2011 Annual Report
1a.Objectives (from AD-416)
To analyze the progeny of rice plants transformed with constructs that result the over-expression and knockdown of the rice PIMT1 and PIMT2 genes.
1b.Approach (from AD-416)
Asparagine and aspartate in proteins can form isoaspartate through deamidation and isomerization in the former, and dehydration and isomerization in the latter. PROTEIN-L-ISOASPARTYL METHYLTRANSFERASE (EC 22.214.171.124) (PIMT) is a repair enzyme that regenerates isoaspartate back to aspartate. In the model plant, Arabidopsis thaliana there are two PIMT genes. PIMT1 is expressed throughout the life cycle of the plant, while PIMT2 is expressed at high levels in dormant seeds where PIMT specific activity is greatest. In addition, PIMT2 has multiple transcriptional start sites and splicing variants allowing for the production of multiple isoforms that are targeted to different subcellular organelles. Two PIMT genes have now been identified in many other plant species suggesting similar strategies in the protection of the proteome. Using sequences of the Arabidopsis thaliana PIMT1 and PIMT2, two genes with homology to the Arabidopsis proteins were identified in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor) and maize (Zea mays) EST databases. We propose to use rice as a model system for monocot analysis due to large genomic resources presently available. Analysis of the rice genomic and EST databases predict that both of the rice PIMT genes potentially encode multiple transcripts from each gene, which is different than the results observed in Arabidopsis. RT-PCR of rice total RNA from different tissues using gene specific primers has been used to amplify the transcripts from each gene. Each cDNA will be cloned into a transformation vector (pCAMBIA1301) for stable over-expression in rice. RNAi constructs will be developed for each transcript where possible to evaluate the knockout/knockdown effect on seed germination. Rice seed dormancy, germination, and seed longevity will be evaluated for each transgenic combination in collaboration with Dr. Bruce Downie (UK Department of Horticulture).
Progress has been made in the construction of the plant transformation vector containing the rice PIMT1 gene for overexpression and expression knock down in rice. Transformation experiments conducted on rice using control vectors have not yet been successful, thus we have not yet obtained transgenic plants over-expressing the rice PIMT gene. Experiments are ongoing to overcome this limitation. We continue with plans to obtain transgenic rice plants, however until the problems with the transformation procedures are overcome, we will not able to evaluate the knock down and over expression in rice. This project was monitored with face-to-face meetings, teleconferences, emails, and involvement in the research.