RESUMO
To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.
Assuntos
Proteínas de Arabidopsis/genética , Morus/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Agrobacterium tumefaciens/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Frutas/genética , Regulação da Expressão Gênica de Plantas , Morus/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plasmídeos , Plântula/genéticaRESUMO
OBJECTIVE: To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses. METHODS: Obtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance. RESULTS: Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity. CONCLUSION: Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/virologia , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/diagnóstico , Sensibilidade e EspecificidadeRESUMO
AIM: Expression and analysis of recombinant chicken IL-18 in Pichia pastoris. METHODS: Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR, and was subcloned into Pichia pastoris expression vector pPICZalphaA to construct the recombinant plasmid pPICZalphaA-ChIL-18. After identified by restriction enzymes digestion analysis, PCR and DNA sequencing, the recombinant plasmid was transformed into Pichia pastoris X-33.Then choosing the multi-copy recombinant strains to be induced for expression.Then the bioactivity of rchIL-18 was analysed by Western blot, ELISA and MTT after purified by Sephadex G-100 column. RESULTS: The chicken IL-18 with the immunogenicity was secreted by Pichia pastoris. It could induce T lymphocytes proliferation and secreting IFN-gamma in vitro. CONCLUSION: The chicken IL-18 with obvious biological activity is secreted by Pichia pastoris X-33.