«IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 7, Issue 5 (Sep. – Oct. 2013), PP 87-95 ...»
IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)
e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 7, Issue 5 (Sep. – Oct. 2013), PP 87-95
Cloning and Expression of a cry III Gene Isolated from the Local
Habitat into a Modified Strain of Bacillus thuringiensis
Kausar Malik*, Mir Muhammad Ali Talpur
Lahore College for Women University Lahore.
Department of Microbiology, Shah Abdul Latif University, Khairpur Mir’s.
Abstract: The cryIII gene, isolated from a locally isolated Bacillus thuringiensis strain,CAMB #30382 (isolated from grain dust of Shakargarh,Punjab,Pakistan), highly effective against red flour beetle Tribolium castaneum, was amplified through Polymerase chain reaction (PCR) by using of specific primers, The cryIII gene was cloned in HindІІІ digested and dephosphorylated expression vector pHB201. For this purpose, the DNA was run through 0.8% agarose gel and was eluted by means of DNA extraction kit.The transformation was done into an acrystalliferous strain of Bacillus thuringiensis, 4D22, by electroporation method. The positive clones were selected onto the petri plates containing LB X-gal/IPTG/Chloramphenicol. The Bacillus thuringiensis containing the gene of interest were also confirmed through PCR, and restriction analysis. Total protein extract from the transformed and non transformed Bt. strains were collected through affinity column chromatography.
Key words: Acrystalliferous strain, Bacillus thuringiensis, Biotoxicity, Expression I. Introduction To increase the efficiency and persistence of Bt. toxins for field use, crystal protein genes are cloned in variety of the microorganisms. A number of toxin genes effective against lepidopteran, coleopteran and diapteran larvae from different strains of Bt. have been cloned & expressed in E. coli, Bacillus subtilis, Pseudomonas, clavibacter, Baculovirus, and nuclear polyhederosis virus (Haider et. al., 1988). Bacillus thuringiensis (Bt.) being a Gram-positive bacterium is widely used in agriculture as a biological pesticide. It is one of the potential and successful biological alternatives of chemical insecticides. Its biological activity mainly resides in a parasporal protein inclusion body, or crystal (Aronson et al., 1986; Hofte and Whiteley, 1989;
Schenpf et al., 1998). Bt. produces several insecticidal crystalline proteins (ICP/Cry proteins) at the time of sporulation. These Cry proteins include alpha (α), beta (β), gamma (γ) and delta (δ) endotoxins. The β-exotoxins and δ-endotoxins are used for the control of pests and vectors of disease. The crystalline delta endotoxins are predominantly synthesized as long, inactive protoxins that are activated by proteolysis in the insect gut. The examples include Cry 1, Cry4A, Cry4B, having molecular weights of 130 to 140 kDa are processed to active 65 to 70 kDa toxins (Gill et.al., 1992,; Hofte and whitely; 1989), while Cry2A, Cry3A, Cry10A and Cry11A are naturally truncated toxins with molecular weights ranging from 65-80 kDa. Sequence analyses of many genes have suggested that significant changes in the activity spectrum can be attributed to comparatively small changes in amino acid sequences (Rahat 1998).
The δ-endotoxins are mostly used in agriculture by organic and other growers to control agronomically important pests (Dulmage, 1981; Guillet et al., 1990; Mulla, 1990).
Most strains are active against lepidopteral larvae, but some are toxic against dipteral (Federici et al., 1990) or Coleopteral species (Kreig et al., 1983).
Protection of storage grains and other food products from different pests has become a serious issue (Haque et al., 2005) Most recently, strains have been identified selectively toxic to different species from several invertebrate phyla arthropods (Mainly insects), nematodes, flatworms and protozoa (Feitelson et al., 1992), and hymenoptran (Garcia-Robles et al., 2001).
Biological control methods being practiced successfully includes the use of pheromones for trapping or disruption of mating behavior, insect growth regulators that interfere with larval development, parasitoids, fungi, viruses and bacteria, which debilitate or cause death in the infected insect (Way and van Emden, 2000).
Chemical insecticides have been valuable in the control of insect pests hence stabilizing even increasing agricultural yields. Besides being uneconomic now-a-days, their indiscriminate and large scale use has given birth to serious problems of pest resistance in plant protection, industry public health and ecology. The exclusive use of non-selective products has disturbed the ecosystem by destroying non-target organisms and killing friendly insects, especially pollinators, raising the need to develop biological control agents.
