Rice Genetics IV - IRRI books - International Rice Research Institute

Ablation of the sexual embryoIsolation of genes capable of ablating the sexual embryoThe gene most commonly used for cellular ablation experiments encodes barnase, abacterial ribonuclease (Goldman et al 1994). We would prefer to enhance the specificityof ablation by using a gene that is more specific for the embryo than an RNAase.The responsibility for the specificity of ablation would thus be shared between thepromoter and the gene. One possibility is to overexpress PKL in the zygotic embryoat the time when PKL transcripts are declining to allow LEC1 to be expressed (Fig. 7).Repression of LEC1 would be expected to block zygotic embryogenesis and result ina gentle and specific form of ablation. Ablation of the sexual embryo requires twopromoters: one driving the meiosis-specific expression of cre recombinase and theother allowing transcription of the gene for ablation. The progress toward these twogoals was described by Kathiresan et al (2001) and therefore is only briefly mentionedhere.Isolation of promoters expressed in the zygotic embryoFollowing a report that REE5, a partial cDNA clone, is expressed only in the embryoand not in the endosperm (Kikuchi et al 1998), we isolated the full-length REE5cDNA and the gene. Our expression studies show that REE5 is expressed in a widerange of tissues including root tips and various developmental stages of panicle andspikelet. Although we would prefer that the promoter allow expression of the gene forablation only in the zygotic embryo, it is not desirable for the promoter to be active inother vital vegetative and reproductive tissues. Therefore, the promoter of REE5 maynot be suitable for driving the expression of the gene for ablation. The first LEC1homologue that we isolated (Fig. 7) appears to be expressed exclusively in the ovule1–4 DAF. To judge by RT-PCR, it is not expressed in calli, roots, coleoptiles, leaves,or anthers. However, in situ hybridization indicates that it is expressed not only in theembryo but also in some neighboring tissues. These additional sites of expressionmay eliminate the LEC1 promoter as a potential promoter for expressing the ablationgene exclusively in the zygotic embryo.Isolation of meiosis-specific promotersSeveral genes of yeast and animal cells are expressed only during meiosis. One of theyeast genes, disrupted meiosis cDNA 1 (DMC1), encodes a DNA strand-exchangeprotein that participates in homologous recombination (Bishop et al 1992). Both yeastand animal DMC1 are meiosis-specific. An initial report on DMC1 in Arabidopsisindicated that the gene is also expressed in a meiosis-specific manner in plants (Klimyukand Jones 1997). However, subsequent studies on Arabidopsis showed clearly thatDMC1 is expressed not only in meiotic tissue but also in certain mitotic tissues suchas root tips (Doutriaux et al 1998). In rice, we have identified two copies of the DMC1gene (DMC1A and DMC1B) by Southern hybridization. Barley, another diploid cereal,contains only one copy of the gene, suggesting that the rice genes diverged afterthe separation of the ancestors of rice from the ancestors of barley. We isolated andMolecular tools for achieving synthetic apomixis in hybrid rice 395