RESUMO
Metallothioneins (MTs) are involved in cellular homeostasis of essential metal ions and detoxification of nonessential metal ions. We report here the identification of four MT genes, CtMT1, CtMT2, CtMT3 and CtMT4, encoding CtMT1, CtMT2, CtMT3 and CtMT4 proteins, respectively, from the industrial guar crop. The primary structures of last three proteins were similar to those of respective MT proteins of other plants but the CtMT1 protein primary structure was different from the other plant MT1 proteins in having an additional C-X-C motif. The four MT genes showed tissue specific expression patterns suggesting their specific roles in different tissues. High expression of CtMT1 gene was observed in roots and nodules whereas CtMT2 and CtMT3 genes showed high expression in leaves. The expression of CtMT4 gene was high in seeds. The qRT-PCR studies revealed upregulation in expression of CtMT1 gene under drought stress. Recombinant CtMT1 protein was produced in E. coli Rosetta cells and purified by metal affinity chromatography. The purified protein showed antioxidant property and the order of its metal ion binding affinities was Cu2+â¯>â¯Zn2+â¯>â¯Fe2+â¯>â¯Cd2+. This information about CtMT1 protein is expected to be useful in understanding its role in drought tolerance and other physiological processes of guar.
Assuntos
Cyamopsis/genética , Cyamopsis/fisiologia , Secas , Metalotioneína/genética , Metais Pesados/metabolismo , Estresse Fisiológico/genética , Regulação para Cima , Motivos de Aminoácidos , Sequência de Aminoácidos , Cyamopsis/efeitos dos fármacos , Cyamopsis/metabolismo , DNA de Plantas/metabolismo , Éxons/genética , Hidróxidos/metabolismo , Hidróxidos/farmacologia , Íntrons/genética , Metalotioneína/química , Metalotioneína/metabolismo , Ligação ProteicaRESUMO
KEY MESSAGE: Two virus inhibitory proteins were purified from Cyamopsis tetragonoloba , induced to resist virus infections by CIP-29, a systemic resistance inducing protein from Clerodendrum inerme, and characterized. One of them shared homology with a lectin. CIP-29, a known 29 kDa systemic antiviral resistance inducing protein isolated from Clerodendrum inerme, has been used to induce systemic resistance in Cyamopsis tetragonoloba against Sunn-hemp rosette virus (SRV). Paper reports the detection of virus inhibitory activity in induced-resistant leaf sap of C. tetragonoloba, and the purification of two virus inhibitory agents (VIAs) thereof. VIA activity was recorded as a reduction in lesion number of SRV, Tobacco mosaic virus, and Papaya ringspot virus, when they were incubated separately with resistant sap and inoculated onto susceptible C. tetragonoloba, Nicotiana tabacum cv. Xanthi-nc, and Chenopodium quinoa, respectively. The two VIAs were isolated from resistant C. tetragonoloba plant leaves using combinations of column chromatography. Both were basic proteins, and since their M r was 32 and 62 kDa, these VIAs were called CT-VIA-32 and CT-VIA-62, respectively, on the basis of their molecular mass and the host. CT-VIA-62 displayed better activity, and was thus studied further. It tested positive for a glycoprotein, and was serologically detected only in leaf tissue post-induction. Tryptic peptides generated in-gel, post SDS-PAGE of CT-VIA-62, were sequenced through LC/MS/MS. All CT-VIA-62 peptides were found to share homologies with proteins from Medicago truncatula that possess a mannose-binding lectin domain.
Assuntos
Antivirais/isolamento & purificação , Cyamopsis/química , Resistência à Doença , Doenças das Plantas/imunologia , Extratos Vegetais/isolamento & purificação , Vírus do Mosaico do Tabaco/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Antivirais/farmacologia , Clerodendrum/química , Cyamopsis/efeitos dos fármacos , Cyamopsis/imunologia , Glicoproteínas , Lectinas/isolamento & purificação , Lectinas/farmacologia , Dados de Sequência Molecular , Peso Molecular , Doenças das Plantas/virologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/imunologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Estrutura Terciária de Proteína , Coelhos , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
A novel combination of plant growth regulators comprising indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and gibberellic acid (GA3) in Murashige and Skoog basal medium has been formulated for in vitro induction of both shoot and root in one culture using cotyledonary node explants of guar, (Cyamopsis tetragonoloba). Highest percentages of shoot (92%) and root (80%) induction were obtained in the medium containing (mg/L) 2 IBA, 3 BA and 1 GA3. Shoot regeneration from the cotyledonary node explants was observed after 10-15 days. Regeneration of roots from these shoots occurred after 20 to 25 days. The regenerated plantlets showed successful acclimatization on transfer to soil. This protocol is expected to be helpful in carrying out various in vitro manipulations in this economically and industrially important legume.