2013 Annual Report
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 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.
The following was completed during FY 2013:
• During this reporting period, 31 Transcription Factor-Open Reading Frames (TF-ORF) from WRKY, bHLH, MYB, bZIP, HD-ZIP, AP2, PHD, PLATZ, and C2H2 (Zn) families related to root development and stress responses were cloned into the Gateway cloning pENTR or pDONR vector and sequences were verified.
• Cloning of 25 more genes with potential role in root development and abiotic stress response mechanisms are in progress.
• To the yeast one hybrid (Y1H) library resources, more than 60 TFs were cloned into pDNOR211 vector. Total, 300 TFs including 100 seed related TFs were transferred into pDEST22 vector as Y1H TF library. Sequence analysis is in progress.
• Five conserved motif elements and ten target gene promoter fragments were cloned and these pGLAC vectors were transformed into yeast strain Y4271. After self-activation test, three of them (including one motif repeats and two original promoters) were used for Y1H screening. We have identified nine TF hits through this screening and these candidates will be further confirmed in-planta.
• Three clusters of 279 stress responsive genes were used for over-represented motif analysis in MEME with soybean genome mining. Two candidates of over-represented motif from each cluster were cloned into pTOPO vector.
• Searching cis element enrichment in targeted set of 279 soybean genes associated with stress resistance is in progress.
• Preliminary quantification of stress responsive elements was done by using Dual-LUC assay with 2X35S promoter, ABRC promoter, SB motif, and Gbx21 promoter. Hairy root transformation was set up for further analysis with pPFR constructs.
• A preliminary ChIP experiment using soybean material has been completed successfully with optimized conditions including cross-linking & de-cross-linking time, sonication. Candidate genes for selected TF direct targets have been identified and primers for detecting the binding regions were developed.
• Generating transgenic soybean roots and evaluation of commercial anti-GFP antibody for ChIP experiments is in progress.