RESUMEN
Genetic variation is crucial for crop improvement. We adopted a gene editing approach to create variations in the rice genome by targeting the mutator locus homolog 2 (MSH2), a DNA mismatch repair gene. The hypothesis is that disruption of the MSH2 gene leads to a reduced DNA mismatch repair that creates INDELs, resulting in altered phenotypes. The Indica rice (IR-64) genotype was transformed with a guide RNA targeted to the MSH2 gene using an Agrobacterium-mediated in planta method. Many plants showed integration of Cas9 and gRNA constructs in rice plants. One of the msh2 mutants showed a superior phenotype due to editing and possible INDELs in the whole genome. The stable integration of the transgene and its flanking sequence analysis confirms no disruption of any gene, and the observed phenotype is due to the mutations in the MSH2 gene. Few transgenic plants showed disruption of genes due to T-DNA integration that led to altered phenotypes. The plants with altered phenotypes having more tiller number, early flowering, and robust growth with a high biomass were identified. These genetically reprogrammed rice plants could be a potential resource to create more segregating population or act as donor lines to stabilize the important agronomic traits that may help in a speed breeding process.
Asunto(s)
Oryza , Sistemas CRISPR-Cas , Reparación de la Incompatibilidad de ADN/genética , Edición Génica , Genoma de Planta , Oryza/genética , Fitomejoramiento , Plantas Modificadas Genéticamente/genéticaRESUMEN
In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta.
Asunto(s)
Aldo-Ceto Reductasas/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Glicina/toxicidad , Resistencia a los Herbicidas/genética , Oryza/efectos de los fármacos , Oryza/enzimología , Oryza/genética , Fotosíntesis/efectos de los fármacos , Fotosíntesis/fisiología , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Unión Proteica/genética , Pseudomonas/enzimología , Pseudomonas/genética , Ácido Shikímico/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/enzimología , Nicotiana/genética , GlifosatoRESUMEN
The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.
