RESUMO
Annexins belong to a multigene family of Ca(2+) dependent, phospholipid and cytoskeleton binding proteins. They have been shown to be upregulated under various stress conditions. We generated transgenic cotton plants expressing mustard annexin (AnnBj1), which showed enhanced tolerance towards different abiotic stress treatments like sodium chloride, mannitol, polyethylene glycol and hydrogen peroxide. The tolerance to these treatments was associated with decreased hydrogen peroxide levels and enhanced total peroxidase activity, enhanced content of osmoprotectants- proline and sucrose in transgenic plants. They showed higher retention of total chlorophyll and reduced TBARS in leaf disc assays with stress treatments, and decreased hydrogen peroxide accumulation in the stomatal guard cells when compared to their wild type counterparts. They also showed significantly enhanced fresh weight, relative water content, dry weight under stress. Treatment with sodium chloride resulted in enhanced expression of genes for Delta-pyrroline-5-carboxylase synthetase in leaves, and sucrose phosphate synthase, sucrose synthase and cellulose synthase A in the leaves and fibers of transgenic plants. The transgenic plants maintained normal seed development, fiber quality and cellulose content under stress.
Assuntos
Anexinas/genética , Fibra de Algodão/normas , Gossypium/genética , Mostardeira/genética , Proteínas de Plantas/genética , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Sequência de Aminoácidos , Anexinas/classificação , Clorofila/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Gossypium/metabolismo , Gossypium/fisiologia , Peróxido de Hidrogênio/metabolismo , Manitol/farmacologia , Dados de Sequência Molecular , Peroxidase/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/classificação , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Defensins are small positively charged, antimicrobial peptides (approximately 5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.
Assuntos
Arachis/genética , Defensinas/genética , Fusarium/patogenicidade , Nicotiana/genética , Phytophthora/patogenicidade , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Arachis/imunologia , Arachis/microbiologia , Sequência de Bases , Southern Blotting , Primers do DNA , Defensinas/química , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.