Detection of protein- protein interactions in planta by BiFC and split luciferase assays

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Title: Detection of protein- protein interactions in planta by BiFC and split luciferase assays
Author: Gao, Song
Contributor: University of Helsinki, Faculty of Agriculture and Forestry, Department of Agricultural Sciences
Publisher: Helsingfors universitet
Date: 2010
Language: eng
Thesis level: master's thesis
Discipline: Växtproduktions biologi (trädgårdsvetenskap)
Plant Production Biology (Horticulture)
Kasvintuotannon biologia (puutarhatiede)
Abstract: Molecular biology has created a new pathway for plant breeding in cut flower industry. It focuses on studying flower gene functions and provides a more direct and effective way of breeding new flower cultivars using genetic transformation. Besides flower color, disease resistance, quality and vase life, modification of flower architecture is an important target for flower breeding. Previous studies have showed that various transcription factors encoded by the corresponding genes are involved regulating flower development and flower architecture. The most studied are MADS domain and TCP domain transcription factors. For targeted breeding, it is crucial to study the functions of the corresponding genes in detail. For both MADS and TCP domain proteins, previous studies have indicated that protein-protein interactions are important for their function. GhCYC1, GhCYC2, GhCYC3 and GhCYC4, isolated from gerbera (Gerbera hybrida), are CYCLOIDEA –like genes affecting inflorescence development. The protein-protein interactions among these four genes have previously been studied by yeast two-hybrid system. The aim of this thesis was to verify the interactions in living plant cells, using both BiFC and split luciferase assays. Protoplast electroporation and agroinfiltration were used to introduce the genes in planta. The results from the two assays were compared in order to find an effective in planta method for detecting protein-protein interactions. The experiment also provided information about DNA transformation efficiency using protoplast electroporation and agroinfiltration. The results of the split luciferase assay showed that GhCYC1+GhCYC4, GhCYC3+GhCYC4 as well as GhCYC4+GhCYC4 interacted quite strongly in plant cells while GhCYC1+GhCYC1, GhCYC2+GhCYC2 as well as GhCYC4+GhCYC2 had almost no interactions. The interactions between GhCYC3+GhCYC4, and GhCYC4+ GhCYC4 were also shown in yeast two-hybrid, but the other results were different. According to the BiFC assay, no signals of interactions were detected from GhCYC2+GhCYC2, while strong signals were observed from GhCYC2+GhCYC3, and weak signals were seen from GhCYC2+GhCYC4. The interactions between GhCYC2+GhCYC3, GhCYC2+GhCYC4 were also observed in yeast two-hybrid, but the other results were unconfirmed. Large standard deviations were observed in the split luciferase assay and thereby reliable conclusions cannot be drawn from it. However, BiFC turned out to be a better method to detect the protein-protein interactions in planta and clear signals from interactions could be observed. Comparison of the transformation methods indicated that agroinfiltration is a better way of introducing DNA into plant cells than protoplast electroporation. For further study, BiFC assay still needs to be repeated to confirm the efficiency of this assay, and factors affecting the transformation efficiency in protoplast electroporation need to be optimized in the future studies.
Subject: protein- protein interactions
split luciferase assay

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