ROOTS TRANSFORMATION OF Glycine max WITH RESPONSIVE PROMOTER FOR NEMATODES INFECTION
Abstract
Successful engineering of transgenic plants for resistance to nematodes requires promoters that over-express resistant genes in the proper cells at the proper time. Gene promoter region controls the temporal and spatial expression of genes. In previous results, Time-course Microarray experiments exhibit the activation of responsive gene in different time points. The data at feeding site showed that the peroxidase class III gene has a stress responsive promoter for nematodes infection and activated upon nematode infection. The transcriptional activation fold change of this gene was tested after 3, 6 and 9 days post nematode infection showing highly responsive and continuous active promoter at the site of infection. The mathematical modeling of its expression is indicated that its expression is prompt and predicts it's declining after ten days. The Pfam domain analysis of peroxidase class III gene showed that this protein is responsible to catalyze a number of oxidative reactions which responsive to many other stresses too. In this investigation we have identified and isolated the promoter region of the peroxidase class III gene which activated in Glycine max during nematode infection as well as infection and propagation of Phytophthora sojae fungus. The promoter region, -1440 bp upstream of the ATG transcription start site were isolated, cloned, sequenced and transferred to a collection of soybean plants roots inside a custom made vector, pJan25T. The Agrobacterium rhizogenes root transformation system was used so that whole plants will not need to be regenerated. The transformed plant roots were screened by infection of the root-parasitic nematode Heterodera glycin using eGFP as visual marker gene for transformation and Gus as a reporter gene for promoter activity. This active and responsive promoter could be used in the future experiments to over-express genes and gene fragments that may inhibit the life cycle of the nematode at the feeding site.References
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