Bacillus thuringiensis based biopesticides are effective in variety of situations. However in terms of reliability, spectrum of activity, speed of action & cost effectiveness, their performance is considered to be poorer than www.iosrjournals.org 87 | Page Cloning And Expression Of A Cryiii Gene Isolated From The Local Habitat Into A Modified Strain Of chemicals. Bacillus thuringiensis is rod-shaped, aerobic, gram positive spore forming, ubiquitous, soil dwelling bacteria which is an insect pathogen and produces highly specific crystal protein called delta endotoxins. These toxins have a great potential for various agricultural & forestry pests as well as human disease vectors. The use of Bt. as biological control agent has several advantages over chemical pesticides. It has a narrow and highly specific host range & it is harmless to non-target insects. Bt. proteins are not harmful to vertebrates and beneficial insects.
There are no receptors in the gut of mammals, including human beings, for delta endotoxins and proteins from Bt. tested so far, are degraded within 20 seconds due to action of digestive enzymes in mammals (Krattiger, 1997).
Although this organism has large variety of field applications, Bt. products are not as potent as chemical products. Because they are rapidly inactivated by exposure to sunlight or other environmental factors, Bt. products act slowly, have narrow spectrum & are not stable in other environment factors (Pustazai, 1991, Tamez Guerra et. al., 2000). It is the potency and spectrum of activity of high yields toxins that is required for economic viability and acceptability rather than their low ecotoxicity & other ecological advantages. The production of antibiotics is usually improved by strain selection and by varying cultural conditions. It is likely that the production of the toxin could similarly be improved (Smith, 1982). A recombinant Bacillus thuringienesis strain expressing additional cryI gene under the control of cry3A gene expression system yields more crystal protein than wild type strain (Sanchis, 1999). It may be possible to increase the total amount of toxin produced in a Bacillus thuringiensis strain (Komono, 2000). Moreover the workers, Sanchis et al., (1999) have reported the development of broad spectrum non viable, asprogenic recombinant strain of Bacillus thuringiensis with greater potency.
To increase the efficiency and persistence of Bt. toxins for field use, crystal protein genes are cloned in variety of the microorganisms. A number of toxin genes effective against lepidopteran, coleopteran and diapteran larvae from different strains of Bt. have been cloned & expressed in E. coli, Bacillus subtilis, Pseudomonas, clavibacter, Baculovirus, and nuclear polyhederosis virus (Haider et. al., 1988).
Amplification techniques can be employed to express foreign genes in a choice host there by offering a technically feasible and commercially viable strain (Jorgensen et. al., 2000). Bhattacharya (2000) isolated the hyper toxic mutant strains of Bacillus thuringiensis var. israeliensis by mutagensis of the parent strain. Coyle et al., 2000, conducted the field experiments to determine the efficacy of two Bacillus thuringiensis Berliner formulations, Novoder and Raven, for controlling cotton wood leaf beetle chrysomella seripta. They reported that both formulations reduced cotton wood leaf beetle defoliation damage after a single application giving high efficacy for control of cotton wood leaf beetle under the conditions and concentrations evaluated.
Nariman (2007), in his study detected and isolated different cry genes toxic towards the larvae of members of the orders lepidopteran, dipteran and coleopteran in some local B. thuringiensis isolates based on PCR method with specific primers, as well as predicted their insecticidal activity.
Managing the insect pests that affect economically important plants and vectors of human diseases is a major concern world wide in food production, storage of various grains and human health.
II. Material and Methods Bacterial Strains and Plasmids An acrystilliferous strain (4D22) of Bacillus thuringiensis used in the present study, was very kindly supplied by the Culture Collection Laboratory, Centre of Excellence in Molecular Biology, Punjab University, Lahore. These samples were collected from different areas of Pakistan. Most of the strains selected for the study were isolated from wheat grain, wheat dust, pulse dust, soil and dead insects.
Bt. plasmid vector p-HB201 was obtained from Promega.
Enzymes and Reagents Restriction enzymes and DNA markers were obtained from New England Biolabs (Beverly, Massachusetts), Bethesda Research laboratories (BRL), Boehringer Mannheim and CAMB Enzyme production lab, HMW and pre-stained protein markers from GIBCO and Bio-Rad. Calf intestine Alkaline Phosphatase was from Boehringer Mannheim and T4 DNA ligase was obtained from New England Biolabs. Qia quick gel extraction Kit was obtained from QIAGEN Inc. Taq DNA polymerase was from CAMB enzyme production laboratory.
Gram Staining To differentiate between Gram +ve and Gram -ve bacteria, staining method of Christian Gram as described by Bortholomew (1962) was used.Thin heat fixed smear from 24 hrs grown Bt. culture was prepared.
