RESUMEN
MYBs are an important family of transcription factors that play significant roles in plant development and stress response in plants. However, knowledge concerning the functions of MYBs in the non-model plants and the target genes is still limited. In this study, we isolated a stress-responsive R2R3-type MYB gene from trifoliate orange (Poncirus trifoliata (L.) Raf.), designated as PtsrMYB. PtsrMYB shares the highest degree of identity with AtMYB109. Subcellular localization using onion epidermal cells indicates that PtsrMYB is localized in the nucleus. Transcript levels of PtsrMYB were up-regulated by abiotic stresses such as dehydration, salt, cold and ABA treatment. Overexpression of PtsrMYB in tobacco confers enhanced dehydration tolerance, as indicated by less water loss, lower levels of malondialdehyde and reactive oxygen species. The transgenic tobacco lines displayed higher mRNA levels of two arginine decarboxylase (ADC) genes before and after dehydration treatment when compared with the wild type, concurrent with the greater levels of polyamines. Several MYB-recognizing cis-acting elements exist on the promoters of PtADC gene. Yeast one-hybrid assay demonstrated that PtsrMYB predominantly interact with two regions of the promoter, indicating the PtADC may be a target gene of PtsrMYB. Take together, PtsrMYB plays a positive role in dehydration tolerance, which may be, at least in part, due to the modulation of polyamine synthesis by regulating the ADC gene.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas de Plantas/genética , Poliaminas/metabolismo , Poncirus/genética , Factores de Transcripción/genética , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Carboxiliasas/genética , Carboxiliasas/metabolismo , Núcleo Celular/metabolismo , Frío , Deshidratación , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Malondialdehído/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Cebollas/citología , Cebollas/genética , Cebollas/metabolismo , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Poncirus/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Nicotiana/genética , Nicotiana/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Exposure of cold-hardy Rubidoux trifoliate orange [Poncirus trifoliata (L) Raf.] plants to temperatures from 28 degrees C to -5 degrees C enabled us to isolate and characterize a novel citrus low-temperature gene (CLT) with two transcripts, called CLTa and CLTb, from leaves and stems. CLTa was produced when plants were subjected to low temperatures (starting at 10 degrees C), while CLTb was constitutively expressed. Both CLTa and CLTb have the same open reading frame (ORF) of 165 nucleotides and encode a small (54 deduced amino acid) protein. However, CLTa has an additional 98 nucleotides in the 3'-untranslated region (UTR) that are absent in CLTb. Expression analysis using relative quantitative RT-PCR demonstrated that CLTa is expressed exclusively at low temperatures, while CLTb is expressed constitutively (expression verified from 33 degrees C to -5 degrees C). A GenBank database search identified 61 nucleotides inside of the ORF that are highly similar to low-temperature-responsive genes from Arabidopsis thaliana and Solanum tuberosum. The deduced amino acid sequence revealed similarity with low-temperature-responsive proteins from A. thaliana, Oryza sativa, and S. tuberosum of 77%, 81%, and 73.9%, respectively. A genomic clone was isolated, and the genome organization revealed the presence of three exons and two introns, the second of which is in the 3' UTR and participates in alternative 3' splice site selection. One of the 3' splice sites of the second intron was located immediately before the additional 98-bp non-coding fragment of CLTa, and the second at the very end of the 98-bp fragment. Additionally, the presence of the tetranucleotides TCTT and TTCT, which are involved in the regulation of transcript processing in animals and possibly also active in peach, was found in this intron. Competition for splicing sites on the pre-mRNA in the spliceosome, which is induced by low temperature, may be involved in the production of the two transcripts of the CLT gene.
Asunto(s)
Aclimatación/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas/genética , Proteínas de Plantas/biosíntesis , Poncirus/genética , Precursores del ARN/genética , Empalme Alternativo/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonación Molecular , Frío , ADN Complementario/análisis , ADN Complementario/genética , Exones/genética , Regulación de la Expresión Génica de las Plantas/genética , Genoma de Planta , Intrones/genética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta/genética , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Poncirus/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Empalmosomas/genéticaRESUMEN
OBJECTIVE: To lay a foundation for the resistant breeding, the anti-virus CTV-cp gene was transformed into the epicotyles mediated by Agrobacterium tumefaciens in Poncirus trifoliata. METHOD: The explants used for the genetic transformation were the epicotyls from P. trifoliata. The Agrobacterum tumefaciens strain was EHA101 containing vector plasmid pGA482GG. The coat protein gene (CTV-cp gene), GUS gene and NPT II gene were introduced into the transformation plasmid. RESULTS: Ceftaxime used as antibiotics was better than carbenicillin. The concentration was 300 mg.L-1; The selection pressure for kanamycin was 50 mg.L-1; 70.0% of the resistant plants were GUS-positive; extra gene was proved to be in P. trifoliata plant by southern blot examination. CONCLUSION: An effective genetic transformation mediated by Agrobacterium tumefacines, which harbours a CTV-cp gene, has been developed in P. trifoliata. Transgenic CTV-cp plants were obtained.
Asunto(s)
Proteínas de la Cápside , Cápside , Virus de Plantas/genética , Plantas Medicinales/genética , Poncirus/genética , Plantas Modificadas Genéticamente , Rhizobium/genética , Transformación GenéticaRESUMEN
OBJECTIVE: Establishing an effective system for the Agrobacterium-mediated genetic transformation of Poncirus trifoliata, so as to lay a foundation for improving breeds and introducing foreign genes. METHOD: The explants used for the genetic transformation were the epicotyls from Poncirus trifoliata. The Agrobacterium tumefaciens strain was EHA101, containing vector plasmid PGA482GG. The GUS gene and NPT II gene were introduced into the transformation plasmid. RESULTS: 20-day epicotyls were suitable for transformation. 2-day precultivation enhanced shoot regeneration. Shoot regeneration frequency was high when the cocultivation lasted 2-3 days. With the presence of acetosyringone during cocultivation, the efficiency of transformation could be greatly enhanced, and the frequency of shoot regeneration rose as high as 27.5%. After the explants were cocultivated with Agrobacterium, 2-day delayed selection could stimulate shoot regeneration. CONCLUSION: An effective genetic transformation system has been established for Poncirus trifoliata on the basis of factors affecting Agrobacterium-mediated genetic transformation.