Gene tagging using TDNA from Agrobacterium tumifaciens Case study of TDNA Activation Tagging in Rice

1. Presented by:
Shivani Upadhyay
TDNA Tagging

2. • Modern genomics depends to a large extent on the identification, isolation
and characterization of genes. Describing the expression, function and
regulation of these genes is crucial in understanding many of the fundamental
questions in development, morphology, pathogen resistance, stress responses,
and other phenomena.
• The classical approaches to cloning require information about the gene
product, i.e., the transcript or polypeptide.This information can be used to
identify cDNA or genomic clones from a DNA library.
• For most of the agronomically important plant genes, very limited information
is available. Often the only character at hand is a phenotype, a feature difficult
to utilize in most of the cloning techniques listed above. Here, gene tagging
offers an attractive alternative.
INTRODUCTION

3. What is Gene tagging?
Gene tagging is a technique to identify and isolate genes via : random or
directed insertion of DNA sequences into a gene.
This technique - mutates and tags the gene.
The insertional element in plant systems is either T-DNA or transposons (or a
combination of both).
Gene tagging method involves insertion of DNA elements, such as T-DNA or
transposons into the genome which we want to tag and track genes of interest.
Example:
T-DNA: Arabidopsis, Snapdragon (Antirrhinum majus)
Transposons mediated gene tagging: Maize (Zea mays)

4. Gene tagging as a perfect alternative is to provide a powerful tool for
understanding the functions and behaviors of specific genes within a cell or
organism.
1. Non-Invasive
2. Specificity
3. Visualization
4. Functional Studies
5. High-throughput Analysis
6. Versatility
7. Ease in integration with other techniques
Gene tagging - A Perfect Alternative

5. TDNA tagging
T-DNA (Transfer DNA) is derived from the Ti (Tumor-inducing) plasmid of
Agrobacterium tumefaciens. T-DNA carries selectable markers, such as
antibiotic resistance genes (i.e. gene of interest), and can be engineered to
contain reporter genes for visual detection. Agrobacterium-mediated
transformation is used to introduce T-DNA into the plant genome, where it
integrates randomly.
T-DNA tagging mutagenesis involves screening of populations by T-DNA
insertional mutations. Collections of known T-DNA mutations provide resources
to study the functions of individual genes, as developed for the model
plant Arabidopsis thaliana.

6. CASE STUDY ON T-DNA TAGGING IN RICE
Jeong, D. H., An, S., Kang, H. G., Moon,
S., Han, J. J., Park, S., ... & An, G.
(2002). T-DNA insertional mutagenesis
for activation tagging in rice. Plant
physiology, 130(4), 1636-1644.

7. Materials and Methods
Construction of Vectors
Plasmid pGA2520 was made by inserting the DNA fragment containing the CaMV 35S
minimal promoter (-90 to -1), the gus-coding region derived from pBI101.1 and the
nopaline synthase (nos) terminator into the HindIII and HpaI sites of Pga1605.Via the same
method, pGA2525 was constructed to contain the minimal nos promoter (-101 to -1)
Production and Growth of Transgenic Rice Plants
Rice was transformed using the Agrobacterium-mediated cocultivation method described
by Lee et al. (1999). Following modifications: (a) cocultivation temperature reduced to
20°C, (b) exclusion of hygromycin in the regeneration media, and (c) direct transfer of the
regenerated plantlets to soil from the shoot induction medium, thereby omitting the rooting
procedure in Murashige and Skoog medium. Plants were grown in the greenhouse at a
minimum night temperature of 20°C, with a day length of at least 14 h, supplemented with
artificial light.

8. GUS Assay
Histochemical GUS-staining was performed according to the method of Jeon et al.
(2000a). Mature flowers of the primary transformants were sampled and stained and
three or four seedlings were dissected and stained as well. Each tissue type was incubated
at 37°C for 12 h; then, the staining solution was replaced with 70% (w/v) ethanol to
remove the chlorophyll. Afterward, examined the tissues under a dissecting microscope
and analyzed their staining patterns.
Isolation of the Sequence-Flanking T-DNA
Extracted genomic DNA from immature leaves. The sequence-flanking T-DNA was
isolated by inverse PCR.
RT-PCR Analysis
Tri-reagent used to isolate total RNA from immature leaves and roots.
Materials and Methods

11. Examination of the 35S Enhancer Element in Rice
 Transformation with vectors pGA2523 and pGA2526, revealed that 35S enhancer
sequence increased the level of gene expression over that found with the minimal
promoters.
 Presence of the tetramerized 35S enhancers upstream of the CaMV 35S minimal
promoter or minimal nos promoter enhanced expression.
 To further evaluate the enhancing activity of the 35S enhancers, vector pGA2524 (with
the enhancer placed upstream of the minimal 35S promoter, in the reverse direction) and
vector pGA2522 (with the enhancer sequences downstream of the nos terminator) were
used.
 In plants transformed with either of those vectors, enhanced GUS activities were
detected in the leaves and flowers of the transformants.
RESULTS

12. Vector construction and Production of T-DNA-Tagged Transgenic Rice Plants
 Plasmid pGA2715 contained the promoter less gus reporter gene with an intron and
multiple splicing donors and acceptors immediately next to the right border.
 The hygromycin-resistant selectable marker gene, with the rice alpha tubulin (OsTubA1-
1) promoter, was placed between the gus gene and the 35S enhancer.
 Thus, this vector can be used to achieve both gene trapping and activation tagging in
rice.
 The other binary vector, pGA2707, resembles pGA2715 except for its lack of enhancer
elements.
 Using Agrobacterium tumefaciens-mediated rice transformation, 13,450 fertile
transgenic plants transformed with pGA2715, and 20,810 fertile transgenic plants with
pGA2707 were generated.
RESULTS

13. Enhancing Gene Expression by ActivationTagging
 Using inverse PCR on 100 lines, 71 sequences flanking the T-DNA were isolated. These
isolated sequences were examined by comparing them with entries in publicly available
DNA databases, and the sequences were then annotated for their identifying open
reading frames.
 28 insertions (39.4%) occurred in the intragenic regions, whereas 43 (60.6%) were
located in the intergenic regions. Insertions of enhancer elements within 4.5 kb upstream
or downstream of the nearest open reading frame occurred in 12 and nine lines,
respectively. These 21 lines (29.6%) were regarded as candidates for activation tagging.
 15 progeny per line were planted and their genotypes by PCR analysis were determined
using a gene-specific primer and a T-DNA primer.
 The expression levels were then examined via reverse transcriptase (RT)-PCR, using
gene specific primers. Among the lines examined, four showed that the levels of product
from the gene near the enhancers were significantly increased in heterozygote and
homozygote plants, compared with those in the wild-type tissue
RESULTS