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1.
Plant Biotechnol J ; 15(10): 1309-1321, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28258966

RESUMO

Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. In Arabidopsis, AtCYP85A2 works as a bifunctional cytochrome P450 monooxygenase to catalyse the conversion of castasterone to brassinolide, a final rate-limiting step in the BR-biosynthetic pathway. Here, we report the functional characterizations of PtCYP85A3, one of the three AtCYP85A2 homologous genes from Populus trichocarpa. PtCYP85A3 shares the highest similarity with AtCYP85A2 and can rescue the retarded-growth phenotype of the Arabidopsis cyp85a2-2 and tomato dx mutants. Constitutive expression of PtCYP85A3, driven by the cauliflower mosaic virus 35S promoter, increased the endogenous BR levels and significantly promoted the growth and biomass production in both transgenic tomato and poplar. Compared to the wild type, plant height, shoot fresh weight and fruit yield increased 50%, 56% and 43%, respectively, in transgenic tomato plants. Similarly, plant height and stem diameter increased 15% and 25%, respectively, in transgenic poplar plants. Further study revealed that overexpression of PtCYP85A3 enhanced xylem formation without affecting the composition of cellulose and lignin, as well as the cell wall thickness in transgenic poplar. Our finding suggests that PtCYP85A3 could be used as a potential candidate gene for engineering fast-growing trees with improved wood production.


Assuntos
Brassinosteroides/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Populus/enzimologia , Madeira/crescimento & desenvolvimento , Sequência de Aminoácidos , Biomassa , Sistema Enzimático do Citocromo P-450/genética , Lycopersicon esculentum , Proteínas de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Populus/genética , Populus/crescimento & desenvolvimento , Árvores/enzimologia , Árvores/crescimento & desenvolvimento , Madeira/citologia
2.
PLoS One ; 11(2): e0148572, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26849212

RESUMO

NHL (NDR1/HIN1-like) genes play crucial roles in pathogen induced plant responses to biotic stress. Here, we report the possible function of NHL6 in plant response to abscisic acid (ABA) and abiotic stress. NHL6 was highly expressed in non-germinated seeds, and its expression was strongly induced by ABA and multiple abiotic stress signals. Loss-of-function of NHL6 decreased sensitivity to ABA in the early developmental stages including seed germination and post-germination seedling growth of the nhl6 mutants. However, overexpression of NHL6 increased sensitivity to ABA, salt and osmotic stress of the transgenic plants. Further studies indicated that the increased sensitivity in the 35S::NHL6 overexpressing plants could be a result of both ABA hypersensitivity and increased endogenous ABA accumulation under the stress conditions. It was also seen that the ABA-responsive element binding factors AREB1, AREB2 and ABF3 could regulate NHL6 expression at transcriptional level. Our results indicate that NHL6 plays an important role in the abiotic stresses-induced ABA signaling and biosynthesis, particularly during seed germination and early seedling development in Arabidopsis.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Membrana/metabolismo , Sementes/fisiologia , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Proteínas de Membrana/genética , Mutação , Pressão Osmótica , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Fatores de Transcrição/genética
3.
Plant Biotechnol J ; 13(7): 962-73, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25641517

RESUMO

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild-type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na(+) accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na(+) /H(+) exchange activity and Na(+) efflux in transgenic plants were significantly higher than those in the wild-type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt-tolerant trees.


Assuntos
Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Populus/genética , Tolerância ao Sal/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Populus/efeitos dos fármacos , Populus/metabolismo , Cloreto de Sódio/farmacologia
4.
Plant Mol Biol ; 86(4-5): 471-83, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25168737

RESUMO

Tubby and Tubby-like proteins (TLPs) play essential roles in the development and function of mammal neuronal cells. In addition to the conserved carboxyl (C)-terminal Tubby domain, which is required for their plasma membrane (PM) tethering, plant TLPs also possess an amino (N)-terminal F-box domain to interact with specific Arabidopsis Skp1-like (ASK) proteins as functional SCF-type E3 ligases. Here, we report the molecular characterization of Arabidopsis TLPs (AtTLPs). ß-Glucuronidase staining showed overlapped but distinct expression patterns of AtTLPs in Arabidopsis. Yeast two-hybrid assays further revealed that AtTLP1, AtTLP3, AtTLP6, AtTLP7, AtTLP9, AtTLP10 and AtTLP11 all interacted with specific ASKs, but AtTLP2, AtTLP5 and AtTLP8 did not. Subcellular localization observations in both Arabidopsis protoplasts and tobacco pollen tubes indicated that all GFP-AtTLP fusion proteins, except GFP-AtTLP8 which lacks the conserved phosphatidylinositol 4,5-bisphosphate binding sites, were targeted to the PM. Detailed studies on AtTLP3 demonstrated that AtTLP3 is a PM-tethered PIP2 binding protein which functions redundantly with AtTLP9 in abscisic acid (ABA)- and osmotic stress-mediated seed germination. Our results suggest that AtTLPs possibly work in multiple physiological and developmental processes in Arabidopsis, and AtTLP3 is also involved in ABA signaling pathway like AtTLP9 during seed germination and early seedling growth.


Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas F-Box/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Pressão Osmótica , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Proteínas F-Box/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido
5.
Yao Xue Xue Bao ; 43(10): 1053-9, 2008 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-19127871

RESUMO

This paper is aimed to develop rapid, sensitive and convenient HPLC-MS/MS methods for the quantification of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma. According to the different natures of the compounds, two sets of liquid chromatography and ionization modes were used for determination the concentration of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma, separately. Following protein precipitation with methanol, the levosimendan and internal standard (rosuvastatin) were separated on a Capcell MG III C18 column (35 mm x 2.0 mm ID, 3 microm) with the mobile phase consisted of methanol-15 mmol x L(-1) ammonium acetate-formic acid (55: 45: 0.02, v/v/v). A tandem mass spectrometer equipped with electrospray ionization source was used as the detector and operated in the negative ion mode. Its metabolites OR-1855, OR-1896 and internal standard doxofylline were extracted from plasma by liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Zorbax Extend C18 column (150 mm x 4.6 mm ID, 5 microm) with the mobile phase consisted of methanol-15 mmol x L(-1) ammonium acetate-formic acid (65 :35 :0.1, v/v/v). A tandem mass spectrometer equipped with electrospray ionization source was used as the detector and operated at the positive ion mode. The linear concentration ranges of the calibration curves for levosimendan and OR-1855 and OR-1896 were 0.10-50.0 ng x mL(-1), 0.20-100 ng x mL(-1), 0.20-100 ng x mL(-1), respectively. The lower limits of quantification of levosimendan and OR-1855 and OR-1896 were 0.10 ng x mL(-1), 0.20 ng x mL(-1), 0.20 ng x mL(-1), respectively. The methods proved to be sensitive, simple and rapid, and suitable for the pharmacokinetic study of levosimendan injection.


Assuntos
Acetamidas/sangue , Hidrazonas/sangue , Hidrazonas/metabolismo , Piridazinas/sangue , Piridazinas/metabolismo , Cardiotônicos/sangue , Cardiotônicos/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Masculino , Simendana , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 23(2): 134-7, 2007 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-17286907

RESUMO

AIM: To express HCV F protein gene fragment in E.coli and detect if there is its antibody in HCV patients. METHODS: RNA of the virus identified as genetype 1b was choosed as template, the F protein gene was amplified by RT-PCR. This gene fragment was inserted into plasmid vector pGEM simple T. F gene was digested by EcoR I and BamH I from pGEM and cloned into plasmid vector pGEX-4T-2. The ligation mixture was transformed into E.coli. TG1 and F fragment expression was induced by IPTG. The expressed protein was purified from lysates with Glutathione Sepharose 4B. The purified protein was used to identify whether there was anti-F antibody in the patients which HCV RNA was positive by ELISA. RESULTS: After IPTG induction, a positive band about 43 kD was detected. 82 of 120 HCV RNA positive's sera had anti-F antibody by ELISA. CONCLUSION: It is possible to efficiently express the HCV F protein in E.coli. The positive rate of anti-F antibody in HCV RNA positive patients was 68%.


Assuntos
Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Animais , Western Blotting , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Genótipo , Anticorpos Anti-Hepatite/sangue , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Core Viral/imunologia
7.
Artigo em Chinês | MEDLINE | ID: mdl-18322585

RESUMO

OBJECTIVE: To investigate whether HV and Ot can coexist in their host (Leptotrombidium scutellare). METHODS: Collecting the separate Leptotrombidium scutellare and the ones from mice in epidemic area. The cells of mites at larva, nymph, and adult stages were cultured and made into smear. In situ RT-PCR and PCR were used to detect and locate HV RNA and Ot DNA in the primary cultured cells. RESULTS: Positive signals of HV RNA and Ot DNA distributed mostly in epithelial cells of digestive system and ovary cells of larva and nymph. The positive rate increased by the generation of passages. CONCLUSION: Coinfection of HV and Ot did exist in wild Leptotrombidium scutellare.


Assuntos
Hantavirus/isolamento & purificação , Ácaros/microbiologia , Ácaros/virologia , Orientia tsutsugamushi/isolamento & purificação , Animais , Células Cultivadas , DNA Bacteriano/análise , Feminino , Camundongos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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