LCI技术.pptVIP

FIREFLY LUCIFERASE COMPLEMENTATION IMAGING ASSAY FOR PROTEIN-PROTEIN INTERACTIONS IN PLANTS Plant Physiology, February 2008 yeast two-hybrid assay(Y2H) ?uorescence resonance energy transfer (FRET) bioluminescence resonance energy transfer (BRET) bimolecular ?uorescence complementation(BiFC) Alternative reporter-based methods for protein-protein interactions have been developed using protein fragment complementation coupled with enzymatic assays. FIREFLY LUCIFERASE COMPLEMENTATION IMAGING(LCI) ASSAY The firefly luciferase (LUC) enzyme is divided into the N- and C-terminal halves that do not spontaneously reassemble and function. LUC activity occurs only when the two fused proteins interact, resulting in reconstituted LUC enzyme. It can be detected by luminometer or a low-light imaging device. PCAMBIA (PUC19) Constructs for LCI assays in plants. A,Schematic diagrams of 35STNLuc and 35STCLuc constructs. L, Gly/Ser linker between LUC fragments and multiple cloning sites (MCS). rbs, Transcription terminator derived from the Rubisco small subunit gene. B, Diagram for LUC complementation resulting from NLuc- and CLuc-fusion proteins. AGROBACTERIUM-MEDIATED TRANSIENT EXPRESSION CLuc NLuc GV3101 Luc RESULTS Interaction between WRKY40 and WRKY18. TheWRKY18D construct lacks the Leu zipper motif. DISCUSSION 1. nonspecific interaction does not impede the proper determination of true interactions. 2. LCI assays are highly quantitative, samples the entire tissue or cell population, not affected by the chlorophyll- and cell wallgenerated autofluorescence,quickly. 3.simultaneously examine a large number of interacting proteins MYB21 AND MYB24 INTERACT WITH JAZ8, AND JAZ11 IN N. BENTHAMIANA. (A) to (D) LCI assays show that Arabidopsis MYB21 and MYB24 interact with Arabidopsis JAZ8 and JAZ11 in N. benthamiana. SPL9 BINDS TO PAP1. Competition of SPL9 and TT8 for PAP1 binding. Constructs were combined at a 1:1:4 ratio for PAP1-LUC-N: LUC-C-TT8: LUC-N, LUC-C,or rSPL9. ABAR INTERA