Cloning of cDNAs and expression of putative insecticidal proteins from Albizzia procera active against cowpea bruchid (Callosobruchus maculatus)
Abstract
Cowpea, Vigna unguiculata (L.) Walp, is an important crop in West Africa and in north-east South America where it is a major source of dietary protein. Upon storage, severe damage to the seeds often occurs due to the infestation by the larvae of the bruchid beetle, Callosobruchus maculatus (F.). As a result, these seeds can no longer be used for planting and for human consumption. Fumigants are used to control the bruchid beetle (cowpea weevils), but their use is limited due to costs. Furthermore, chemical control in general causes environmental damage and may evoke a producer and consumer risk. An alternative to chemical control is to use a biotechnological approach, which involves the transformation of genes encoding proteins that have toxicity to cowpea weevil. This approach first requires the isolation of novel proteins that show in vitro activity against cowpea weevil. We collected various seeds from different non-host plant species from several locations worldwide. Crude extracts of these seeds were screened for insecticidal activity toward cowpea weevil. This screening resulted in the isolation and identification of two insecticidal proteins from Albizzia procera seeds. The corresponding cDNAs were cloned and subsequently analyzed. One cDNA clone encodes a cysteine proteinase-like protein, while the other cDNA clone encodes a Kunitz-type trypsin inhibitor. Incorporation of recombinant Kunitz-type trypsin inhibitor into an artificial seed diet caused a delay in the growth and development of cowpea weevils. From this we can conclude that at least one of the two cDNAs isolated has a very high potential to be used successfully in the production of cowpea plants that are resistant to cowpea weevil. We also are interested in the possibility of using multiple insecticidal proteins that exhibit synergistic (multiple mechanisms) anti-insect activities because of the risk of selection pressure resulting in resistant insect population. We therefore tested whether purified GS-II (legume lectin from Griffonia simplicifolia II) and recombinant N2 (cysteine proteinase inhibitor from soybean) have synergistic effects on the growth and development of cowpea weevil.
Degree
Ph.D.
Advisors
Bressan, Purdue University.
Subject Area
Entomology|Molecular biology|Genetics
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