoint in the Ds6-like element resident as a molecular signature in IPCR (http:/ /www.plantgdb.org/prj/AcDsTagging/). Two component systems have been used for gene characterization extensively in Arabidopsis, tomato and rice (for example, Bancroft et al 1993;Jones et al 1994;James et al 1995;Sundaresan et al 1995;Chin et al 1999;Meissner et al 1999). However, in maize only two genes, Indeterminate1 and Knotted1, have been cloned using a two-component directed tagging approach with Ds (Hake et al 1989;Colasanti et al 1998).…”

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

“…Ac/Ds tagging systems have been utilized to isolate a number of genes (Bancroft et al 1993;Jones et al 1994;James et al 1995;Bhatt et al 1996;Meissner et al 1999), to define promoter and enhancer elements (Sundaresan et al 1995;Chin et al 1999;Greco et al 2003;Wu et al 2003;Jin et al 2004), and to create genetic mosaics for clonal analysis (Peng and Harberd 1997;Jenik and Irish 2001). The tendency of Ds to transpose to linked sites has also been exploited to disrupt genes present in tandem arrays (Takken et al 1999;Tantikanjana et al 2004) and to perform localized saturation mutagenesis (Nishal et al 2005).…”

“…The tendency of Ds to transpose to linked sites has also been exploited to disrupt genes present in tandem arrays (Takken et al 1999;Tantikanjana et al 2004) and to perform localized saturation mutagenesis (Nishal et al 2005). Ac/Ds mutagenesis platforms in Arabidopsis (Sundaresan et al 1995;Ito et al 1999;Muskett et al 2003;Kuromori et al 2004), tomato (Healy et al 1993;Knapp et al 1994;Meissner et al 2000), tobacco (Scofield et al 1992), rice (Chin et al 1999;Zheng-Ge et al 2003;Kim et al 2004;Kolesnik et al 2004), and barley (Koprek et al 2000;Cooper et al 2004) have been developed. Yet, despite the demonstrated utility of Ds in gene tagging in maize (Hake et al 1989;Colasanti et al 1998), a two-component Ac/Ds genetagging program for this important crop plant has not yet been developed.…”

Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

“…Slides were covered with a precleaned lifter slip (Erie Scientific Company, Portsmouth, NH) and placed in a hybridization cassette with 15 l of distilled water in each chamber to maintain humidity. The cassette was sealed and placed in a water bath at 42°C for [16][17][18] h. The slides were rinsed at room temperature in 2ϫ SSC and 0.2% SDS for 3 min, followed by a 2-min rinse in 1ϫ SSC, 1 min in 0.2ϫ SSC, and, finally, a 15-sec rinse in 0.05ϫ SSC. The slides were spun-dried and scanned immediately in an Axon scanner (Axon Instruments, Foster City, CA).…”

“…Phenotypic information about gene function often is sought through the analysis of loss-or gain-of-function mutations resulting from DNA insertions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Transposons, reporter cassettes, gene traps, promoter traps, and Agrobacterium T-DNAs all have been used as insertional mutagens in different organisms (1,2,5,6,(17)(18)(19).…”