RESUMEN
We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.
Asunto(s)
Agrobacterium tumefaciens/genética , Cromosomas Artificiales Bacterianos/genética , ADN de Plantas/genética , Genoma de Planta , Magnoliopsida/genética , Southern Blotting , Clonación Molecular , ADN de Plantas/análisis , Escherichia coli/genética , Vectores Genéticos , Reacción en Cadena de la Polimerasa , Transformación GenéticaRESUMEN
Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar encodes for Phosphinothricin Acetyl Transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenic L. japonicus plants resistant to PPT were positive upon PCR by bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA insertion into the plant genome. All regenerated plants were fertile and void of visible somaclonal abnormalities contrary to 14% infertility when antibiotic selectable markers were used. The lack of somaclonal variation, ease of PPT application and low cost of PPT makes this protocol an attractive alternative for the regeneration of transgenic L. japonicus. The production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production.
Asunto(s)
Acetiltransferasas/genética , Aminobutiratos/metabolismo , Fabaceae/genética , Herbicidas/farmacología , Plantas Medicinales , Acetiltransferasas/metabolismo , Técnicas de Cultivo , ADN Bacteriano/genética , ADN de Plantas , Resistencia a Medicamentos/genética , Fabaceae/efectos de los fármacos , Genes de Plantas , Marcadores Genéticos , Herbicidas/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Transformación GenéticaRESUMEN
The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen-fixing symbiosis. Here, we demonstrate that plant-produced isoflavones induce nodulation gene expression in a population density-dependent fashion. Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures. A quorum signal molecule in the conditioned medium of B. japonicum cultures mediates this repression. Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression. Consistent with this, nolA-lacZ and nodD2-lacZ expression increased with increasing population density. Unlike the wild type, the ability to induce nodY-lacZ expression did not decline with population density in a NolA mutant. Normally, nod gene expression is repressed in planta (i.e. within nodules). However, expression of a nodY-GUS fusion was not repressed in a NolA mutant, suggesting that quorum-sensing control may mediate in planta repression of the nod genes. Addition of conditioned medium to cultures significantly reduced nod gene expression. Treatment of inoculant cultures with conditioned medium also reduced the ability of B. japonicum to nodulate soybean plants.