RESUMO
Gene duplication could be beneficial by functional division but might increase the risk of genetic load. The dynamics of duplicated paralogs number could involve recombination, positive selection, and functional divergence. Duplication of DIHYDROFLAVONOL 4-REDUCTASE (DFR) has been reported in several organisms and may have been retained by escape from adaptive conflict (EAC). In this study, we screened the angiosperm DFR gene focusing on a diversified genus Scutellaria to investigate how these duplicated genes are retained. We deduced that gene duplication involved multiple independent events in angiosperms, but the duplication of DFR was before the divergence of Scutellaria. Asymmetric positive selective pressures resulted in different evolutionary rates between the duplicates. Different numbers of regulatory elements, differential codon usages, radical amino acid changes, and differential gene expressions provide evidences of functional divergence between the two DFR duplicates in Scutellaria, implying adaptive subfunctionalization between duplicates. The discovery of pseudogenes accompanying a reduced replacement rate in one DFR paralogous gene suggested possibly leading to "loss of function" due to dosage imbalance after the transient adaptive subfunctionalization in the early stage of duplication. Notwithstanding, episodic gene duplication and functional divergence may be relevant to the diversification of ecological function of DFR gene in Scutellaria.
Assuntos
Oxirredutases do Álcool/genética , Genes Duplicados/genética , Variação Genética , Proteínas de Plantas/genética , Seleção Genética , Códon/genética , Evolução Molecular , Duplicação Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Magnoliopsida/classificação , Magnoliopsida/enzimologia , Magnoliopsida/genética , Modelos Genéticos , Filogenia , Elementos Reguladores de Transcrição , Scutellaria/classificação , Scutellaria/enzimologia , Scutellaria/genéticaRESUMO
Arginase II has recently reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. In the course of screening plants used in natural medicines as arginase II inhibitory activity, a methanol extract of Scutellaria indica showed significant inhibitory effect. Further fractionation and repeated column chromatography led to the isolation of a new flavan-type (1), and seven known compounds (2-8). The chemical structures of isolated compounds were elucidated based on extensive 1D and 2D NMR spectroscopic data. The isolates 1-8 were investigated in vitro for their arginase II inhibitory activity using enzyme solution prepared from kidney of anesthetized C57BL/6 mice. Compounds 3 and 5 significantly inhibited arginase II activity with IC50 values of 25.1 and 11.6 µM, respectively, whereas the other compounds were apparently inactive.
Assuntos
Arginase/antagonistas & inibidores , Arginase/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Scutellaria/enzimologia , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Flavonoides/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Componentes Aéreos da Planta , Extratos Vegetais/isolamento & purificaçãoRESUMO
Anthocyanidin is a group of flavonoid compounds used as a vegetable pigment and plays an important role in flower coloration and environmental adaptations of the Chinese ornamental plant Scutellaria viscidula. We determined the cDNA sequences of phenylalanine ammonia-lyase (SvPAL), flavanone 3-hydroxylase (SvF3H) and dihydroflavonol 4-reductase (SvDFR) genes in S. viscidula. Comparative analysis showed that the protein products of these three genes did not have a transit peptide at their N-terminal portion, which indicated that these enzymes were directly involved in the substrate conversion in the cytoplasmic matrix. Bioinformatic analysis further revealed that Svpal, Svf3h and Svdfr were the members of flavonoid biosynthetic genes with highly conserved motifs. Based on phylogenetic tree analysis, it appears that PAL, F3H or DFR from different plants might have originated from the same ancestor. This study can help to map and regulate the important stages involved in anthocyanidin biosynthesis by genetic engineering to diversify flower color and improve the ornamental value of S. viscidula.
Assuntos
Oxirredutases do Álcool/genética , Antocianinas/genética , Genes de Plantas/genética , Oxigenases de Função Mista/genética , Fenilalanina Amônia-Liase/genética , Scutellaria/genética , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Sequência de Aminoácidos , Antocianinas/biossíntese , Sequência de Bases , Vias Biossintéticas/genética , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/genética , Evolução Molecular , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/isolamento & purificação , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Scutellaria/enzimologia , Alinhamento de SequênciaRESUMO
The conversion of flavonoid aglycones to their glycosides by plant glycosyltransferases may affect a wide range of outcomes, including stability, solubility and bioavailability. Scutellaria barbata, rich in flavonoid glycosides, is widely used as a traditional Chinese herbal medicine. In this study, a flavonoid glycosyltransferase cDNA (SbUGT) and its promoter from S. barbata were cloned and characterized as a flavonoid glycosyltransferase using whole-cell biotransformation. Fragments of different lengths of the 5'-flanking region of the SbUGT gene were fused to the beta-glucuronidase (GUS) gene and analyzed with transgenic Arabidopsis plants using histochemical and fluorometric assays. GUS activity in transgenic plants carrying the SbP-850U construct (-850 to +86 relative to the transcription start site) displayed the highest level and was enhanced by salt and methyl jasmonate, similar to the expression patterns of the endogenous SbUGT. GUS activity disappeared when the promoter was deleted to -98, and deletion analyses indicated the existence of positive and negative regulatory element(s). Unexpectedly, plants carrying the construct SbP-102U (-102 to +86) exhibited strong GUS activity exclusively in the roots. Our experiments revealed that the specific expression is mediated by different promoter regions and the unique region driving root-preferred expression can be used as a root-specific promoter.
Assuntos
Glicosiltransferases/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Scutellaria/enzimologia , Dados de Sequência MolecularRESUMO
OBJECTIVE: To get Scutellaria viscidula CHS gene sense and antisense plant expression vectors,and explore the mechanism of CHS's effect on the flavonoid biosynthetic pathway through the transformation of Scutellaria viscidula. METHODS: CHS gene fragment was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) from total RNA of Scutellaria viscidula and cloned into PMD18-T vector, sequenced and bioinformaticly analyzed. RESULTS: The DNA sequence was correct and we simultaneously guessed the amino acid sequence and the three-dimensional structure of CHS. Cloned fragments and pCAMBIA1304+ vector were digested using Blg II and BstE II at the same time, then they were connected with CHS gene fragment and pCAMBIAI304+ big skeleton fragment to get CHS sense and antisense plant expression vectors. CONCLUSION: The successful construction of Scutellaria viscidula CHS gene sense and antisense plant expression vectors, and the transformation into C58C1 and LBA4404 through the freeze-thaw method, lay the foundation for Agrobacterium-mediated transformation of Scutellaria viscidula CHS gene in plant heredity.
Assuntos
Aciltransferases/genética , Genes de Plantas , Vetores Genéticos , Scutellaria/enzimologia , Scutellaria/genética , Aciltransferases/química , Sequência de Aminoácidos , Clonagem Molecular , Primers do DNA , DNA de Plantas/genética , Dados de Sequência Molecular , Plasmídeos/genética , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Recombinant chalcone synthase (CHS) from Scutellaria baicalensis and stilbene synthase (STS) from Arachis hypogaea accepted CoA esters of long-chain fatty acid (CHS up to the C12 ester, while STS up to the C14 ester) as a starter substrate, and carried out sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide alpha-pyrones. Interestingly, the C6, C8, and C10 esters were kinetically favored by the enzymes over the physiological starter substrate; the kcat/KM values were 1.2- to 1.9-fold higher than that of p-coumaroyl-CoA. The catalytic diversities of the enzymes provided further mechanistic insights into the type III PKS reactions, and suggested involvement of the CHS-superfamily enzymes in the biosynthesis of long-chain alkyl polyphenols such as urushiol and ginkgolic acid in plants.
