genetic engineering

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“T-DNA & Transposon Tagging”
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 INTRODUCTION
 ROLE OF GENE TAGGING
 TRANSPOSON TAGGING
 TRANSPOSON TAGGING OF PLANT GENES
 DIFFICULTIES IN TRANSPOSON TAGGING
 T-DNA TAGGING
 SUMMARY
 CONCLUSION
 REFERENCES

3. • 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.
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4. • 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.
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5. • 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
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6. “T-DNA & Transposon Tagging”
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Flow chart 1 - A generalized scheme for gene tagging

7. • 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.
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8. “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.
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9. “T-DNA & Transposon Tagging”
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Fig. 2- Transposons tagging

10. • 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.
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11. • 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.
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Fig. 3 - Ti plasmid

12. • 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.
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13. • 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.
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14. • 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.
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15. 15
Primrose S. 2004 Principle Of Gene Manupulation
6th Edition
Lewin Benjamin 2007 Gene IX
9th Edition
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16. THANK
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