บทคัดย่องานวิจัย

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Molecular biological investigations of ACC oxidase and its expression attending apple fruit ripening.

Dilley, D. R.; Kuai, J.; Wilson, I. D.; Pekker, Y.; Zhu, Y.; Burmeister, D. M.; Beaudry, R. M.;

Acta Horticulturae Year: 1995 Issue: No. 379 Pages: 25-39 Ref: 24 ref.

1995

บทคัดย่อ

Molecular biological investigations of ACC oxidase and its expression attending apple fruit ripening.

 

Protein changes during the development of the ethylene climacteric were studied in harvested apple fruits of several cultivars. Using polyclonal antibodies and Western blot analysis, ACC oxidase was not detectable in fruits prior to the onset of the climacteric but increased rapidly as the climacteric developed. A quantitative ELISA for ACC oxidase revealed that the enzyme's induction preceded increasing ACC content and the ethylene climacteric by 4-7 days in all cultivars. Western blot analyses and immunoprecipitation of in vitro translation products of poly A+mRNA indicated that the translation of ripening-related ACC oxidase results in accumulation of the enzyme prior to the ethylene climacteric as a consequence of an increase in its translatable mRNA transcript. Escherichia coli BL21 (DE3) pLysS were transformed with the cDNA pAP4 known to encode apple ripening-related ACC oxidase, the insert being fused to a His-Tag leader sequence with an intervening thrombin proteolytic site on the N-term

inal methionine of ACC oxidase, thus yielding a clone (pETAOEx2a) with acquired ability to convert ACC to ethylene under induction by isopropyl-b-D-thiogalactoside (IPTG). This was correlated with the synthesis of a novel 1.2-kb mRNA that hybridized with the pAP4 cDNA. The His-Tag-ACC oxidase fusion protein (37.7 kDa) was purified and proteolytically cleaved with thrombin to yield a recombinant enzyme with a mass of 35.4 kDa. The His-Tag-ACC oxidase fusion protein and the recombinant ACC oxidase exhibited enzymatic properties similar to those of the native apple fruit enzyme (35.3 kDa) in all respects (including kinetic properties, CO2 activation, and substrate and inhibitor specificity).