15A-18 |
Sequencing of the first Bowman-Birk trypsin inhibitor cDNA clone in peanut (Arachis hypogaea L) |
H. W. DODO, Dept. of Food & Animal Sciences, Alabama A&M Univ., Food Biotechnology Lab., PO Box 1628, Normal, AL 35762 Proteinase inhibitors are ubiquitous in legumes and other plants species. They are said to be natural defense compounds that help to protect plants from attack by insects, pathogens and other predators. At least 37% of the peanut crop is lost to insect predation. In designing a genetic engineering approach to increase peanut natural resistance to insects, it is crucial to isolate and molecularly characterize cDNA clones encoding the trypsin inhibitors. The objective of this investigation was to isolate and sequence a peanut Bowman-Birk trypsin inhibitor cDNA clone. A peanut cDNA library constructed in a lambda gt-11 vector, was screened with synthetic oligonucleotides #10 and #17 which were constructed based on a partial amino acid sequence of a peanut Bowman-Birk trypsin inhibitor. The DNA of putative positive clones were isolated, purified, transferred to a nylon membrane, autoradiographed and confirm by Southern hybridization using standard molecular biology procedures. A positive lambda clone with a 4 KB insert was isolated, digested with HindIII, subcloned into a pBluescript vector and sequenced. Sequence analysis revealed a cDNA sequence of 792 bp in length which has 47.5% homology with a cowpea Bowman-Birk trypsin inhibitor gene and 41.7% homology with a soybean Bowman-Birk trypsin inhibitor gene. The sequence also showed a 95% homology to a peanut allergen gene Ara h4 and a 93% homology to another peanut allergen gene Ara h3. This is the first time that a sequence homology has been shown between a peanut trypsin inhibitor and allergens.
Session 15A, Biotechnology
|