Get the answer of: How terminator gene works ?
The key to the efficacy of terminator technology is the ability to make abundant toxin confined to seeds and kill the embryo in last stages of development. To accomplish this, the strategy to select the promoter from gene normally activated late in seed development. This promoter is fused with coding sequence for a protein that will kill an embryo going through the last stages of development.
The promoter selected for this work is from a cotton LEA (Late Embryogenesis Abundant) gene. Its protein will not be produced until the seed is full-sized, accumulated its storage oil and protein and is drying down in preparation for the dormant period between leaving parent and germination in the soil.
The toxin used in this process, is a Ribosome Inhibitor Protein (RIP) from the plant Saponaria officinalis. This protein at mearge quantities can inhibit synthesis of all proteins. However, the RIP is non-toxic to organism other than plants.
The terminator patent displays a novel strategy for keeping the toxin gene from being active until farmers sow the seeds. The trick is accomplished by inserting a piece of DNA (buffer DNA) in between the seed. Specific promoter and the toxin coding sequence that blocks it from production of protein. At either end of the blocking DNA are insertions of special DNA pieces that can be recognised by an enzyme known as recombinase.
These DNA pieces are cut precisely by recombinase and the cut ends of the DNA fuse together, consequently and blocking DNA is removed. Several sequence-specific recombinases are cre-lox system from bacteriophage and flp-frt system from the yeast. In the cre-lox system, the phage encoded recombinase, CRE recognises the LOX DNA sequence and finally get excised.
As a result, the seed specific promoter comes right next to the toxin coding sequence and is able to produce toxin. But this does not happen immediately. Because production of toxin takes place only at the end of the next round of seed development when the LEA promoter is active.
After the recombinase enzyme does its work, the plant grows normally passes all the growth stages i.e. flower formation, pollination and most of seed development. Then, the seeds die to the production of toxin protein.
When all this accomplished, there remains question: How to grow several generation of the genetically engineered plants and how company can save the seeds for several generations so that its seeds can be collected at every season and sell to farmer.
According to companies key terminator strategy, it deliberately preventing recombinase from acting until just before farmers plant their seeds. To achieve this, recombinase coding sequence is put next to a promoter that is always active in all the cells, at all times promoter of recombinase is always repressed. Regulation of repressed or depressed gene can be accomplished by a chemical treatment like tetracycline.
A gene for the production of repressor protein all the time is introduced into the plant. Once repressor protein is produced, binds specifically the promoter of recombinase and blocks the production of recombinase and consequently toxin gene would also block. Therefore no toxin would be made, even during seed development, where the LEA promoter normally would be active (Fig. 20.9).
In order to activate the toxin gene, seeds are treated with tetracycline before they are sold to farmers. The tetracycline acts as inducer, would interact with repressor protein and facilitate the expression of recombinase gene.
Once recombinase enzyme is made, which recognises the DNA sequence (excising sequence) inserted on either side of the spacer or blocking sequence cut and completely removes them along blocking sequence which then flank the toxin gene is now comes next to the LEA promoter and would now be capable of making the toxin, but would not actually do so because the LEA promoter activity stage has already passed when tetracycline treatment were given.
Therefore, only the next generation would be killed when transgenic plant is armed with terminator gene. Three engineered gene components are introduced into plant DNA.
The overall process is summarized in the following steps:
(1) A toxin or killer gene (RIP) controlled by seed-specific promoter (LEA).
(2) A repressor gene controlled by constitutive promoter.
(3) A recombinase gene controlled by a promoter—repressed by repressor protein, which can be depressed by tetracycline.