I= CLONING AND SEQUENCING OF THE DELTA ENDOTOXIN GENE FROM LOCALLY ISOLATED BACILLUS THURINGIENSIS TOXIC AGAINST SPOTTED BOLLWORM
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Title of Thesis
CLONING AND SEQUENCING OF THE DELTA ENDOTOXIN GENE FROM LOCALLY ISOLATED BACILLUS THURINGIENSIS TOXIC AGAINST SPOTTED BOLLWORM

Author(s)
Rahat Makhdoom
Institute/University/Department Details
University of the Punjab/ National Centre of Excellence in Molecular Biology
Session
1998
Subject
Molecular Biology
Number of Pages
160
Keywords (Extracted from title, table of contents and abstract of thesis)
bacillus thuringiensis, cotton spotted bollworm (earias sp.), gene, clone, proteins, bt. toxins, dna, dna sequencing

Abstract
The study presented in this thesis is an attempt to explore the potential and diversity of Bacillus thuringiensis from the local environment for the control of cotton spotted bollworm (Earias sp.), a major pest of cotton. Two hundred and ninety eight samples of soil, grain dust, wild animal dung, bird dropping, decaying leaves and dead insect were collected from different ecological environments of Pakistan yielding 438 B. thuringiensis isolates that produce parasporal crystalline inclusions. In this study the soil samples were found to be the richest source for B. thuringiensis. Further characterization of 350 isolates for cry gene content divulged cry4 genes as the most prevalent followed by cry1A genes. The entomocidal activities of representative isolates were examined against Earias vitella larvae reared on locally developed synthetic diet. On the basis of bioactivities, an isolate JR6.3, separated from a soil sample, was selected as source material for gene cloning studies. This isolate was found highly effective against Earias vitella giving LC50 value 11.22 ng/mg in comparison with 25.12 ng/mg of diet for B. thuringiensis kurstaki HD1, which was used as a reference standard.

The gene cloned from isolate JR6.3 was characterized to be homologous to Lepidoptera active c1yAC gene as judged by Southern hybridization, PCR and restriction pattern analyses. Further, alignment of the complete nucleotide sequence of the coned gene with holotype cry1AC sequence from B.t.k. strain HD-73 showed 10 difference, with one difference resulting in change in amino acid residue from phenylalanine to serine at position 440. Full-length sequencing of a previously cloned and partially sequenced cry gene from a local B. thuringiensis isolate CEMB1, reported to be toxic against cotton spotted bollworm and rice leaf folder, was accomplished and sequence homology analyzed. The nucleotide sequence of cry gene from isolate CEMB1 was found to be 100% homologous with a nematode toxic cry6B gene sequence reported in the gene data bank.

Expressions of Histidine tagged 133kDa Cry1Ac and 44kDa Cry6B protenins were obtained in E. coli under T7 promoter. The crude lysate of E. Coli expressing Cry1Ac protein exhibited 100% larval mortality when fed to E. vitella larvae mixed in the artificial diet. The expressed proteins were purified through Ni+2 affinity column and thereafter subjected to trypsin digestion analysis. After incubation with trypsin 133kDa Cry1Ac protein was converted into 65kDa Cry6B protein was completely hydrolyzed showing its sensitivity towards serine proteases. The protease susceptibility of Cry6B protein was corroborated by computer aided analysis of the amino acid sequence.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
84.85 KB
2 1 Introduction 1
347.19 KB
  1.0 Literature Review 7
  1.1 Bacillus Thuringiensis, The History 7
  1.2 Pathogenicity Of Bacillus Thuringiensis 8
  1.3 Diversity And Classification Of Bt. Crystal Proteins 12
  1.4 The Bt. Toxin Structure 25
  1.5 Mode Of Action Of Bt. Toxins 27
  1.6 Screening For Novel Activity 32
  1.7 Development Of Insect Resistance To Bt. 33
  1.8 Managing Resistance To Bt. 35
  1.9 Role Of Bt. In Integrated Pest Management System 36
3 2 Materials And Methods 39
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  2.1 Bacterial Strains And Plasmids 39
  2.2 Enzymes And Reagents 39
  2.3 Isolation Of B. Thuringiensis 39
  2.4 Toxin Extraction From B. Thuringiensis 40
  2.5 Determination Of Toxin Concentration 41
  2.6 Activation Of Solubilized Protoxins 41
  2.7 Rearing And Maintenance Of Spotted Bolloworm 42
  2.8 Diet Preparation 42
  2.9 Biotoxicity Assays Of Bt. Against Spotted Bollworm Larvae 42
  2.10 SDS-Polyacrylamide GEL Electrophoresis 43
  2.11 Larger Plasmid DNA Purification 43
  2.12 Mini Preparation Of Plasmid DNA By Alkali Lysis Method 44
  2.13 Mini Preparation Of Plasmid DNA By PEG Precipitation 45
  2.14 Restriction Endonuclease Digeston Of DNA 46
  2.15 Purification Of DNA Fragment From Agarose Gel 46
  2.16 Ligation 47
  2.17 Transformation Of E. Coli 47
  2.18 DNA Labelling 48
  2.19 Colony Hybridization 49
  2.20 Southern Blotting 49
  2.21 Oligonucleotide Purification 50
  2.22 Polymerase Chain Reaction 51
  2.23 Production Of Antibodies 53
  2.24 Western Blot Analysis 54
  2.25 DNA Sequencing 55
4 3 Results 61
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  3.1 Isolation And Characterization Of B. Thuringiensis 61
  3.2 Southern Blot Analysis To Characterize The Genes In Local Isolates 68
  3.3 Rearing And Maintenance Of Spotted Bollworm 71
  3.4 Biotoxicity Assays 74
  3.5 Characterization Of B. Thuringiensis Isolate JR6.3 77
  3.6 Cloning Of Delta-Endotoxin Gene In E. Coli 79
  3.7 Cloning Of Full-Length Cry1ac Gene 84
  3.8 Sequencing Of Cry1ac Gene 86
  3.9 Expression Of Cry1ac Gene In E. Coli 94
  3.10 Sequencing Of Cry6b Gene 98
  3.11 Expression Of Cry6b Gene In E. Coli 102
  3.12 Trypsinization Of Cry6B Protein 107
5 4 Discussion 113
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  4.1 Isolation And Characterization Of B. Thuringiensis 113
  4.2 Bioactivity Of Local B. Thuringiensis Isolates 116
  4.3 Cloning, Sequencing And Expression Of Cry1ac Gene 117
  4.4 Studies On Cry Gene From Bt. Isolate CEMB-1 121