I= CLONING AND EXPRESSION OF BT δ-ENDOTOXIN GENE, CONTAINING LARVICIDAL ACTIVITY AGAINST PECTINOPHORA GOSSYPIELLA (SAUNDER), IN E. COLI
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Title of Thesis
CLONING AND EXPRESSION OF BT δ-ENDOTOXIN GENE, CONTAINING LARVICIDAL ACTIVITY AGAINST PECTINOPHORA GOSSYPIELLA (SAUNDER), IN E. COLI

Author(s)
Shahid Karim
Institute/University/Department Details
Centre of Excellence in Molecular Biology/ University of the Punjab
Session
1998
Subject
Molecular Biology
Number of Pages
270
Keywords (Extracted from title, table of contents and abstract of thesis)
cloning, bt δ-endotoxin gene, pectinophora gossypiella, larvicidal activity, saunder, e. coli, bacillus thuringiensis, pesticidal proteins

Abstract
Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. A study have conducted to obtain a quantitative comparison of locally isolated B. thuringiensis toxins specificity with a focus on insect pink bollworm (Pectinophora gossypiella) and cotton bollworm (Helicoverpa zea) were also examined. P. gossypiella was susceptible to Cry1 Aa, Cry1 Ab, Cry1 Ac, Cry2A toxins. To H. zea, Cry1Ac and Cry1Ab were more potent than Cry1Aa and Cry2A. Cry1 B, Cry1 C, Cry1 D, Cry1 E, Cry1 F, Cry1 G, Cry1 Hand Cry2B were not potent against both pests. Binding assays were performed with 1251-labeled toxins (Cry1 Aa, Cry1 Ab, Cry1 Ac and Cry2A) and brush border membrane vesicles (BBMVs) prepared from H. zea and P. gossypiella midguts. Heterologous competition binding assays were performed to investigate the binding site cross reactivity. The results showed that Cry1Aa and Cry1Ab recognize the same binding site which is different from Cry2A. Ligand blot assay showed that Cry1 Ac toxin binds to 120 KDa BBMV protein in R. gossypiella and Cry1Ab binds to a major 210 KDa protein. our data suggest that development of transgenic cotton plant with toxin combinations which recognize different binding sites may be useful in implementing deployment strategies that decrease the pest adaptation to transgenic Bt cotton.

Biological activity of different Bacillus thuringiensis δ-endotoxins, Cry1 Aa, Cry1 Ab, Cry1 Ac and Cry2A was also determined by using force feeding bioassay method to 4th instar larvae of H. zea. Helicoverpa zea was susceptible to Bt toxins in order of Cry1Ac>Cry1Ab>Cry1Aa>Cry2A with 63.60, 89.04, 159.65 and 375.78 concentration 16.7ng/ml, lag time 4 min, at higher concentration, 333.3 ng/ml decay was -2.44pA/min, lag time 1 min. The inhibition of Isc (-1.10 µA/min, lag time 25) at lower concentration (33.3ng/ml) of Cry2A and higher concentration (500ng/ml), decay (-2.38µA/min, lag time 5 min), showing a correlation between toxin concentration and inhibitory response. The lag time decreased with increasing concentration of toxin applied which is an additional evidence of dose response beside direct correlation of toxicity assays and Isc.

To understand the mechanism of action of these pesticidal proteins to the agronomically important insect pests is of immense commercial importance. In this study, two most potent toxins, Cry1 Ab and Cry1 Ac were compared, which show 98% amino acid identity in domain I and domain II but differ significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's (368RR369) of Cry1Ab and Cry1Ac toxins were replaced with Ala (368RR369 , 368AA369) Glu (368EE369), Phe(368FF369), His(368HH369), and Lys(368KK369), Effect of these mutants on structural stability, larvicidal potency, receptor binding and ionic permeability towards two important cotton pests, cotton pink bollworm (Pectinophora gossypiella) and cotton bollworm (Helicoverpa zea) were analysed. All the seven mutants of Cry1Ab excluding only 368AR369 which failed to produce stable protoxin, whereas for Cry1 Ac, all the seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similar to their corresponding wild-type on incubation with trypsin and gut juice of both insect larvae. The Cry1Ab mutants, 368AR369, 368AA369, 368FF369,and 368HH369 lost the toxicity, 368EE369 reduced the toxicity, whereas a more conserved change 368KK369 retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1 Ac, 368AA369 and 368FF369 abolished their toxicity. Double mutant 368KK369retained its toxicity against P. gossypiella whereas single mutants 368AR369, 368RA369 and 368HH369 retained only reduced toxicity. All the mutants of Cry1 Ab lost their toxicity against H. zea except 368KK3699. Cry1 Ac single mutants, 368AR369 and 368RA369, reduction in the toxicity was observed. A double mutant of Cry1Ac, 368KK369, also retain a reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence that the insecticidal property, inhibition of Isc across the transepithelial membrane of the insect midgut.

Download Full Thesis
3991.99 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
240.86 KB
2 1 Introduction 17
1393.11 KB
  1.1 Literature Review 23
  1.2 Experimental Procedures 55
3 2 Search And Cloning Of Cry 1a Gene 82
324.68 KB
  2.1 Introduction 82
  2.2 Results 84
  2.3 Discussion 96
4 3 Binding To Insect Midgut Rceptors 100
676.96 KB
  3.1 Introduction 100
  3.2 Results 104
  3.3 Discussion 120
5 4 Ion Channel Activity Of Bt 127
232.81 KB
  4.1 Introduction 127
  4.2 Results 130
  4.3 Discussion 135
6 5 Studies On The Binding Sites 140
776 KB
  5.1 Introduction 144
  5.2 Results 144
  5.3 Discussion
7 6 General Discussion 168
619.74 KB
  6.1 List Of References 173