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Cloning, Prokaryotic Expression And Purification Of Gibberellin Oxidase GA2ox1

Posted on:2011-01-18Degree:MasterType:Thesis
Country:ChinaCandidate:L ChangFull Text:PDF
GTID:2120360308968659Subject:Biochemistry and Molecular Biology
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GA (gibberellin) is a class of tetracyclic diterpenoid carboxylic acid, is one of endogenous hormones in plants, which has the largest species and the most extensive physiological function. People have discovered 136 kinds of GAs up to now, and a small number of them are biologically active, which affects all stages of higher plants life cycle. With the rapid development of technology and access to a variety of mutant materials, greater progress had been made in studying biosynthesis and regulation of GA, especially cloning key enzyme genes in GA synthesis. GA2-oxidase is encoded by multiple genes dioxygenase. It regulates GA metabolism, changes bioactive GA1 and GA4 into inactive GA8 and GA34 and reduces the activity of GAs in plants, so plants showed dwarfing, internode shortening phenotype. GA2-oxidase is also involved in plant photomor-phogenesis. Light induces GA2-oxidase gene expression, and plants reduce the biological activity of GA4, so that promote the completion of photomorphogenesis. However, there is no report about GA2-oxidase antibody currently. Therefore, this thesis predicted and analysed GA2ox1 gene encoding protein by bioinformatics, then cloned GA2ox1 from the Arabidopsis thaliana genome, constructed prokaryotic expression vector pDEST17-GA2ox1, expressed it in host strain E.coli BL (21) star, and optimized the expression condition; we also purified fusion protein GA2ox1 by means of affinity chromatography, Western Blot identificated whether is the target protein or not. The specific results of this study are as follows:(1) Bioinformatics analysis showed GA2ox1 encoded 329 amino acids. This protein had no obvious hydrophobic and its molecular weight was 36.7314kD, isoelectric point was 8.5793. It was a soluble protein with a potential transmembrane domain. It had not N-terminal signal peptide sequence. Its secondary structure enriched as well as contained a number of protein modification and activation of sites, such as the phosphorylation sites, N-glycosylation sites, N-myristoylation sites, ATP/GTP binding site and so on.The tertiary structure prediction showed that the protein was asymmetric and was an loosely folded globular protein.(2) We took columbia wild-type Arabidopsis thaliana as experimental material, Arabidopsis thaliana total RNA as template, and amplified GA2ox1 gene ORF by RT-PCR, which was about 990bp, as expected.(3) We successfully constructed prokaryotic expression vector pDEST17-GA2ox1, and confirmed by sequencing and PCR, it turned out the target gene was correctly inserted into the vector cloning site.Then we transformed it into host bacteria E. coli BL(21)star, we got prokaryotic-expressed engineering strain BL(21)star-pDEST17-GA2ox1.(4) We successfully inducted GA2ox1 to express in the host bacteria E.coli BL(21)star with IPTG. SDS-APGE analysis reviewed its expression mainly in the form of inclusion bodies and the molecular weight was about 40kD as we expected. The optimal conditions as follows:IPTG concentration was 0.2mmol/L, temperature was 30℃, the inducted time was 6 h.(5) We explored the expression of protein purification conditions, and obtained high purity GA2oxl fusion protein. Western Blot further analysis confirmed that the fusion protein was our target protein. This study establishes a basis for GA2oxl polyclonal antibody preparation, further studying GA2ox1 protein's function and researches on gibberellin regulation of plant growth.
Keywords/Search Tags:Arabidopsis thaliana, GA2ox1 gene, Bioinformatics, Prokaryotic expression, Purification, Western Blot
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