RESUMO
A cDNA clone designated arf1 was isolated from a physic nut (Jatropha curcas L.) endosperm cDNA library which encodes a small GTP-binding protein and has significant homology to ADP-ribosylation factors (ARF) in plants, animals and microbes. The cDNA contains an open reading frame that encodes a polypeptide of 181 amino acids with a calculated molecular mass of 20.7 kDa. The deduced amino acid sequence showed high homology to known ARFs from other organisms. The products of the arf1 obtained by overexpression in E. coli revealed the specific binding activity toward GTP. The expression of arf1 was observed in flowers, roots, stems and leaves as analyzed by RT-PCR, and its transcriptional level was highest in flowers. In particular, the accumulation of arf1 transcripts was different under various environmental stresses in seedlings. The results suggest that arf1 plays distinct physiological roles in Jatropha curcas cells.
Assuntos
Fatores de Ribosilação do ADP/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Jatropha/crescimento & desenvolvimento , Jatropha/genética , Proteínas de Plantas/genética , Fatores de Ribosilação do ADP/metabolismo , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Guanosina Trifosfato/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de Proteína , Estresse Fisiológico/genéticaRESUMO
A novel 1 L-myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) gene, designate rcMIPS, was cloned from Ricinus communis. It contained an open reading frame (ORF) of 1669 bp coding for a peptide of 510 amino acids with a molecular mass of 56 kDa. Sequence anaylsis showed high homology compared to other plant MIPS genes, because it contained typical domains owned by MIPS enzymes. The transcript levels of the rcMIPS gene in leaves, stems, and roots were examined after drought stress for 24, 48, and 72 h. The transcript levels in the leaves, stems, and roots increased significantly compared to the control. Results of the enzyme assay showed a significant correlation between the changes of enzyme activity and the transcript levels of the rcMIPS gene in different organs. Decreased relative water contents (RWC) and increased malondialdehyde (MDA) contents in the leaves represented a stress response against drought stress. Our findings suggest that MIPS plays an important role in the defensive mechanisms of R. communis against drought stress.
Assuntos
Mio-Inositol-1-Fosfato Sintase/genética , Oryza/enzimologia , Oryza/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , DNA de Plantas/genética , Secas , Malondialdeído/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To date, two types of ribosome-inactivating proteins (RIPs) have been found in Jatropha curcas. One is curcin, which has been isolated from the endosperm, and the other is curcin-L, which is expressed in leaves upon stress treatment. Phylogenetic analysis of the predicted amino acid sequences of the RIPs in plants revealed that these belong to a major subfamily and are close to trichosanthin (TCS). Studies on the mRNA and protein levels showed that both curcin and curcin-L have an organ-specific expression pattern. Curcin is only expressed and accumulated in the endosperm; its expression begins in the globular embryo period and peaks during the mature embryo period. In contrast, curcin-L is only expressed in the leaves, but its expression is induced by certain conditions such as treatment with phytohormones or polyethylene glycol, exposure to high and low temperatures, and fungal infection. Analysis of the 5' flanking regions of curcin and curcin-L revealed that the 5' flanking region of curcin-L has three major inserted fragments, which are not present in the corresponding region of curcin. Comparison of characteristic cis-elements suggests the presence of several motifs that are involved in the endosperm-specific expression in the 5' flanking region of curcin, while in curcin-L some stress- and defense-responsive motifs are found to be mainly located in the three inserted fragments. Comparison of the antifungal activity of the two RIPs showed that the one of curcin-L is higher than that of curcin. Differences in the expression and activity of curcin and curcin-L suggest that these two RIPs have different functions.