15 酵母Tree-hybrid法によるオーキシン受容体のクローニング

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In order to clarify the signal transduction mechanisms by which plant hormones control various physiological events in plans, it is very important to isolate the receptors of plant hormones. Our objective in this study is the cloning of an auxin receptor cDNA with a yeast three-hybrid system, which was adapted from the yeast two-hybrid system to detect small ligand-protein interactions. As a third hybird ligand, a synthetic auxin, 1-naphtalene acetic acid (1-NAA), was covalently linked to dexamethasone (Dex). The yeast expression plasmids for the three-hybrid system were constructed as followed; the plasmid to express the fusion protein of the GAL4 DNA binding domain and the rat glucocorticoid receptor (GR), and the plasmids in which an Arabidopsis cDNA library was fused to the GAL4 transcriptional activation domain. Yeast cells transformed with both the plasmids were plated on medium containing the synthesized hybrid ligand, and the screening of an auxin receptor is now in progress. Next, the combination of His-tag and Ni-NTA, which form a stronger interaction than that of GR and Dex, was adapted to improve the three-hybrid system. The Ni-NTA was covalently corsslinked to Dex. The yeast cells expressing the His-tagged GAL4 DNA-binding domain and the fusion protein of the GAL4 activation domain and GR activate reporter genes when plated on medium containing the synthesized heterodimer of Ni-NTA and Dex. This new method is now under construction.
植物化学調節学会の論文
1999-10-01

15 酵母Tree-hybrid法によるオーキシン受容体のクローニング

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