RESUMO
A beta-1,3-glucanase gene from Hordeum vulgare was isolated by a PCR strategy, cloned and subsequently sequenced. The amplified sequence contained the entire coding region of the isoenzyme II, which is interrupted by a 165 bp intron at 73 bp downstream the starting codon. This intron contains all the elements required for the processing mechanism in monocots: a high A + U content, the appropriate splice sites in the 5' and 3' ends and four typical YUNAN consensus sequences. Transient transformation of wheat protoplasts with the complete beta-1,3-glucanase gene under the control of maize polyubiquitin promoter revealed that the intron sequence was spliced out. The gene was also expressed at high levels, probably due to an enhancer-like sequence found near the 3' end of the intron.
Assuntos
Genes de Plantas/genética , Hordeum/genética , beta-Glucosidase/genética , Sequência de Bases , Northern Blotting , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Éxons , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucana 1,3-beta-Glucosidase , Hordeum/enzimologia , Íntrons , Isoenzimas/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Protoplastos/enzimologia , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Triticum , Ubiquitinas/genética , Zea maysRESUMO
Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.