Gatehouse, Laurence Neil (1995) Novel genes for insect resistance in transgenic plants. Doctoral thesis, Durham University.
From a cDNA library of potato tuber, cDNAs for the potato carboxypeptidase inhibitors were isolated and characterised by DNA sequencing. One full length clone of each type was used to make plant expression constructs, and these constructs used to transform tobacco. An insect bioassay, conducted using the self crossed progeny from the highest expressing transgenic line, revealed that the expression of potato carboxypeptidase inhibitor in tobacco increased the susceptibility of the plants to attack by Heliothm virescens .A cDNA library of whole Manduca sexta larvae was constructed in XZAP II. Oligos were designed to fit the strongly conserved region of insect haemolymph Q-ypsin inhibitor protein sequences and to a region of published protein sequence from Manduca haemolymph trypsin inhibitor A ( MHTl A ) and these were used to PGR a fragment of a MHTI A cDNA. This fragment was used to screen the cDNA library and a number of clones for MHTI A were isolated, along with cDNAs for a previously unknown related protein. These cDNAs were characterised by DNA sequencing. One of the MHTI A cDNAs was used to make plant and E. coli expression constructs and these were sent, for subsequent bioassays of the resultant transgenic plants and of bacterially expressed protein, to Horticulture Research International. While these assays were seriously flawed, there were strong indications from both the plant and the artificial diet bioassays that MHTI expression did enhance insect resistance. A cDNA library of whole Diabrotica undecimpunctata larvae was constructed in XZAP II. Oligos were designed to fit each of the three strongly conserved regions of protein sequence of mammalian and nematode microsomal aminopeptidases. These were used to PGR fragments from both Manduca and Diabrotica cDNA templates. These PGR products were characterised by DNA sequencing and the Diabrotica PGR products used to screen the cDNA library. Two cDNAs were isolated, neither of which were full length, but which were of sufficient length for protein expressed from them to be likely to be functional as an aminopeptidase. E. coli expression constructs were made from each cDNA and bacterial expression was demonstrated. Pilot work on the feasibility of using antibodies as anti-insect proteins was conducted and the antibodies shown to be reasonably resistant to Diabrotica gut proteases. It was also demonstrated that antibodies could be produced that were active at the extreme pHs (3.5- 11) found in insect guts. While many questions have been left unanswered, this project has successfully demonstrated the viability of such novel approaches to the enhancement of insect resistance in plants by genetic engineering.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||24 Oct 2012 15:11|