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Biotechnology Home

Identification of Bt genes for control of parasitic nematodes in banana

X-Q. Li, J-Z. Wei, A. Tan, R.V. Aroian

Plant-parasitic nematodes cause significant crop loss worldwide, including to banana and plantain in eastern, central and western African regions. Crystal (cry) proteins made by the bacterium Bacillus thuringiensis (Bt) have been used for over 50 years to control insect pests and during the past decade have been successfully expressed in transgenic crops, notably cotton and corn. These Bt crops demonstrate excellent protection against insect pests and allow for reduction in chemical pesticide use. Our laboratory has demonstrated that some cry proteins are toxic to diverse free-living nematode species. Thus we decided to test whether expressing these proteins in transgenic plants might afford protection against plant-parasitic nematodes. We synthesized ‘plant-friendly’ versions of the nematicidal cry protein Cry6A by assembling the entire genes de novo from 70-90-mer oligonucleotides with high-fidelity polymerase chain reaction (PCR). About 25% of the nucleotides have been altered relative to the bacterial Cry6A gene and the G-C content has been increased from 27 to 44%. The synthesized cry6A gene driven by enhanced 35S promoter was introduced into tomato hairy root via Agrobacterium rhizogenes–mediated transformation, since the hairy root system is simple and has been used to study transgenic resistance to various nematodes. After trouble-shooting, we further modified the original synthesized cry6A with point-mediated mutagenesis. The highest Cry6A expression level was about 0.25% of the total soluble protein from hairy root lines either transformed with full-length (1425 nucleotides) or truncated (1158 nucleotides) constructs. Northern blot results revealed that no aberrant transcripts were detected from cry6A transgenic plants. Sequencing of RT-PCR products has indicated that transcripts from transgenic lines have correct coding sequence, terminator sequence and polyA tail. We are testing these transgenic hairy root plants for biocontrol of the endoparasitic nematode Meloidogyne incognita. Our results measuring changes in infection rate and production of progeny will be presented, as well as results studying the uptake of the protein by the nematode.

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