T dna & transposone tagging 1 (2)

genetic engineering

1. T-DNA AND TRANSPOSON TAGGING By Dr Arunima Karkun 1

2. 2 “T-DNA & Transposon Tagging” S Y N O P S I S 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. “T-DNA & Transposon Tagging” I N T R O D U C T I O N 3

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. “T-DNA & Transposon Tagging” R O L E O F G E N E T A G G I N G 4

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 “T-DNA & Transposon Tagging” R O L E O F G E N E T A G G I N G 5

6. “T-DNA & Transposon Tagging” R O L E O F G E N E T A G G I N G 6 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. “T-DNA & Transposon Tagging” T R A N S P O S O N E T A G G I N G 7

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. 8 T R A N S P O S O N E T A G G I N G OF PLANT GENES

9. “T-DNA & Transposon Tagging” 9 T R A N S P O S O N E T A G G I N G OF PLANT GENES 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. “T-DNA & Transposon Tagging” D I F F I C U L T I E S 10

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 . “T-DNA & Transposon Tagging” T- D N A T A G G I N G 11 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. “T-DNA & Transposon Tagging” A P P L I C A T I O N S 12

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. “T-DNA & Transposon Tagging” S U M M A R Y 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. 14 “T-DNA & Transposon Tagging” C O N C L U S I O N

15. 15 Primrose S. 2004 Principle Of Gene Manupulation 6 th Edition Lewin Benjamin 2007 Gene IX 9 th Edition “T-DNA & Transposon Tagging” R E F E R E N C E S

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