1a.Objectives (from AD-416):
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.
1b.Approach (from AD-416):
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 Transcription Factors (TF) 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 polymerase chain reaction (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.
Twenty Transcription Factors (TF) Open Reading Frames (ORFs) from bHLH, MYB and AP2 families related to root development and stress responses have been cloned. Cloning of seventeen more genes is in progress. In total, 68 TF-ORFs are in the Gateway cloning pENTR vector. Primer sets for ~30 more genes related to root development and drought resistance have been developed and the cloning process is in progress. The GmERF3 antibody has been generated and purified and is under quality check using Western blotting. After the evaluation of GmERF3 antibody, ChIP, ChIP-Seq and RNA-Seq will be performed using GmERF3 transgenic Arabidopsis plants generated in the lab. Antibody production for another TF, GmbZIP1, is in progress. We have completed third biological replicate of the abiotic stress experiments in controlled conditions and collected tissues for various objectives of the project including the RNA seq and smallRNA seq. The slow wilting tolerant and high yielding population (NAM 10; IA3023/LD00-3309) was selected for the drought experiments under the controlled conditions. A preliminary study was conducted with the parental lines to analyze permanent wilting point and fix the field capacity of soil medium to be used in the main experiment. Seventy RILs developed from IA3023 and LD00-3309 along with four checks (W82, U06-100052, LD04-13265, and LD04-11056) was sown in two pots, one for control and one for stress conditions respectively. Leaf samples of the RILs (70) along with parents and checks (W82, U06-100052, LD04-13265, and LD04-11056) were collected at permanent wilting point for various genomic studies including RNA-Seq, smallRNA-Seq and methylome profiling experiments. These leaf tissues were shipped to various Co-PI laboratories for the above analyses. Two replications of this population were completed and based on the results a third replication will be conducted after this summer.