Makhdoom, Rahat (1998) CLONING AND SEQUENCING OF THE DELTA ENDOTOXIN GENE FROM LOCALLY ISOLATED BACILLUS THURINGIENSIS TOXIC AGAINST SPOTTED BOLLWORM. PhD thesis, University of the Punjab, Lahore.
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.
|Item Type:||Thesis (PhD)|
|Uncontrolled Keywords:||bacillus thuringiensis, cotton spotted bollworm (earias sp.), gene, clone, proteins, bt. toxins, dna, dna sequencing|
|Subjects:||Biological & Medical Sciences (c) > Biological Sciences(c1) > Biology (c13)|
|Deposited By:||Mr. Muhammad Asif|
|Deposited On:||08 Aug 2006|
|Last Modified:||04 Oct 2007 21:01|
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