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Reporter
Genes -
Some genes being transferred produce enzymes whose activities can be easily detected or used as a basis of selection for the transformed cells, e.g., genes for herbicide resistance.
However, most genes need to be tagged with another gene, called reporter gene, whose expression is easily detected either through highly sensitive enzyme assays (scorable reporter genes) or through expression of resistance to a toxin (selectable reporter genes).
Some commonly used easily detectable enzyme producing genes are, nos (nopaline synthase, from Agrobacterium), lux (luciferase from bacteria or firefly), cat (chloramphenicol acetyltransferase from bacteria), and gus (β-glucuronidase from bacteria), etc.
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Activities of the enzymes produced by scorable reporter genes are determined either in situ, i.e., in the transformed tissues, or by in vitro assays using plant tissue extracts. In addition, immunological methods may also be used to detect the protein products of marker genes either in situ, in plant extracts or by western blotting.
The essential features of an ideal reporter gene are:
(1) lack of endogenous activity in plant cells of the concerned enzyme,
(2) an efficient and easy detection, and
(3) a relatively rapid degradation of the enzyme.
Each reporter gene has some advantages and some disadvantages and none of them are ideally suited for all plant species. For example, there is little or no endogenous luciferase activity in plant cells, the enzyme serves as a visual marker, the assay is quite sensitive, but the enzyme is highly stable.
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Similarly, the Gus enzyme serves as visual marker and the assay is highly sensitive so that expression in individual cells can be monitored, but the enzymes is highly stable and many plant species show variable endogenous Gus activity.
Reporter genes, more particularly, scorable reporter genes have been extensively used to assay the function of promoters and other regulatory sequences, and also to demonstrate the transformation of plant cells.
A key step in gene transfer is the selection of few transformed cells from among the nontransformed ones. This is usually achieved by the use of selectable markers, which are genes that confer resistance to various compounds toxic to normal plant cells. The best selection agents are those that either inhibit growth or slowly kill the nontransformed cells so that the dying cells do not overwhelm the transformed ones.
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The commonly used selectable marker genes include those conferring resistance to the antibiotics kanamycin (nptII, encoding neomycin phosphotransferase) and hygromycin (hptIV, encoding hygromycin phosphotransferase, isolated from E. coli); and broad range herbicides glyphosate (modified versions of the enzyme EPSPS, 5-enolpyruvate shikimate-3-phosphate synthase, isolated from E. coli or Salmonella typhimurium), phosphinothricin (bar, isolated from Streptomyces hygroscopicus, codes for phophinothricin acetyltransferase), etc.
Each selectable marker presents some favourable and some unfavourable features. Therefore, the choice of a marker should be based on the plant species and other considerations in the study. The marker gene of choice is fused with a suitable plant promoter and is placed in a suitable vector along with the gene being transferred.
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The bar gene confers resistance to the herbicide phosphinothricin, which inhibits the enzymes glutamine synthetase and, thereby, leads to an accumulation of ammonia to lethal levels. The bar product, phosphinothricin acetyl transferase, catalyzes acetylation of phosphinothricin leading thereby to its inactivation.
Transformed cells expressing bar gene almost normally grow on lethal concentrations of phosphinothricin, while nontransformed plant cells stop to grow, gradually become necrotic and die within 10-21 days. Phosphinothricin is used at 1- 10 mg/l for selection of transformed cells.
The nptII gene from transposon Tn5 confers resistance to the aminoglycoside antibiotics kanamycin, neomycin and G418. The nptII gene product, neomycin phosphotransferase, inactivates these antibiotics through their phosphorylation. Kanamycin resistance due to nptII has been widely used as a selectable marker in many dicot plant species.
But this gene is of limited value in monocots due to their tolerance to relatively high levels of kanamycin. The selection of
transformed cells is done on a medium containing 50-350 mg/l of kanamycin sulphate in case of tobacco.
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