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Durham e-Theses
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Biochemical basis of insect resistance in winged bean (Psophocarpus tetragonolbus): characterisation of insecticidal proteins and their encoding genes

Howe, David S. (1991) Biochemical basis of insect resistance in winged bean (Psophocarpus tetragonolbus): characterisation of insecticidal proteins and their encoding genes. Doctoral thesis, Durham University.



Many pulses and beans grown for human comsumption are susceptible to insect attack. Winged bean, a high protein crop of the tropics, yield seeds which appear to be immune to infestation by the storage bruchid Callosobruchus maculatus. In this thesis the biochemical basis of this resisitance was investigated. Insect bio-assays were carried out in which protein fractions from seeds of winged bean were incorporated at a range of concentrations into artificial seeds, and their effects upon the development of C.maculatus determined. Both albumin and globulin fractions were toxic to the developing larvae and their toxicity correlated with their haemagglutination activity. Assay of psophocarpin fractions A, B and C found the fraction psophocarpin B to be most insecticidal. On further purification this fraction yielded two lectin fractions and a protease inhibitor fraction. Purified basic lectin was highly insecticidal to C. maculatus larvae with an LC(_50) value of 0.35%. The physiological level of this protein in winged bean seeds is sufficient to account for their resistance to attack by C maculatus. Winged bean trypsin inhibitor was also purified and tested in artificial seeds against C maculatus. However, even at concentrations in excess of twice the physiological concentration it had no deleterious effects upon development. Winged bean protein fractions, incorporated in artificial diets, proved toxic to the Lepidopteran pests Heliothis virescens and Spodoptera littoralis in bio-assays, but it appeared that the basic lectin was not responsible for toxicity towards these insects. Attempts to clone the gene encoding the winged bean basic lectin were made by constructing cDNA and genomic libraries, and heterologous lectin genes from soybean and Phaseolus were investigated as possible probes for the basic lectin gene. Purification of the basic lectin B3 and sequencing of 44% of its primary protein structure, along with comparisons with other legume lectin sequences allowed the synthesis of oligonucleotide primers for use in polymerase chain reaction experiments. However, all the PGR products obtained were shown to be the result of non-specific amplification. Further work needed to obtain the basic lectin gene is discussed.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Thesis Date:1991
Copyright:Copyright of this thesis is held by the author
Deposited On:18 Dec 2012 12:04

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