A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro.
Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. 'Sponsor' were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.
Hassan
Fathi
F
Institute of Plant Genetics, Leibniz University of Hannover, Herrenhäuserstr. 2, D-30419 Hannover, Germany.
Meens
Jochen
J
Jacobsen
Hans-Jörg
HJ
Kiesecker
Heiko
H
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015139:Blotting, Southern; D015153:Blotting, Western; D002688:Chitinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D025301:Hyphae / Q000187:drug effects / Q000254:growth & development; D007525:Isoelectric Focusing; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014162:Transfection; D014242:Trichoderma / Q000187:drug effects* / Q000254:growth & development
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002688:Chitinases