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Descripción
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| The objective of our project is to test whether the plastids of plant cells are organelles capable of sustaining the activity of the nitrogenase Fe protein for the future engineering of nitrogen fixing plants. To test this ability, we studied the transient expression and import to chloroplasts of four genes from the model diazotroph Azotobacter vinelandii (nifH, nifM, nifS and nifU) in Nicotiana benthamiana leaves. In-silico synthetic gene design was performed on the four nif genes, generating versions that are codon optimized, have a GC and GC3 overall content very similar to that of the tobacco genes and where known dicotyledonous polyadenylation sequences and RNA instability motifs have been avoided. In addition, we screened 12 previously characterized chloroplast transit peptides (CTPs) to test their ability to drive each specific Nif protein to the chloroplast stroma in tobacco mesophyll cells. For this purpose, we built libraries with each nif gene fused to one of the 12 CTPs in two different versions: i) a longer one with proven import ability and ii) a minimal one up to the predicted cleavage site by the Stromal Processing Peptidase. If this minimal version work properly, no residues would be left in the N-terminus of Nif proteins after cleavage, what would be optimal to assure their activity. Our results show that a set of CTPs led to the immuno-detection of a band corresponding to the size of Nif proteins, suggestive of correct import to the chloroplast and subsequent processing. In fact, the presence of Nif proteins in the chloroplast stroma in these cases was proved by chloroplast isolation and confocal microscopy experiments. However, in other cases we could observe lower mobility bands suggestive of unprocessed CTP-Nif fusions or of Nif proteins with extra residues in their N-terminus left behind after the CTP cleavage. Interestingly, CTPs show different results depending on the Nif protein fused, supporting an important role for cargo proteins in their functionality during import. These experiments pave the way for further work on the co-expression of Nif genes targeted to the chloroplasts of tobacco mesophyll cells and the assessment of their functionality. | |
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Internacional
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Nombre congreso
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XVII National Meeting of the Spanish Society of Nitrogen Fixation and VI Portuguese-Spanish Congress on Nitrogen Fixation |
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Tipo de participación
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970 |
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Lugar del congreso
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Madrid |
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Revisores
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ISBN o ISSN
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DOI
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Fecha inicio congreso
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10/07/2019 |
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Fecha fin congreso
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12/07/2019 |
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Título de las actas
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