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EXPRESSION OF PROTEINASE INHIBITOR GENE IN COTTON

Majeed, Asifa (2005) EXPRESSION OF PROTEINASE INHIBITOR GENE IN COTTON. PhD thesis, University of the Punjab, Lahore.

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Abstract

The work presented in this thesis carries the objective to find more new ways to develop sustainable resistance in plants especially against those insects resistant to Bt genes. The present study was carried out to find what role potato proteinase inhibitor-II could play in the protection of cotton against Helicoverpa armigera. Potato proteinase inhibitor-II expression was first studied in tobacco against Helicoverpa armigera through the particle bombardment method. Pin2 had demonstrated enhanced resistance to Helicoverpa armigera both in feeding trial and artificial diet. Significant insect mortality up to 70% was achieved during 7-days assay. Expression of Histidine tagged 18kDa Pin2 protein was obtained in E. coli under T7 promoter. Pin2 protein exhibited significant weight reduction and larval mortality at concentrations IOO-500ng/mg mixed in artificial diet. The larval mortality was up to 60% at highest concentration 500ng/mg of diet. On the basis of bioactivities, pin2 gene was cloned under CaMV35S promoter and two plant expression vectors pAR2 containing 35S+pin2 gene and pAR3 comprised pin2+ClJIIAc were constructed for Agrobacterium transformation. Cotton CIM-446 was selected for transformation on the basis of favorable regeneration variety. Particle bombardment and Agrobacterium method in combination was applied using mature embryos and seedling tissues. The transformation efficiency was 2% with mature embryos and 7.5% with seedling transformation after two months selection. Molecular analyses were performed to confirm the presence of pin2 & pin2+crylAc genes. Western analysis confirmed the expression of transformed genes. Pin2 protein accumulation level in cotton was identified as 0.05%-1 % of total soluble protein. The biotoxicity assay of wounded and non-wounded leaves indicated pin2 a potential insect control agent. There was significant difference between the H. armigera larvae applied to transgenic and non-transgenic leaves in their growth and development. The plants expressing pin2 gene were normal in phenotype. The antifungal behavior of Pin2 was examined against Fusarium and Rhizoctonia to ascertain the nature and expression of this protein and its role in protection against pathogens. Pin2 was found highly effective against Rhizoctonia and growth inhibition was much pronounced with increase of pin2 protein in comparison to Fusarium in vitro assay.

Item Type:Thesis (PhD)
Uncontrolled Keywords:bt genes, cotton, helicoverpa armigera, potato proteinase inhibitor-ii, camv35s promoter, cotton cim-446, toxicity
Subjects:Biological & Medical Sciences (c) > Biological Sciences(c1) > Biology (c13)
ID Code:459
Deposited By:Mr. Muhammad Asif
Deposited On:11 Aug 2006
Last Modified:04 Oct 2007 21:01

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