Yeast Two-Hybrid Assay for GI Interactions

We used the ProQuest Two-Hybrid System (Invitrogen) as previously described (28 (link)). Specifically, the cDNA encoding full-length GI was transferred from the pENTR/D-TOPO vector (Invitrogen) into the pDEST32 vector by Gateway LR recombination reaction (Invitrogen) to generate the bait plasmid; the pDEST22 prey plasmids containing the sequences encoding RGA, GAI, RGL1, RGL2, and RGL3 have been previously described (45 (link)). Empty pDEST22 and the pExpAD502 plasmids were used as negative controls. Quantitation of β-galactosidase activity was performed in a 96-well format as previously described (47 (link)).

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Nohales M.A, & Kay S.A. (2019). GIGANTEA gates gibberellin signaling through stabilization of the DELLA proteins in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 116(43), 21893-21899.

Publication 2019

ProQuest Two-Hybrid System pENTR/D-TOPO vector Gateway LR recombination reaction

Cdna Hybrid Plasmids Recombination Topo Vector β galactosidase

Corresponding Organization :

Other organizations : University of Southern California

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Variable analysis

independent variables

GI (full-length)

dependent variables

β-galactosidase activity

control variables

Empty pDEST22
PExpAD502

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