RESUMEN
KEY MESSAGE: The glyphosate resistance in Escherichia coli and Arabidopsis was due to D-amino acid oxidase expression. Transgenic glyphosate-resistant crops have a high percentage in the total area devoted to transgenic crops worldwide. D-amino acid oxidase (DAAO) can metabolize glyphosate by oxidative cleavage of the carbon-nitrogen bond on the carboxyl side and yield aminomethyl phosphonic acid and glyoxylate, which are less toxic to plants than glyphosate. To date, reports on the use of DAAO to enhance glyphosate resistance in plants are lacking. In this paper, we report synthesis, and codon usage optimization for plant expression, of the DAAO gene by successive polymerase chain reaction from Bradyrhizobium japonicum. To confirm the glyphosate resistance of the DAAO gene, the recombinant plasmid pYPX251 (GenBank Accession No: AY178046) harboring the wild-type DAAO gene was transformed into DH5α. The positive transformants grew well both on solid and in liquid M9 medium containing 200 mM glyphosate. The optimized DAAO gene was transformed into Arabidopsis and 9 days after application of 10 mM glyphosate, the 4-week-old wild-type plants all died; by contrast, transgenic plants could grow normally. The proline content and peroxidase activity showed that glyphosate could induce proline accumulation and produce reactive oxygen species.
Asunto(s)
Arabidopsis/fisiología , Bradyrhizobium/enzimología , D-Aminoácido Oxidasa/genética , Glicina/análogos & derivados , Resistencia a los Herbicidas , Herbicidas/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Bradyrhizobium/genética , Codón/genética , D-Aminoácido Oxidasa/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/fisiología , Expresión Génica , Glicina/farmacología , Plantas Modificadas Genéticamente , Prolina/análisis , Plantones/efectos de los fármacos , Plantones/genética , Plantones/fisiología , GlifosatoRESUMEN
Quantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1-S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including 'Hakuho' (melting type), 'Xiacui' (stony hard type), 'Fantasia' and 'NJC108' (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in 'Total' set, 'Hakuho' samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including 'Fantasia', 'NJC108', S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, 'NJC108' and S2 sets, while showed the highest stability in 'Xiacui' samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of 'Hakuho'. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.
Asunto(s)
Proteínas de Plantas/genética , Prunus persica/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/normas , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Proteínas de Plantas/metabolismo , Prunus persica/crecimiento & desarrollo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de ReferenciaRESUMEN
Ammonium (NH4 +) plays key roles in plant growth, development, fruit quality, and yield. In plants, NH4 + uptake and transport are facilitated by NH4 + transporters (AMT). However, molecular mechanisms and physiological functions of type-II AMT (AMT2) transporters in fruit trees are still unclear, especially in peach. In this study, we cloned and characterized an AMT2 family gene from peach, PpeAMT3;4, and determined its function in yeast mutant. Expression analysis showed that PpeAMT3;4 was majorly expressed in peach roots and significantly decreased by NH4 + excess but had no response to NH4 + deficiency. Functional determination and 15nitrogen-labeled NH4 + uptake assay in yeast cells implied that PpeAMT3;4 was a typical high-affinity transporter, with a K m value of 86.3 µM, that can uptake external NH4 + in yeast cells. This study provides gene resources to uncover the biological function of AMT2 transporters and reveals molecular basis for NH4 + uptake and nitrogen (N) nutrition mechanisms in fruit trees.