Pakistan Research Repository

CLONING AND EXPRESSION OF THE DELTA-ENDOTOXIN GENE FROM LOCALLY ISOLATED BACILLUS THURINGENSIS.

Khan, Esther (1994) CLONING AND EXPRESSION OF THE DELTA-ENDOTOXIN GENE FROM LOCALLY ISOLATED BACILLUS THURINGENSIS. PhD thesis, University of the Punjab, Lahore.

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Abstract

Research presented in this thesis is aimed to investigate the potential of Bt toxins for the control of rice stem borers (Scirpophaga incertulus) and leaf folders (Cnaphalocrocis medinalis) which are potent pests of rice crop in various parts of the world including Pakistan, India, far east USA. The reported investigations include a search in different ecological regions of Pakistan for different isolates of Bacillus thuringiensis, study diversity in their gene content and efficacy against rice insect pests. 157 samples were collected to yield 314 B. thuringiensis isolates. 150 isolates were further characterized for gene content, on the basis of SDS-PAGE analysis, DNA-DNA hybridization studies and ELISAs. The most frequently occurring crystal protein combinations were Cry IV, CryIA(a)/CryA(c) and CryIA(a)/CryIA(a)/CryIIA. Soil samples were found to contain the highest percent of B. thruingiensis (53%). One isolate was characterized to contain a novel CryIA related protein. Toxicity of epresentative isolates were tested against the yellow stem borer and leaf folder in laboratory biotoxicity assays developed during the course of this study. Kurstaki HD-1 was used as a reference standard. Three isolates JR6.3 PR17.4 and KM9.5 were most effective against the yellow stem borer giving LC50 values in the renge of 10ug 73ug/ml respectively. The toxicity level of these isolates was 20 fold higher than that of B. thuringiensis subsp. Kurstaki HD-1. Bioassays against the leaf folder identified the novel isolate, CEMB-1 to be highly toxic (LC50 value of 0.48ug/ml). Isolates D1.1, JR6.3 and PR17.4 showed the same level of toxicity as the reference standard giving LC50 values in the range of 2.5 3.5uh/ml. One local isolate D4.10, showing activity against the yellow stem borer, was chosen for further studies. Two proteins with 130kd and 72Kd were purified by electroelution from SDS polyacrylamide gels. Amino terminal sequence analysis was used to read 16 terminal amino acids of the 72Kd protein. Based on the sequence and codon usage in other toxin genes, a 33mer degenerate Oilgonucleotide probe was synthesized and used to isolate a gene encoding the 72 Kda crystal protein from a large plasmid (70-100 Mds). The cloned gene was characterized by extensive restriction mapping and partial sequencing, to show 100% homology with CryIVD gene. Antibodies were generated against the 130Kd and 72Kd proteins of the D4.10 isolate. They were purified by ammonium sulphate precipitation and affinity purification from nitrocellulose membrane. Western blot analysis was done to check the titer of the generated antibodies. CryIVD gene under its own promoter did not produce detectable levels of the 72 KDa protein in Escherichia coli cells. The CryIVD gene was then subcloned in PmK4 shuttle vector and pDH32 integration vector for expression in B. subtilis.

Item Type:Thesis (PhD)
Uncontrolled Keywords:rice stem borers (scirpophaga incertulus), bt toxicity, cloning, delta-endotoxin gene, bacillus thuringensis, dna, hybridization
Subjects:Biological & Medical Sciences (c)
ID Code:414
Deposited By:Mr. Muhammad Asif
Deposited On:23 Sep 2006
Last Modified:04 Oct 2007 21:00

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