Of the classes of organisms, insects have the largest number of described species. Insects negatively affect humans in a variety of ways; they cause massive crop damage and act as vector for both human and animal diseases. Therefore a number of chemical insecticides were developed to control the harmful proliferation of insect population. For example: Use of chlorinated hydrocarbons, DDT etc… These insecticides disturb the functioning of motor neurons and brain neurons of the insects. But eventually the scientists realized that chemical pesticides not had side effects on insect ecosystem but where also effecting the humans. That’s where the use of recombinant DNA technology came. Researchers started using this technology to increase the effectiveness of microbial pesticide.
Bacillus thuringiensis is a Gram positive, rod shaped, soil dwelling bacteria that is normally used as a natural insecticide. A microbial insecticide can be an organism that either produces a toxic substance that kills an insect species or has ability to fatally infect a specific target species of insect. Bacillus thuringiensis comprises a number of different strains and subspecies each of which can produce a different toxin that can kill certain specific insects , for example Bt kurstaki is a toxic to Lepidopteran larvae including moth butterfly, Bt subspecies israelensis kills Diptera such as mosquito, black fly etc.
The insecticidal activity of Bt toxin is contained within a very large structure called parasporal crystal which is synthesized during bacterial sporulation. The parasporal crystal comprises approximately 20-30% of dry weight of the sporal culture and usually consists mainly of protein and small amount of carbohydrates. The crystal is an aggregate of protein that can generally be dissociated by mild alkali treatment into subunit. The subunits can be further dissociated invitro by treatment of β-mercaptoethanol which reduces di-sulphide linkage. The insecticidal toxin is classified into 4 major classes Cry I, Cry II, Cry III and Cry IV. Cry protein are generally toxic to Lepidoptera, Cry II to Lepidoptera and Diptera, Cry III Coleoptera and CryIV Diptera. These proteins are further arranged in classes and sub classes according to the DNA sequence of the toxin gene.
MODE OF ACTION
Parasporal crystals don’t contain active form of the insecticide rather once crystal has been solubilized the protein is released and this inactive form of protein is called as protoxin. When the insect ingests the parasporal crystal the protoxin is activated within its gut by combination of alkaline pH(7.5-8)and specific digestive protease which converts protoxin to an active toxin with a molecular mass of approximately 68 KDa. In its active form toxic protein inserts itself into the membrane of the gut epithelial cells of the insect and creates ion channels through which there will be an excessive loss of cellular ATP. After this ion channels are formed, cellular metabolism stops and the insect stops feeding which leads to dehydration and eventually death. Since the activation of protoxin requires both an alkaline pH and the presence of specific protease it is unlikely the non-targeted species such as humans and farm animals will be affected.
The mode of action of Bt toxin imposes certain constrains on its application. To kill an insect pet Bt must be ingested by the insect, a simple contact of the bacteria or the insecticidal toxin with the surface of the insect has no effect on target organism. Bt is generally applied by spraying so it is usually formulated with insect attractants to increase the probability that the targeted insect will ingest the toxin. But insects that bore into the plants or attack the plant root are less likely to ingest the toxin that has been sprayed. So, different strategies are being employed to cover all types of insects. One such approach is to create transgenic plants that carry the Bt toxin gene.
TOXIN GENE ISOLATION
To develop Bt based insecticide that have higher potencies and broader host ranges. It is necessary as a first step to isolate and characterize the protoxin gene. The first aspect of this problem is to determine whether the toxin genes are located on a plasmid or on chromosomal DNA. To test for plasmid born toxin genes, the source Bt strain can be conjugated with a strain that lack insecticidal activity. If the latter strain acquires the ability to synthesize the insecticidal toxin then it can be concluded that the toxin genes are most likely present on plasmid as transfer of chromosomal DNA during conjugation is very rare.
PROCEDURE FOR ISOLATING PROTOXIN ENCODING GENE
Bt cells are grown in a lab culture and lysed. The cellular DNA is isolated and separated into plasmid and chromosomal DNA fractions by Cesium chloride gradient centrifugation. Since the toxin is encoded by the genes in the plasmid, the plasmids are further centrifuged (Sucrose gradient centrifugation) which separates different plasmids further according to their size. Let’s take an example, in Bt subspecies kurstaki after sucrose gradient centrifugation the plasmid is divide into three fractions that contain the small (2Kb),medium(7.4-14.4Kb) and large (45-71KB) plasmids, the fraction with the smaller plasmid is discarded as these plasmids are too small to encode the protoxin protein equivalent to 113KDa .The medium and the large plasmids fractions are partially digested with restriction enzymes Sau3A1 and then ligated into BamH1 sites of pBR322. These clone banks were transformed to E.coli and screened immunologically by the following procedure
- Colonies are transferred from agar plate to a nitrocellulose membrane.
- The transferred colonies are partially lysed with organic solvents.
- All available sites on the membrane to which primary and secondary antibodies could bind are blocked by treating the membrane with BSA.
- The bovine serum albumin treated membrane is next treated with rabbit antiserum that contains antibodies against insecticidal toxin.
- The membrane is then washed to remove unbound antibodies and then treated with I125 labeled staphylococcus aureus protein A which binds to Fe portion of the bound antibody.
- Spots on the membrane corresponding to colonies that actively synthesizes the insecticidal toxin are visualized by autoradiography.
ENGINEERING OF Bt TOXIN GENE
Once isolation and sequencing of the toxin gene was accomplished the complete amino acid sequence was deduced. Therefore it can be further used for genetic manipulation. Most Bt toxin are synthesized only during the sporulation phase of growth. In other words only a portion of growth cycle of the organism is devoted to parasporal crystal production. Therefore it might be an advantage in terms of increased yield and lower production time to have the toxin gene transcribed and translated during vegetative growth.
During the sporulation of Bt a specific transcription initiation factor (sigma) interacts with the promoter of the that are active only during the sporulation phase of the life cycle. The factor turns on the transcription of the m-RNA’s that are unique to sporulation. Thus to express the Bt insecticidal toxin it is essential to place it under a promoter that is active during all phases of the life cycle.The gene has to be designed so that it can be successfully inserted into the plants.
Selectable marker gene:Hygomycin
The gene can be transferred into the plant by two methods
- Agrobacterium mediated transfer
Use of Bacillus thuringiensis as a natural insecticide can be very effective in insect targeting as well will not have any side effects on nature or humans.Let’s hope that we switch to natural methods than rely on chemical alternatives.