Location: Corn Insects and Crop Genetics Research
Project Number: 5030-21220-005-05-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Dec 6, 2011
End Date: Sep 30, 2015
1: ORFeome clones will be cloned into Gateway vectors. The focus of the clones will be on roots and transcription factors (gene regulators). We will target 300 clones; 2. Antibodies will be created for transcription factor proteins; 3. A CHIP will be developed for the analysis of DNA binding targets; 4. Selected transcription factors identified in stressed roots will be functionally characterized.
Cloning of approximately 300 open reading frames (ORFs) with a primary focus on root development and biotic and abiotic stress responsive transcription factors. This will occur over a period of three years with approximately 100 cloned per year. We will mine newly available transcriptome data generated by RNA seq and will select root related and biotic and abiotic stress related candidates for ORFeome cloning. Sequences (Open Reading Frame, ORF) of these potential TFs will be obtained from the soybean genome information (www.phytozome.net) and the cloning primers will be designed. Total RNA extracted from soybean tissues will be used for the gene isolation followed by cloning utilizing the Gateway cloning technology. PCR will be performed using Pfu polymerases and the PCR products will be purified and cloned into the Gateway cloning vector system (Invitrogen, CA), upon which the sequence will then be verified. We will use appropriate techniques for interaction studies of selected candidates after testing “TF-centered” and “Gene-centered” methods. Following sequence verification, candidate gene function will be tested in the model plant Arabidopsis using specific promoters. Transgenic soybean lines with selected genes will be generated through the Agrobacterium-mediated transformation technology developed at the Plant Transformation Core Facility at the University of Missouri. The transgenic plants will be analyzed for transgene integration and segregation followed by the extensive physiological and biochemical evaluation for the specific traits.