Browsing by Subject "BiFC"

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  • Gao, Song (Helsingfors universitet, 2010)
    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.
  • Vasupalli, Naresh; Hou, Dan; Singh, Rahul Mohan; Wei, Hantian; Zou, Long-Hai; Yrjälä, Kim; Wu, Aimin; Lin, Xinchun (2021)
    Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1, PheCAD6, PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2, PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1, PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.
  • Streng, Janne (Helsingfors universitet, 2013)
    RNA silencing is a sequence specific RNA degradation mechanism which is used by plants to regulate gene expression and to combat virus infections. However, viruses have developed so called silencing suppressors, which can prevent and interfere silencing reaction by many ways. For example, virus proteins can bind to maintaining proteins of the silencing reaction or to molecules which are responsible for signaling of the silencing reaction. This thesis focused on the study of protein-protein-interactions between known silencing suppressors of crini- and potyviruses and four maintaining plant proteins of RNA silencing. Protein-protein-interactions were studied using the yeast two-hybrid system (YTHS) and the bimolecular fluorescence complementation assay (BiFC). The latter method enables visualization of the studied protein interactions in plant cells. Protein expression of the cloned genes in yeast vectors were studied by using western blot. BiFC analysis was focused on protein interactions which were found by YTHS. This study detected three previously unknown protein interactions. Two virus proteins were found for the first time to bind directly to silencing maintaining proteins that are known to be targets of other silencing suppressors. Because the functions of these silencing maintaining proteins are known, it is possible that the three interactions described in this study interfere RNA silencing by impeding the functions of the plant proteins.