Gene Isolation by TDNA Insertion | Free PPT

What is TDNA insertion?

T-DNA (Transfer DNA) is a segment of DNA found in the Ti (tumor-inducing) plasmid of certain strains of Agrobacterium, a type of soil bacterium. Agrobacterium tumefaciens and Agrobacterium rhizogenes are two common species known to carry T-DNA.

T-DNA plays a significant role in the natural genetic transformation process carried out by Agrobacterium. It has the ability to transfer itself from the bacterium to the DNA of a host plant cell, leading to the integration of the T-DNA into the genome of the plant cell. This natural transformation phenomenon has been exploited for genetic engineering purposes, particularly in the field of plant biotechnology.

TDNA insertion refers to the process by which the T-DNA molecule from Agrobacterium is integrated into the genome of a host plant cell during genetic transformation. It involves the transfer of the T-DNA segment from the bacterium to the plant cell, followed by its integration into the plant’s DNA.

Components of TDNA

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1. Border Sequences: The T-DNA is flanked by two identical border sequences known as the left border (LB) and right border (RB). These border sequences serve as recognition sites for the transfer process and are required for the integration of T-DNA into the plant genome.
2. Promoter and Terminator Regions: The T-DNA often contains its own promoter and terminator sequences. The promoter region is responsible for initiating the transcription of the genes present within the T-DNA, while the terminator region marks the end of transcription.
3. Selectable Marker: The T-DNA often carries a selectable marker gene, which confers resistance to specific antibiotics or herbicides. The presence of the selectable marker allows transformed plant cells to be identified and selected during the transformation process.
4. Vir region: The Vir region, also known as the virulence region, is a crucial component of the T-DNA (Transfer DNA) system found in Agrobacterium tumefaciens and related species. The Vir region consists of a set of genes that are responsible for the transfer of T-DNA from the bacterium to the host plant cell.

Vir regions play vital roles in transferring tdna into host genome. For example virA is a kinase protein which can sense phenolic compounds is released by wounded plant and act as a receptor for that. VirD1 is a topoisomerase, it helps virD2 to cut tdna at right border. VirD2 is an endonuclease, it cuts tdna at right border and helps in the integration of tdna into host chromosome.

TDNA insertion into plant cell

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1. Agrobacterium Recognition and Attachment: Agrobacterium detects a wound or a susceptible plant tissue and attaches to the plant cell surface. This initial recognition involves specific molecular interactions between the bacterium and the plant.
2. Virulence Gene Activation: Once attached to the plant cell, specific compounds released by the wounded plant trigger the activation of the virulence genes located in the Vir region of the Agrobacterium Ti plasmid. These genes initiate the transfer process.
3. T-DNA Processing and Formation of T-DNA-Protein Complex: The activated virulence genes lead to the excision of the T-DNA segment from the Ti plasmid. The T-DNA is processed and protected by VirD proteins, which bind to the T-DNA ends. The T-DNA, along with associated Vir proteins, forms a T-DNA-protein complex within the Agrobacterium.
4. T-DNA Transfer: The T-DNA-protein complex is transported from the Agrobacterium into the plant cell through a specialized structure called the type IV secretion system. This system spans the bacterial envelope and forms a channel through which the T-DNA complex is delivered into the plant cell cytoplasm.
5. Nuclear Import: Once inside the plant cell, the T-DNA complex moves toward the nucleus. The exact mechanisms of nuclear import are still not fully understood, but it is believed to involve interactions with host cell factors. The T-DNA complex reaches the nuclear envelope and enters the plant cell nucleus.
6. Integration into Plant Genome: Once inside the nucleus, the T-DNA integrates into the plant genome through recombination events. The T-DNA border sequences, which flank the T-DNA segment, recombine with the plant genomic DNA, leading to the stable integration of the T-DNA into the plant genome. The exact integration site can be random and can vary among transformed cells.
7. Gene Expression and Plant Transformation: After integration, the T-DNA becomes a part of the plant genome and is subject to the regulatory mechanisms of the host plant. The genes carried by the T-DNA can be expressed under the control of plant regulatory elements, such as promoters and terminators. This expression leads to the production of specific proteins and the development of transformed characteristics in the plant.

It’s important to note that the TDNA insertion process can vary depending on the specific experimental conditions, Agrobacterium strain, and plant species being used for transformation.

Identification of TDNA insertion

There are different methods for screening recombinant Ti  plasmids, but one common approach is to use antibiotic  resistance markers. The Ti plasmid contains a selectable marker gene, usually for resistance to an antibiotic such as kanamycin.

The foreign DNA is inserted into the plasmid in a way that  disrupts the function of the marker gene, so that cells that have taken up the recombinant plasmid will not be able to  survive in the presence of the kanamycin.

Therefore, cells that are successfully transformed with the  recombinant Ti plasmid will only survive on selective media  where kanamycin is absent. Cells that have not taken up the plasmid or have  taken up a non-recombinant plasmid will survive in the presence of the kanamycin antibiotic.

Applications of TDNA insertion

1. Genetic Modification of Crop Plants: TDNA insertion allows for the introduction of specific genes of interest into crop plants, leading to the development of genetically modified (GM) or genetically engineered (GE) crops. This technology has been used to confer traits such as insect resistance, herbicide tolerance, disease resistance, improved nutritional content, and enhanced yield in various crop plants.
2. Functional Genomics: TDNA insertion can be used as a tool to study gene function and elucidate gene regulatory networks in plants. By randomly inserting T-DNA into the plant genome, researchers can generate a library of mutant plants, each with a TDNA insertion disrupting a different gene. This enables the identification of genes involved in specific biological processes, development, or response to environmental stimuli.
3. Plant Transformation for Basic Research: TDNA insertion is used as a method to introduce foreign genes or modify endogenous genes in plants for basic research purposes. This includes studying gene expression, protein localization, protein-protein interactions, and understanding plant development and physiology.
4. Production of Recombinant Proteins: TDNA insertion can be employed to produce recombinant proteins of interest in plant cells or tissues. The T-DNA is designed to carry genes encoding the desired proteins, and the transformed plants are cultivated to express and accumulate these proteins. Plant-based production systems offer advantages such as scalability, cost-effectiveness, and potential post-translational modifications.
5. Crop Improvement and Trait Stacking: TDNA insertion allows for the stacking of multiple genes in a single plant to combine desirable traits. This facilitates the development of crop varieties with improved agronomic characteristics, such as multiple pest resistances or enhanced stress tolerance. Trait stacking through TDNA insertion can help address challenges in agriculture, such as yield improvement, disease resistance, and climate resilience.
6. Creation of Model Organisms: TDNA insertion has been used to generate mutant or transgenic model organisms in plant research, such as Arabidopsis thaliana. These model organisms enable researchers to study gene function, developmental processes, signaling pathways, and interactions with pathogens or symbiotic organisms.

These applications demonstrate the versatility of TDNA insertion in plant biotechnology and its potential to contribute to crop improvement, functional genomics, and fundamental research in plant biology.

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