RESUMO
Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.
Assuntos
Clonagem Molecular , Isatis/enzimologia , Fenilalanina Amônia-Liase/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Isatis/genética , Dados de Sequência Molecular , Família Multigênica , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.
Assuntos
Aciltransferases/genética , Clonagem Molecular , Isatis/enzimologia , Proteínas de Plantas/genética , Aciltransferases/química , Aciltransferases/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Isatis/química , Isatis/classificação , Isatis/genética , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ácido Quínico/metabolismo , Alinhamento de Sequência , Ácido Chiquímico/metabolismoRESUMO
The experiment included three potassium levels (K0 0 g x kg(-1), K1 0.33 g x kg(-1), K2 0.67 g x kg(-1)) and two water gradients (well watered and drought stress), then measured growth indicators, SOD, POD, CAT activities and concents of osmotic regulation substances. To explore the effects of K fertilizer and water on growth and physiological characteristics of Isatis indigotica, providing reference for improving drought resistance of I. indigotica. The result showed drought stress inhibited the growth and decreased the biomass of I. indigotica but K fertilizer can alleviate the drought stress. Compared with K0 treatment, K1, K2 treatment increased the biomass of overground part of by 89. 13% ,60. 87% under drought stress. The corresponding increase in soluble sugar content was 16.67%, 5.00%, and in proline content was 42.41%, 65.62%, respectively. SOD,POD and CAT activities was significantly improved in K1, K2 treatment in comparison with K0 treatment under drought stress, but soluble protein content significantly reduced. The conclusion is that appropriate amount of K fertilizer can increase the activities of antioxidase and the content of osmoregulation substance under drought stress, and improve drought resistance of I. indigotica.
Assuntos
Fertilizantes/análise , Isatis/crescimento & desenvolvimento , Isatis/metabolismo , Potássio/metabolismo , Água/metabolismo , Isatis/química , Isatis/enzimologia , Peroxidase/metabolismo , Proteínas de Plantas/metabolismo , Potássio/análise , Plântula/química , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Superóxido Dismutase/metabolismo , Água/análiseRESUMO
In plants, stomata play a pivotal role in the regulation of gas exchange and are distributed throughout the aerial epidermis. SDD1, a gene isolated from Arabidopsis thaliana has been demonstrated to specialize in stomatal density and distribution. In our present study, a comprehensive survey of global gene expression performed by using an A. thaliana whole genome Affymetrix gene chip revealed SDD1 tends to be significantly lower in tetraploid Isatis indigotica than in diploid ones. To intensively investigate different SDD1 expression in response to polyploidy, a full-length cDNA clone (IiSDD1) encoding SDD1 was isolated from the traditional Chinese medicinal herb I. indigotica cDNA library. IiSDD1 shared a high level of identity with that from A. thaliana, containing some basic features of subtilases: D, H and S regions, as well as a substrate-binding site. Real-time quantitative PCR analysis indicated that IiSDD1 was constitutively expressed in all tested tissues, including roots, stems and leaves, both in tetraploid and diploid I. indigotica, and with the highest expression in leaves. In addition, IiSDD1 was also found to be down-regulated by signalling molecules for plant defence responses, such as abscisic acid (100 microM) and gibberellin (100 mg/L), as well as by environmental stresses including salt, darkness, coldness and drought. Our study, for the first time, indicates SDD1 participates not only in the defense/stress responsive pathways, but also probably involves in plants polyploidy evolution.
Assuntos
Regulação da Expressão Gênica de Plantas , Isatis/genética , Poliploidia , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Clonagem Molecular , Diploide , Secas , Meio Ambiente , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas , Giberelinas/farmacologia , Isatis/enzimologia , Isatis/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Sais/farmacologia , Homologia de Sequência , Estresse Fisiológico/genéticaRESUMO
OBJECTIVE: To study the effect of NH4(+)-N/NO3(-)-N ratios in the applied supplementary fertilizer on the growth, nitrogen metabolis related enzymes activity and photosynthetic characteristics of Isatis indigotica. METHOD: The sand culture experiment was conducted, and seedling of I. indigotica was fertilized with the mixed nutrition that containing the Hoagland's macro elements and the Aron's micro elements, the additional 63 mmol N was supplementary with the NH4(+)-N/NO3(-)-N ratio of 100:0, 75:25, 50:50, 25:75 and 0:100. RESULT: The biomass of I. indigotica increased at first when the supplementary N of NH4(+)-N/NO3(-)-N ratio changed from 100:0 to 50:50 and decreased afterwards. The maximum value was at 50:50 and the minimum at 100: 0. With increasing the ratio of NO3(-)-N, the activity of nitrate reductase and glutamine synthetase increased and then decreased and the relationship between the activity and the ratio could be described with an approximate parabola curve. The net photosynthetic rate of I. indigotica was the highest at the NH4(+)-N/NO3(-)-N ratio of 75:25 and the lowest at 100:0. CONCLUSION: Increasing the NO3(-)-N ratio properly was beneficial to promote the growth and improve the activity of nitrate reductase and glutamine synthetase and net photosynthetic rate of I. indigotica.
Assuntos
Fertilizantes/análise , Isatis/crescimento & desenvolvimento , Isatis/metabolismo , Nitrogênio/metabolismo , Fotossíntese , Biomassa , Isatis/química , Isatis/enzimologia , Nitrato Redutase/metabolismo , Nitrato Redutases/metabolismo , Nitratos/análise , Nitratos/metabolismo , Nitrogênio/análise , Proteínas de Plantas/metabolismo , Compostos de Amônio Quaternário/análise , Compostos de Amônio Quaternário/metabolismoRESUMO
Isatis indigotica carried by the Chinese first spaceship "Shenzhou" was studied in order to find the mutation after spaceflight. TLC differentiation experiments showed no distinct discrepancy among the samples of spaceflight and non-space-flight, and the same color spot appeared corresponding to the location of the arginine. Isoenzymes of esterase and peroxidase were studied with PAGE. Isoenzymes of esterase were difference among the samples. To peroxidases, little difference was found with them. The ratio of dry weight and extract contents showed out the mutation has emerged after spaceflight, but some characters were unstable. It is necessary for further study.
Assuntos
Esterases/metabolismo , Isatis/enzimologia , Peroxidases/metabolismo , Plantas Medicinais/enzimologia , Voo Espacial , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Esterases/análise , Isoenzimas/análise , Isoenzimas/metabolismo , Peroxidases/análise , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Sementes/enzimologiaRESUMO
A novel calcium-dependent protein kinase gene (designated as IiCPK2) was cloned from tetraploid Isatis indigotica. The full-length cDNA of IiCPK2 was 2585 bp long with an open reading frame (ORF) of 1878 bp encoding a polypeptide of 625 amino acid residues. The predicted IiCPK2 polypeptide included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs. Further analysis of IiCPK2 genomic DNA revealed that it contained 7 exons, 6 introns and the length of most exons was highly conserved. Semi-quantitative RTPCR revealed that the expression of IiCPK2 in root, stem and leaf were much higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin (GA3), NaCl and cold treatments could upregulate the IiCPK2 transcription. All our findings suggest that IiCPK2 might participate in the cold, high salinity and GA3 responsive pathways.