Slide was flooded with crystal violet or methylene blue for 1min, Stain was poured off and washed with mordant (iodine solution) for 30 seconds. Slides were rinsed with slow running water and then decolorized with www.iosrjournals.org 88 | Page Cloning And Expression Of A Cryiii Gene Isolated From The Local Habitat Into A Modified Strain Of alcohol at a slanting position.The slide was counterstained for 1min with safranin solution and then were washed with tap water and observed under microscope with oil immersion at 100X.Gram positive bacteria gave a blue or purple color, while Gram negative gave a red or pink color.
Malachite Green and Fuschin Staining Bt cultures grown for 72 hrs. were stained for visualizing spores and crystals with malachite green and fuschin stain by a modified spore- staining procedure of Shaeffer and Fulton (1993) as described by E. Khan (1994, Ph.D Thesis). Smears of sporulated cultures containing spores and crystals were air-dried and heat fixed.
Slides were placed above a boiling water bath and flooded with 5% aqueous malachite green. As soon as they started to dry, more were added and after 15 min., they were washed away and blotted off. The slides were seen under microscope at 100X.The spores appeared green while the vegetative cells and crystals deep pink.
SDS Polyacrylamide Gel Electrophoresis SDS-PAGE was performed in a Bio-Rad Mini protein-ii gel apparatus by the method of Laemli (1970).
The acryl amide concentration for the resolving gel was 10% (W/V) and that for stacking gel was 4% (W/V).
Purified and crude toxins were run on the gel and stained with commasie stain (0.25% (w/v) commasie brilliant blue R-250, 45.5% (v/v) methanol, 9%(v/v) glacial acetic acid) for 30-60 min. at 65 0C or overnight at room temperature (sambrook et al.1989). The gel was destained in 25% (w/v) ethanol and 7% glacial acetic acid with several changes until the blue background disappeared.
Total cell DNA Purification For total DNA isolation, the procedure described by Kronstad et.,al. (1983), was adopted. An overnight culture from a single colony of bacterial cells was diluted in SPY medium in 1:100 ratio. Cells were grown at 37 0 C with shaking to optical density 0.8 at 600 nm in 2.8-litre flask with shaking at 200 rpm. Cells were harvested by centrifugation at 4 0C. at 7K for 10 min. The cell pellet was washed with solution containing 100 mM NaCl, 10 mM Tris-HCl (pH7.9) and 10 mM EDTA. Lysozyme was added to give a concentration of 0.25 mg/ml.
Mixture was incubated at 37 0C for 20 min. 6.25 ml of a solution containing 100 mM Tris-HCl (pH 7.5), 100 mM NaCl and 2% SDS were added to lyse the cells. Preparation was mixed by gently inverting the tubes several times. Incubation was given at 60 0C, till the preparation was clear. Extraction was done with PhenolChloroform mixture. Aqueous layer was removed with the wide bore pippette. 2.5 volume of chilled Ehanol was added and DNA was spooled out by means of a glass rod. DNA was rinsed with 70% Ethanol, air dried and resuspended in 500 ul to 1ml of T.E (10mM Tris-HCL pH 7.9, 1mM E.D.T.A). DNA was dialyzed extensively against cold TE buffer at 4C and concentration measured by taking O.D.260.
Mini Preparation of Plasmid DNA by Alkali lysis method Plamid DNA was isolated by alkaline lysis method (Brinboin and Dolly, 1979). Single bacterial colony was used to inoculate 5 ml of LB medium containing the appropriate antibiotic and grown at 37 0C with vigorous shaking for 12-16 hours (overnight). 1.5 ml of overnight culture was shifted to a micro centrifuge tube and centrifuged at 12000 rpm for 3 minutes. The supernatant was decanted and the pellet was re-suspended in 100 ul of ice cold cell suspension buffer (25M Tris Hcl pH 8.0,10 mM EDTA, 50mM glucose,2 mg/ml lysozyme) and incubated for 5 minutes. Then 200 ul of a freshly prepared cell lysis solution (1% SDS, 0.2 % NaOH) was added. After immediately mixing by inverting the tube several times and 5 minutes incubation of lysate at room temperature, 150 ml of ice-cold 3M potassium acetate solution (pH 4.8) was added and again mixed by inversion followed by 15 minutes incubation on ice. Centrifugation was done at 12000 rpm for 15 minutes and clear supernatant was extracted with an equal volume of Phenol: Chloroform: Isoamylalcohol (25:24:1) mixture. Extracted aqueous phase was carried out at -20 0C for 15 minutes followed by centrifugation at 12000xg for 15 minutes. The pellet was rinsed with ice-cold 70% Ethanol, air dried and re-suspended in 25 ul of nuclease free water.