I= EXPRESSION OF PROTEINASE INHIBITOR GENE IN COTTON
Pakistan Research Repository Home
 

Title of Thesis
EXPRESSION OF PROTEINASE INHIBITOR GENE IN COTTON

Author(s)
Asifa Majeed
Institute/University/Department Details
University Of Punjab/ National Centre Of Excellencein Molecular Biology
Session
2005
Subject
Molecular Biology
Number of Pages
186
Keywords (Extracted from title, table of contents and abstract of thesis)
bt genes, cotton, helicoverpa armigera, potato proteinase inhibitor-ii, camv35s promoter, cotton cim-446, toxicity

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.

Download Full Thesis
3151.33 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 1 Introduction 1
126.09 KB
2 2 Review Of Literature 11
525.67 KB
  2.1 Proteinase Inhibitor 11
  2.2 . Structure Of Proteinase Inhibitor Genes 11
  2.3 Proteinase Inhibitors Regulation And Expression 15
  2.4 Mechanism Of Toxicity 30
  2.5 Plant Proteinase Inhibitors And Transformation. 32
  2.6 Bacillus Thuringiensis 38
  2.7 Mechanism Of Action 38
  2.8 B.T In Agronomical Crops 39
  2.9 B. T & Proteinase Inhibitor 43
  2.10 Agrobacterium And Plant Transformation 44
3 3 Materials And Methods 51
335.86 KB
  3.1 Expression Of Potato Proteinase Inhibitor-II (Pin2) Gene In Tobacco 51
  3.2 Nicotiana Tabacum Cv.Xanthi Callus Initiation And Maintenance 51
  3.3 Optimization Of Different Parameters For Nicotialla Tabacum
  3.4 Cv Xanthi Transformation 51
  3.5 Biolistic Transformation Of Nicotialla Tabacum Cv.Xanthi With Prwl Plasmid 51
  3.6 Expression Studies Of Potato Proteinase Inhibito--II Gene Against Helicoverpa Armigera 52
  3.7 Molecular Analysis 53
  3.8 Genomic Dna Isolation 53
  3.9 Polymerase Chain Reaction 53
  3.10 Dot Blot Analysis 54
  3.11 Cloning 54
  3.12 Cloning Of Pin2 Gene Under Camv35s Promoter 54
  3.13 E.Coli DH5α Electroporation Competent Cells 55
  3.14 Mini Scale Dna Isolation 55
  3.15 Restriction Endonuclease Digestion 56
  3.16 Polymerase Chain Reaction 56
  3.17 Transformation Into DH5 α Cells 58
  3.18 Probe Labeling And Estimation 59
  3.18 Colony Hybridization 60
  3.19 Southern Hybridization 61
  3.20 Wizard Mini Preparation Of Plasmid DNA 62
  3.21 Pin2 Gene Sequencing 63
  3.22 Construction Of Plant Expression Vectors 64
  3.23 Sub-Cloning Of Pin2 Into Agrobacteril/Ill Biliary Vector 64
  3.24 . Expression Of Pin2 In E.Coli 65
  3.25 . Protein Puritication 66
  3.26 Transformation 67
  3.27 Agrobacterium tumefaciens Competent Cells Preparation 67
  3.28 Transformation Of pAR2 And pAR3 Into Agrobacterium tumefaciens Cells 67
  3.29 Bacterial Strains Long And Short Term Storage 67
  3.30 Particle And Agrobacterium Mediated Transformation Of Gossypium hirsutum L . 68
  3.31 Mature Embryo Transformation 68
  3.32 Bombardment With Tungsten Particles 68
  3.33 Agrobacterium-Mediated Transformation 69
  3.34 Seedling Transformation 70
  3.35 Molecular Analysis Of Transgenic Plants 70
  3.36 Genomic DNA Isolation 70
  3.37 Polymerase Chain Reaction 72
  3.38 Southern Hybridization 72
  3.39 Dot Blot 73
  3.40 Immunological Assay Of Transgenic Plants 74
  3.41 Isolation Of Proteins From Plant Materials 74
  3.42 Insects Bioassay 75
  3.42 Expression Of Pin2 Against Cotton Fungal Pathogen 76
4 4 Results 77
676.49 KB
  4.1 . Expression Of Potato Proteinase Inhibitor-II (Pin2) Gene In Tobacco 77
  4.2 Optimization Of Different Parameters For Nicotiana Transformation 80
  4.3 Transformation Of Tobacco With Pin2 82
  4.4 Expression Studies Of Potato Proteinase Inhibitor-II Gene Against Helicoverpli Armigera 85
  4.5 Molecular Analysis 87
  4.6 Cloning 89
  4.7 Construction Of Plant Expression Vectors 92
  4.8 Expression Of Pin2 In E.Coli 98
  4.9 Transformation 104
  4.10 Mature Embryos: 108
  4.11 Agrobacterium And Biolistic Transformation Of CIM-446 Using Shoot Tip (Seedling). 109
  4.12 Molecular Analysis 112
  4.13 Cotton Genomic DNA Isolation 114
  4.14 Polymerase Chain Reaction 118
  4.15 Southern Blotting 121
  4.16 Dot Blot Analysis 123
  4.17 Western Analysis 124
  4.18 Insect Bioassay 127
  4.19 . Potato Proteinase Inhibitor-II Expression Against Cotton Fungal Pathogen 130
5 5 Discussion 139
144.85 KB
6 6 References 150
482.37 KB
7 7 Appendices 186
997.63 KB