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“T-DNA & Transposon Tagging”
SYNOPSIS
INTRODUCTION ROLE OF GENE TAGGING TRANSPOSON TAGGING
TRANSPOSON TAGGING OF PLANT GENES DIFFICULTIES IN TRANSPOSON TAGGING
T-DNA TAGGING SUMMARY CONCLUSION REFERENCES
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• Gene tagging strategies are used to isolate those genes that produce a detectable phenotype.
• Gene tagging broadly involve the insertion of a recognizable DNA fragment with a gene.
• T-DNA is the part of ti-plasmid , DNA found in the soil bacterium.
• Transposons are mobile genetic element that can move from one place to another place in a DNA molecule.
• T-DNA and transposons can be used in gene tagging and gene analysis.
“T-DNA & Transposon Tagging”
INTRODUCTION
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• Gene tagging is the method of insertion a recognizable DNA fragment with in a gene which a gene such that the function of the gene is distrupted.
• Gene can be easily recognize by virtue of the inserted fragment.
• The inserted fragment is usually a well characterized transposable element, most of which has been sequenced.
“T-DNA & Transposon Tagging”
ROLE
OF
GENE
TAGGING
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• These DNA inserts are then analyzed to identify and isolate the gene responsible for the mutant phenotype produced by insertional mutagenesis.
• The tag may be based on – T-DNA of Agrobacterium – A transposable element– A retroviral genome
“T-DNA & Transposon Tagging”
ROLE
OF
GENE
TAGGING
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“T-DNA & Transposon Tagging”
ROLE
OF
GENE
TAGGING
Flow chart 1 - A generalized scheme for gene tagging
• Transposon tagging describes isolation of genes using transposable elements as gene tags.
• This strategy is applicable to both plants as well as animals, but we shall confine on discussion to their use in plants.
• A transposable element is a DNA sequence that has the ability to change its location in the genome, i.e., it can transpose from one location to another in the genome.
• Some transposable elements behave like retroviruses and, for this region, they are called retrotransposons.
“T-DNA & Transposon Tagging”
TRANSPOSONE
TAGGING
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“T-DNA & Transposon Tagging”
• When a transposon integrates within a gene, the gene function is lost.
• But when the transposon move out of the gene, the gene function may be partially of fully restored.
• Thus when a Ds element integrates within a gene, the gene function may be partially or fully restored.
• Thus when a Ds element integrates within the gene C1 on chromosome 9 of maize, C1 function is suppressed and colourless kernels are produced.
• Several maize inbred lines having colourless kernels are of this type.
• The Ds may also transpose in the germline; in such a case, the functional C1 gene will be transmitted to the next generation.
TRANSPOSONE
TAGGING
OF
PLANT
GENES
• It is necessary to establish that the mutational event is due to transposon insertion; in this respect, the instability of mutant phenotype may serve as a useful marker.
• Species like maize and snapdragons carry several different transposon, and each transposon, and may be parent in more than one copy per genome.
• A major limitation of the method is the low frequency of transposition.
• In addition, most species lack active transposons.
“T-DNA & Transposon Tagging”
DIFFICULTIES
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• Agrobacterium T-DNA integrates into plant genome at random sites.
• Agrobacterium mediated transformation can be used to produce a large number of independent transformants.
• Insertion of T-DNA within a gene would generates a mutant phenotype.
• The transformants are screened for mutant phenotype, and the genomic DNAs of the selected mutants can then be used for isolation of the concerned gene by using the T-DNA sequence either as a probe or as primers.
“T-DNA & Transposon Tagging”
T-
DNA TAGGING
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Fig. 3 - Ti plasmid
• Transposon tagging has been used to isolate several genes in maize (e.g. A1, A2, BZ2, C1, C2, opaque2, R, P, etc.), tomato (cf-9, Dem, etc.), tobacco (cf-4A), rice (Ei-Ef-1), etc.
• In gene expresion.
• In gene silencing.
• In knockout gene.
• In site directed mutagenesis.
“T-DNA & Transposon Tagging”
APPLICATIONS
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• Gene tagging strategies are used to isolate those genes that produce a detectable phenotype, but whose base sequences or protein products are not known.
• The tag may be based on – T-DNA of Agrobacterium – A transposable element– A retroviral genome
• Transposon tagging has been used to isolate several genes in maize (e.g. A1, A2, BZ2, C1, C2, opaque2, R, P, etc.), tomato (cf-9, Dem, etc.), tobacco (cf-4A), rice (Ei-Ef-1), etc.
“T-DNA & Transposon Tagging”
SUMMARY
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• T-DNA and transposon tagging are very important technique for isolation of unknown DNA sequences.
• These strategies are used for the construction of genomic libraries.
“T-DNA & Transposon Tagging”
CONCLUSION
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Primrose S. 2004 Principle Of Gene Manupulation6th Edition
Lewin Benjamin 2007 Gene IX9th Edition
“T-DNA & Transposon Tagging”
REFERENCES