ABSTRACT
Gene manipulation allows the isolation of discrete pieces of a genome from its host organism, and its propagation in the same or different host, a technique known as cloning. This in turn enables the DNA segment to be sequenced [1]. A typical cloning experiment requires the isolation of the DNA of interest (sometimes called foreign, passenger, or target DNA), a cloning vector, restriction endonucleases, DNA ligase, and a prokaryotic or eukaryotic cell to serve as the biological host. Once the vector and foreign DNA have been isolated, they are treated with the same restriction endonuclease to produce site-specic scission in the DNA. Because both vector and foreign DNA have sticky ends when they are generated by the same restriction enzyme, they can be ligated, thus producing a recombinant molecule. This recombinant molecule can be physically inserted into the appropriate host by transformation [2]. Once a gene has been isolated and cloned (multiplied in a bacterial vector), it must undergo several modications to create a construct that can then be inserted into a plant cell, as described in the following:
1. A promoter sequence must be added for the gene to be expressed (i.e., translated into a protein product). The promoter is the on/off switch that controls how much, when, and in what plant tissues the gene will be expressed. To date, most promoters in transgenic crop varieties have been “constitutive,” that is, triggering gene expression throughout the life cycle of the plant and in most of the tissues. The most commonly used constitutive promoter is CaMV35S, which was isolated from the cauliower mosaic virus. This promoter generally results in a high degree of expression in plants. Other promoters are more specic and respond to cues in the plant’s internal or external environment. An example of this is the lightinducible promoter from the cab gene, encoding the major chlorophyll a/b binding protein. On the other hand, the cloned gene is sometimes modied to achieve a large expression in a plant. For example, the genes encoding toxins from the bacteria Bacillus thuringiensis, which confers insect resistance in plants, have a higher percentage of A-T nucleotide pairs compared with plants in which coding regions show a higher degree of G-C nucleotide pairs. In a clever modication, researchers substituted A-T nucleotides with G-C nucleotides in the Bt gene without inducing a signicant change in the protein amino acid sequence. The result was an enhanced production of the gene product in plant cells.
