|GAFFNEY, BOBBY - University Of Kentucky|
|HUNT, A - University Of Kentucky|
Submitted to: North American Alfalfa Improvement Conference
Publication Type: Abstract Only
Publication Acceptance Date: 6/27/2010
Publication Date: 7/27/2010
Citation: Gaffney, B., Dinkins, R.D., Hunt, A.G. 2010. CPSF30 in medicago. 42nd North American Alfalfa Improvement Conference and 21st Trifolium Conference.
Technical Abstract: CPSF30 is a component in 3’ RNA processing that has been well characterized in the model species Arabidopsis thaliana. In this study the CPSF30 gene has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thaliana and both Medicago species. This area of the gene was cloned out and 5’ and 3’ RACE were used to clone each full length gene. The Medicago genes were cloned into a protein expression vector. The vector was used to translate each gene into its corresponding protein and each protein was purified and dialyzed. CPSF30 in A. thaliana binds calmodulin and it was hypothesized that CPSF30 from both the Medicago species would also bind calmodulin based on the conservation of the amino acids between the model species and the Medicago species. This study shows that CPSF30 from both Medicago species does bind calmodulin. CPSF30 from A. thaliana also has been shown to possess three CCCH type zinc fingers, with the first implicated in RNA binding and the third in RNA cleavage. Amino acid alignments showed the zinc finger regions were conserved and it was hypothesized that CPSF30 from both Medicago species would bind RNA and cleave RNA. Electromobility shift assays were used to determine that RNA binding did occur. RNA nuclease activity in Medicago is still being tested. CPSF30 knockout studies in A. thaliana have yielded some consistent phenotypes, such as a decrease in lateral roots and an elevated resistance to oxidative stress. CPSF30 M. sativa knockout/knockdown plants have been recovered, and the transgenic T0 plants exhibit aberrant root phenotypes. Additional analyses are ongoing.