RESUMO
Hydroxy-methylglutaryl coenzyme-A synthase (HMGS) is a rate-limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non-coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three-dimensional structural studies highlighting the structural positioning of non-synonymous SNPs from different HMGS haplotypes revealed that the ligand-binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.
Assuntos
Evolução Molecular , Variação Genética , Hevea/enzimologia , Hidroximetilglutaril-CoA Sintase/genética , Alelos , Haplótipos , Hevea/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Borracha/metabolismo , Análise de Sequência de DNARESUMO
Copper is an essential micronutrient that functions as an enzymatic cofactor in a wide range of cellular processes. Although adequate Cu levels are essential for normal metabolism, excess Cu can be toxic to cells. Cellular responses to copper deficiency and overload involve changes in the expression of genes directly and indirectly involved in copper metabolism. However little is known on the effect of physiological copper concentration on gene expression changes. In the current study we aimed to establish whether the expression of genes encoding enzymes related to cholesterol (hmgcs1, hmgcr, fdft) and fatty acid biosynthesis and LDL receptor can be induced by an iso-physiological copper concentration. The iso-physiological copper concentration was determined as the bioavailable plasmatic copper in a healthy adult population. In doing so, two blood cell lines (Jurkat and THP-1) were exposed for 6 or 24 h to iso- or supraphysiological copper concentrations. Our results indicated that in cells exposed to an iso-physiological copper concentration the early induction of genes involved in lipid metabolism was not mediated by copper itself but by the modification of the cellular redox status. Thus our results contributed to understand the involvement of copper in the regulation of cholesterol metabolism under physiological conditions.
Assuntos
Colesterol/biossíntese , Cobre/farmacologia , Expressão Gênica/efeitos dos fármacos , Histidina/análogos & derivados , Compostos Organometálicos/farmacologia , RNA Mensageiro/genética , Colesterol/genética , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Histidina/farmacologia , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Células Jurkat , Metabolismo dos Lipídeos/efeitos dos fármacos , Oxirredução , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS), EC 4.1.3.5, is an essential enzyme in rubber biosynthesis in Hevea brasiliensis. We have isolated a new cDNA encoding HMGS in H. brasiliensis. The full-length hmgs2 consists of 1,916-bp and encodes a protein of 464 amino acids with a predicted molecular mass of 51.27 kDa and an isoelectric point of 6.02. In comparison, HMGS1 and HMGS2 show 92% and 94% nucleotide and amino acid sequence identities, respectively. Semiquantitative RT-PCR analysis indicates that the hmgs2 is more highly expressed in laticifer and petiole than in leaves. Sequence searching and alignment revealed that HMGS is a distant relative of the condensing enzyme; beta-ketoacyl acyl carrier protein synthase III (ACP synthase III), EC 2.3.1.41, identified three completely conserved residues; Cys(117), His(247), and Asn(326). The relationship was greatly strengthened by making a proper alignment of numerous sequences of both HMGS and ACP synthase III. The same Cys(117), His(247), and Asn(326) absolutely conserved in both groups play a catalytic role in ACP synthase III, while such a role of Cys and His has only been reported for HMGS. According to site-directed mutagenesis, the expressed wild-type enzyme shows comparable level with mutant proteins. The mutation of Cys(117) and Asn(326) affects the HMGS activity, indicating that Cys(117) and Asn(326) are important amino acids for the catalytic activity of HMGS. A phylogenetic tree constructed on the basis of proper multiple alignment indicates that HMGS1 and HMGS2 result from recent gene duplication. This is also the case for HMGS and ACP synthase III, which appear to have arisen from an ancient gene duplication event of an ancestral condensing enzyme. Therefore, a possible secondary structure of HMGS could be predicted based on the Protein Data Bank information of ACP synthase III.