Transgenic Bt Technology: 3. Expression of Transgenes
Prof. C Kameswara Rao
Foundation for Biotechnology Awareness and Education,
Bangalore, India krao@vsnl.com, www.fbae.org, www.fbaeblog.org

Genes and gene expression:

  Genes determine the characteristics and life processes of organisms.   Each species contains several thousand genes.   A gene, the basic unit of inheritance and diversity, is a segment of DNA containing a specific sequence of nucleotides (the building blocks of DNA).   Most genes occur in one of three combinations of two variant forms (for example, AA, Aa or aa).   The genotype of an organism is the specific and characteristic combination of variants of all genes the organism carries.  

A gene expresses through the synthesis of a protein or an enzyme (most proteins are enzymes), which is the means of functioning of a gene.   Gene expression varies with the nucleotide sequence of the gene, its promotor, and the point of insertion of the gene in the DNA of the transgenic variety, the internal cell environment, as well as several external factors in the environment.

Quantification of gene expression: 
It is necessary to know how a Bt gene is expressing in a transgenic variety, in order to evaluate its effectiveness against the targeted pest.   Comparing the density, morbidity and mortality of pest populations, on the Bt and its isogenic non-Bt variety, is one way of doing this.   But a more direct way is to accurately quantify gene expression in terms of the protein/enzyme it helps to synthesize.   There must be a certain minimum quantity of the Bt toxin in the plant parts, particularly during the more vulnerable phases of the crop, to control the pest.   The quantity Bt protein present in different parts of the plant during the crucial phases of pest damage such as the boll formation in cotton, would give an idea of the effectiveness of the technology in a particular Bt variety.  

Field kits have been developed to quantify Bt protein in transgenic varieties.   The Bt gene construct is introduced into the experimental bacterium Escherichia coli, so that the gene product is more easily purified from the transgenic bacterium, than from a transgenic crop variety.   Antibodies are raised against this purified protein, and these antibodies are used to quantify the Bt toxin in the transgenic variety, through an enzyme-linked immuno-assay method.   This procedure results in a colour reaction whose intensity gives the measure of the quantity of the protein involved.   Quantification of Bt toxin by this procedure is relatively simple and with little instruction and minimal facilities, a semi-skilled worker can conduct the test.   However, the simplicity of the test itself is its Achilles’ heel.   The test is expected to work with a little bit of hand-crushed tissue of the Bt transgenic plant.   Unfortunately, quantification of expression of the Bt gene is sensitive to the following factors:

a)      Kits from different sources vary in their details, such as whether the antibodies used were monoclonal or polyclonal (see the article on Immunology and Immunotechnology, www.fbae.org, for details).   Kits based on polyclonal antibodies are good enough to find out if any Bt toxin is present in the tissue, but are not very exact to quantify the toxin that occurs in microgram quantities.   Though monoclonal antibodies provide for a more accurate quantification, some kits are based on polyclonal antibodies, as the production of monoclonal antibodies is more technically involved and so more expensive.   There have been complaints on the accuracy and consistency of several of these kits, but authentic data are unavailable.   Actually it is necessary that the kits available on the market were assessed for their reliability. 
 
b)      The tissue should be properly homogenized and the protein extracted in a proper solvent, an appropriate buffer.   Crushing a bit of a tissue is not an exact scientific way of extracting even most of, if not all of, the protein in the tissue.

c)      The excised plant part should be used immediately for assay.   The rate of protein degradation is quite rapid in excised and stored tissue. 
    
d)      There would be differences in the toxin content depending upon whether the part used for assay was from a plant in the vegetative or the reproductive phase.   Hence the results can be compared only between similar parts of similar age taken from plants that were in a comparable physiological state of development.

e)      Mature leaves, bolls and seeds are more fibrous and harder, and contain several chemical compounds such as resins, oils, phenolics, etc., all of which may interfere with the extraction of all the protein in the tissue.      

Not observing these precautions would result in incomparable, unreliable and misleading data.