RESUMO
Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1-7) of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment. These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production.
Assuntos
Hevea/genética , Liases/genética , Sequência de Aminoácidos , Clonagem Molecular , Etilenos/farmacologia , Genes de Plantas , Hevea/enzimologia , Liases/classificação , Liases/metabolismo , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Regulação para Cima/efeitos dos fármacosRESUMO
Calcium-dependent protein kinases (CDPKs), as major primary Ca(2+) sensors, have been implicated in the regulation of stress and developmental signals in plants. In this study, a novel CDPK gene, designated HbCDPK1, was isolated from Hevea brasiliensis. The HbCDPK1 cDNA had 2,400 bp with an open reading frame of 1,671 bp encoding 556 amino acids, and the deduced HbCDPK1 protein contained four characteristic domains identified in CDPKs, showing a high level of sequence similarity to CDPKs from other plants. Expression analysis revealed more significant accumulation of the transcripts of HbCDPK1 in latex than in the leaves, bark, and roots in H. brasiliensis. In addition, transcription of HbCDPK1 was strongly induced by mechanical wounding, jasmonic acid (JA), and ethephon. Recombinant HbCDPK1 was expressed in E. coli, and its activity was assayed. The assay indicated that HbCDPK1 had the kinase and Ca(2+)-binding activity in vitro as a calcium-dependent protein. The potential roles of the HbCDPK1 are discussed as to latex production and rubber biosynthesis.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hevea/enzimologia , Compostos Organofosforados/farmacologia , Proteínas Quinases/genética , DNA Complementar/isolamento & purificação , Perfilação da Expressão Gênica , Genes de Plantas , Hevea/genética , Látex/biossíntese , Fases de Leitura Aberta/genética , Reguladores de Crescimento de Plantas , Estruturas Vegetais/química , RNA Mensageiro/análise , BorrachaRESUMO
Hsp70s have been shown to play important roles in helping cells to cope with adverse environments, especially in response to temperature. In this study a novel ethephon-induced Hsp gene, designated as HbHsp70, was isolated from Hevea brasiliensis. The HbHsp70 cDNA contained a 1965 bp open reading frame encoding 655 amino acids. The deduced HbHsp70 protein showed high identities to Hsp70s from other plants. Expression studies revealed more significant accumulation of HbHsp70 transcripts in leaves and stems than in roots, barks and latex. The transcription of HbHsp70 was induced by ethephon, heat treatment and low temperature stress, whereas jasmonic acid had little effects. Recombinant HbHsp70 was expressed in Escherichia coli and purified by Ni-NTA affinity chromatography. Measuring the light scattering of luciferase (Luc) revealed that HbHsp70 prevents the aggregation of luc during high-temperature stress. In vitro experiments showed that HbHsp70 had protective functions not only against heat stress but also against chilling stress. All these data suggest that HbHsp70 may play roles in responses to heat shock and low temperature in H. brasiliensis.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Hevea/genética , Compostos Organofosforados/farmacologia , Proteínas de Plantas/genética , Sequência de Aminoácidos , Clonagem Molecular , Temperatura Baixa , DNA Complementar/química , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Hevea/metabolismo , Temperatura Alta , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A simple and sensitive method is described for determination of jasmonic acid (JA) in plant tissues. The method is based on derivatization of JA with 5-bromomethylfluorescein (5-BMF) and separation and quantification of the resulting 5-BMF-JA derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). The derivatization conditions were studied in detail. Our results indicated that 5-BMF-labeled JA could be well separated from other plant hormones present in the sample by use of 20 mmol L(-1) borate buffer (pH 8.5). The response to JA was a linear function of concentration in the range 1 to 100 micromol L(-1), with a correlation of 0.9986. Our preliminary work showed that the proposed method had fairly good selectivity and sensitivity. Only small amounts of plant sample are needed to complete the analysis. This described method enables the analysis of JA in crude extracts without extra purification and enrichment procedures.