Kathryne J. Blair worked with Dr. David Robinson and Dr. Joann Lau in the Bellarmine Department of Biology for more than two years. Katie’s project started out to be a bioinformatic approach to analyzing a cDNA library made from Giant Ragweed pollen. She analyzed 75 different genes from this library. One of the pollen genes she discovered was for the enzyme, Pectate Lyase. This protein is believed to be one of the primary causes of pollen allergies in Ragweed, so it is quite important. It has never been found in this species of Ragweed before. Because of the size of this gene Ms. Blair carried out ‘primer walking’ to attain the full-length cDNA sequence. She used the cDNA information to design primers to sequence the genomic Pectate Lyase gene, as well. This allowed her to identify the exon/intron structure of the Pectate Lyase gene.
After that, Katie collected various tissues from Giant Ragweed plants growing on campus. She collected fresh roots, leaves, female flowers and male flowers and extracted mRNA. Ms. Blair used Reverse Transcriptase-PCR to examine the expression of Pectate Lyase in these different tissues. She also inserted the Pectate Lyase cDNA into an expression vector to induce the cells to synthesize this enzyme. Kathryne Blair has presented her research at numerous national, regional and state conferences, and has won two awards for her presentations. She enters medical school at Marshall University, West Virginia in the fall of 2009.
Abstract
Over 36 million Americans suffer from allergies and hay-fever as a result of pollen released by grasses, trees, and weeds. A major source of this allergenic pollen is Giant Ragweed (Ambrosia trifida L.), a common weedy plant in the U.S. When this project was initiated there were only four nuclear DNA sequences from Giant Ragweed published in the NCBI GenBank. Our objective is to contribute to this database by sequencing and analyzing Giant Ragweed genes. A cDNA library was constructed from mRNA isolated from dehiscing male flowers. To date, 153 clones have been characterized according to their biological, cellular, and molecular functions using NCBI BLASTX searches. Of the known cellular locations, the most common for these gene products was in the nucleus. The most frequent biological roles appear to be involved with transcription, post-transcriptional processing, transport, and cell division/development. At least seven potential allergenic protein sequences were also isolated, including a pectate lyase protein. Pectate lyase is an enzyme responsible for the degradation of cell walls and pectin. Primer walking was performed to determine the entire cDNA sequence, and PCR primers were designed to isolate the genomic sequence for further analysis. RT-PCR was performed on 11 different tissues and the highest expression appeared to be in pollen, with the lowest expression in female flowers. Recent studies have shown that global climate change might increase pollen production in Ragweed, thus the likelihood of pollen allergies could increase. A better understanding of pollen biology may enable researchers to ameliorate the problem.
Presentations
Blair KJ, Robinson DL, and Lau JM. 2008. The ambrosia project: analysis of a cDNA library from giant ragweed (Ambrosia trifida L.) pollen and initial examination of allergenic pectate lyase. American Institute of Biological Sciences, 58th Annual Meeting, Arlington, VA. May 12-14.
Blair KJ, JM Lau DL Robinson. 2008. The Ambrosia project: Characterizing a pollen cDNA library from giant ragweed. April 18, 2008, Butler University Undergraduate Research Conference, Butler University, Indianapolis, IN.
Blair KJ, JM Lau DL Robinson. 2008. Initial analysis of a giant ragweed (Ambrosia trifida L.) cDNA library from pollen and characterization of allergenic pectate lyase. April 19, 2008, Math, Engineering, Science Conference (MESCON), University of Evansville, Evansville, IN. Awarded 2nd place in the Biology presentation competition.
Blair KJ, JM Lau, DL Robinson. 2007. Characterization of a pollen cDNA library from giant ragweed (Ambrosia trifida L.), and analysis of allergenic pectate lyase. 93rd annual meeting of the Kentucky Academy of Science, Louisville, KY, Nov. 8-10. Awarded 1st place in the Undergraduate Research Competition (Botany Section).
Manning, CN, KJ Blair, JM Lau, DL Robinson. 2007. The Ambrosia project: Student characterization of a cDNA library derived from ragweed pollen. Annual meeting of the American Society of Plant Biologists, Chicago, IL, July 7-11. p. 260.