Use of Marker Genes

Rhizobium and Legume Root Nodulation
Leguminous Plants
The Discovery of Symbiotic Nitrogen Fixation
Rhizobium Classification
Cultural Characteristics
Genetics
Flavonoids Induce Nodualation
Novel Approaches to Extend Root Nodulation
Ecology
Infection
Lectins
Structure of the Nodule
Function of the Nodule
Nitrogenase Hydrogenase Interrelationship
Stem Nodulating Legumes
Factors Affecting Nodulation
Temperature and Light
Hydrogen Ion Concentration
Mineral Nutrition

Use of Marker Genes

	Use of Marker Genes The methods using marker genes are simple, inexpensive and involve observation of colours by the unaided eye. The marker must be highly sensitive, affordable and usable in the field with no background activity either in the bacterium or in the phase in which they are studied such as soil or plant roots. The marker gene selected must have no or very little interference with the physiology or genetics of the host.
The strains constructed with marker genes are indeed genetically engineered organisms (GEOs) and must not possess characteristics that are prone to spread in any environment. In other words, the construct must pass the legislative regulatory measures of GEOs of a country in which they are used. A number of marker genes are available for their potential use in rhizobial competition studies. They include the Gus A gene coding for B-glucuronidase (GUS) hydrolysing a variety of glucuronide substrates to give coloured fluorescent products; the LacZ (YA) gene coding for B-galactosidase hydrolysing a variety of galactoside substrates to give coloured. fluorescent compounds; the phoA gene coding for alkaline phosphatase hydrolysing a variety of phosphate substrates to give coloured fluorescent compounds; the XylE gene coding for Catechol 2,3-dioxygenase capable of converting colourless catechol to 2-hydroxymuconic semialdehyde which is bright yellow in colour;
Vio-operon coding for violacein biosynthetic enzymes capable of producing violacein, a purple pigment from endogenous tryptophan; tfd A gene coding for 2,4-dichlorophenoxyacetate monoxygenase capable of converting phenoxyacetate to phenol which can be measured by gas chromatography or by the development of a red dye on reaction with 4-aminoantipyrene; luxAB, luc genes coding for luciferase enzyme (firefly enzyme) whose activity leads to ,light production on an aldehyde substrate containing oxygen and reducing equivalents.
Among these marker genes, the gusA gene merits consideration because the method involving this gene satisfies the several criteria listed above to qualify as an easy and acceptable tool for rhizobial ecological studies. Gus marker gene can be introduced into Rhizobium through a plasmid or as a piece of DNA (transposon) the latter becoming integrated into the chromosome of the bacterium. The procedure for assay can be outlined as follows: A donor plasmid or transposon element having Gus A gene is mated with a pure culture of desired rhizobial strain → plate on selective media → the marked rhizobial strain grows on selected plates → use the marked rhizobial strain as an inoculant → grow plants → harvest nodulated roots → place washed nodulated roots in a solution of 5-bromo-4chloro-3-indolyl-D-glucuronide (X-glc A).
	The GUS enzyme cleaves X-glcA to release an indoxyl derivative which on dimerization gives rise to a stable indigo precipitate that is blue in colour → count blue nodules. The method is useful for quantitative enumeration of selected rhizobial strains in a mixture of strains in the laboratory or in the field and to track inoculant strains on seeds or in carriers.

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