engleski

Auxin-amidohydrolase in wheat: cloning, expression and substrate specificity

The wheat IBA-amidohydrolase, TaIAR3, was investigated. The TaIAR3 gene is an ortholog of the Arabidopsis IAA-amidohydrolase and has been isolated from winter wheat (Triticum aestivum L. cv. Caledonia). The complete cDNA of TaIAR3 was cloned into the pETBlue-2 vector and protein expression was obtained in E. coli. In order to determine enzyme activity and substrate specificity, the bacterial cell lysate was processed for biochemical reaction with the substrate, IBA-Ala, for 1, 2, 4, and 6 hours, extracted with ethyl acetate, evaporated to dryness in a stream of nitrogen and resuspended in methanol for HPLC analysis. The HPLC separation confirmed hydrolase activity in the bacterial homogenate, Furthermore, it was shown that specific cleavage of IBA-Ala, which is an inactive form of auxin, affords free, physiologically active, IBA. This leads to the conclusion that auxin-amidohydrolase may be involved in auxin metabolism, specifically controlling the release of active auxin.

auxin; amidohydrolase; indole-3-acetic acid; IAA; indole-3-acetic acid; IBA; indole-3-butyric acid; gene cloning

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