t dna & transposone tagging 1 (2)

T-DNA AND TRANSPOSON TAGGING

By Dr Arunima Karkun

1

2

“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

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.


INTRODUCTION

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.


ROLE

OF

GENE

TAGGING

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


ROLE

OF

GENE

TAGGING

6


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.


TRANSPOSONE

TAGGING

7

8


• 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

9


TRANSPOSONE

TAGGING

OF

PLANT

GENES

Fig. 2- Transposons tagging

• 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.


DIFFICULTIES

10

• 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 TAGGING

11

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.


APPLICATIONS

12

• 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.


SUMMARY

13

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.


CONCLUSION

15

Primrose S. 2004 Principle Of Gene Manupulation6th Edition

Lewin Benjamin 2007 Gene IX9th Edition


REFERENCES

THANK YOU

THANK YOU

t dna & transposone tagging 1 (2)

Education