Publicação
The role of OsRMC gene in the JA signaling pathway: a transgenic approach in Arabidopsis and by Y1H assay
| Resumo: | Jasmonic acid is a plant hormone that has been involved in several biological processes such as development, biotic and abiotic stress. Previously, it was found in the host lab that a rice RNA interference (RNAi) – OsHOS1 line had up-regulation of OsRMC gene expression. In rice, this gene seems to be involved in the modulation of abiotic stress and root curling, acting as a negative regulator of the jasmonic acid signaling. OsRMC does not have a known homologue in Arabidopsis which makes this model species an important system to study the function of this gene. To achieve this goal, we overexpressed OsRMC in Arabidopsis in two different systems either in a cell suspension cultures (to study gene expression changes of JA-responsive genes) or whole plants (phenotypic changes induced by JA). We were able to obtain stable transgenic cell suspension culture overexpressing OsRMC as well for the empty vector (negative control). However, we were not able to see gene expression changes induced by JA even in untransformed cell suspension cultures for the analyzed genes (PDF1.2, VSP1 and MYC2). Regarding the transformation of whole plants, we generated three independent lines overexpressing OsRMC. These lines were characterized for transgene expression level and corresponding protein (OsRMC-RFP). We also performed a direct yeast one hybrid (Y1H) assay to complement the work done with Arabidopsis plants and cell suspension cultures regarding JA-signaling. In this assay, the promoter sequences of three genes involved in JA-signaling from rice (JAZ-like 1, JAZ-like 2 and JAZ-like 3) were analyzed. Promoter fragments corresponding to 1000bp upstream of the ATG codon of each gene were used as bait sequences. As prey, we used two transcription factors from the EREBP subfamily, previously identified as binding to the OsRMC gene promoter. However, we couldn’t detect interaction between the two TFs analyzed and the bait sequences from the JAZ genes. As future work, these baits strains will be screened by Y1H with cDNA libraries induced by salt or JA. As main achievements of this work, we were able to produce transgenic Arabidopsis plants overexpressing OsRMC which were fully characterized at transgene expression and protein level. These lines will be used in further studies such as to monitor expression changes in JA-responsive genes, determination of the sub-cellular localization of the OsRMC-RFP, and JA-induced phenotypic characterization. |
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| Autores principais: | Cortes, João Guilherme Costa de Alvarez, 1986- |
| Assunto: | Biologia molecular Expressão génica Stress Arroz Transgénicos Teses de mestrado - 2012 |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Jasmonic acid is a plant hormone that has been involved in several biological processes such as development, biotic and abiotic stress. Previously, it was found in the host lab that a rice RNA interference (RNAi) – OsHOS1 line had up-regulation of OsRMC gene expression. In rice, this gene seems to be involved in the modulation of abiotic stress and root curling, acting as a negative regulator of the jasmonic acid signaling. OsRMC does not have a known homologue in Arabidopsis which makes this model species an important system to study the function of this gene. To achieve this goal, we overexpressed OsRMC in Arabidopsis in two different systems either in a cell suspension cultures (to study gene expression changes of JA-responsive genes) or whole plants (phenotypic changes induced by JA). We were able to obtain stable transgenic cell suspension culture overexpressing OsRMC as well for the empty vector (negative control). However, we were not able to see gene expression changes induced by JA even in untransformed cell suspension cultures for the analyzed genes (PDF1.2, VSP1 and MYC2). Regarding the transformation of whole plants, we generated three independent lines overexpressing OsRMC. These lines were characterized for transgene expression level and corresponding protein (OsRMC-RFP). We also performed a direct yeast one hybrid (Y1H) assay to complement the work done with Arabidopsis plants and cell suspension cultures regarding JA-signaling. In this assay, the promoter sequences of three genes involved in JA-signaling from rice (JAZ-like 1, JAZ-like 2 and JAZ-like 3) were analyzed. Promoter fragments corresponding to 1000bp upstream of the ATG codon of each gene were used as bait sequences. As prey, we used two transcription factors from the EREBP subfamily, previously identified as binding to the OsRMC gene promoter. However, we couldn’t detect interaction between the two TFs analyzed and the bait sequences from the JAZ genes. As future work, these baits strains will be screened by Y1H with cDNA libraries induced by salt or JA. As main achievements of this work, we were able to produce transgenic Arabidopsis plants overexpressing OsRMC which were fully characterized at transgene expression and protein level. These lines will be used in further studies such as to monitor expression changes in JA-responsive genes, determination of the sub-cellular localization of the OsRMC-RFP, and JA-induced phenotypic characterization. |
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