Poster: Plant insect-nematode interactions
Abs #
492: Enzymatic activity, cloning, and comparison of hydrolytic enzymes found in Nepenthes burkei
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Presenter: |
Stephenson, Paul T., PStephenson@Rollins.edu |
Authors | Stephenson, Paul T. (A) Hogan, Jamie (A) Butendieck, Ronald (A) Tucker, Kevin (A) Andretta, Anthony (A) | | Affiliations: |
(A): Rollins College, Department of Biology
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The pitchers of the carnivorous plant Nepenthes burkei contain an aqueous solution of hydrolases enabling the plant to digest trapped prey and obtain supplemental nitrogen. Non-denaturing SDS-PAGE activity gels were used to characterize proteinase activity in pitcher fluid samples, where best activity was observed at pH 3. Activity was examined in pitchers during a 22 day time course experiment in which the plants had been provided either with 0.1 mg/mL BSA as a digestible substrate or sterile water as a control. Newly opened pitchers were also assayed to determine whether pitcher fluid possessed proteolytic activity at opening. The results of this study suggest N. burkei pitchers produce at least five proteinases of differing molecular weights (126, 64.6, 50.3, 35.5, and 31.5 kD), with consistent activity from samples of individual pitchers, but variant activity among pitchers (even those on the same plant) suggesting that different pitchers secrete different enzymes. Inhibition of activity was observed when pitcher fluid samples were treated with either pepstatin or E-64. In addition, a single 21.4 kD band was observed in ribonuclease activity gels. Using degenerate oligonucleotide primers based on sequences of known RNases, cysteine proteinases, and aspartic proteinases we performed RT-PCR and cloned three partial gene sequences with homology to known hydrolases. A putative aspartic proteinase clone (NbAP1) was 97% homologous to an aspartic proteinase previously cloned from Nepenthes alata. A putative cysteine proteinase clone (NbCP1) showed 76% homology to a cysteine proteinase in Zinnia elegans and a putative RNase clone (NbRN1) showed 80% homology to an S-type RNase in Prunus dulcis.
