A vector is a DNA molecule used to carry a gene of interest from one organism to another. A gene is a basic unit of inheritance containing hereditary genetic material, i.e., DNA. A gene of interest can be integrated into the genetic material of the vector, which then introduces it into another organism. There are two commonly used vectors: plasmids and virus-based vectors.
Plasmids are the most common vectors used in plant transformation. They are used to multiply the gene of interest and to transfer foreign genes into the recipient organism. Plasmids are small circular pieces of DNA found in almost all bacteria and in some fungi, protozoa, plants and animals. Plasmids are separate from the chromosome. Chromosomes are the primary structures containing DNA in cells (Figure 1). Plasmids replicate independently of the chromosome.
Figure 1: Bacterium cell illustration of the chromosome and plasmid DNA
Plasmids used for plant transformation contain three key elements:
- An origin of replication: This is a DNA sequence within the plasmid that indicates the location for DNA replication to begin. DNA replication is a process by which new copies of DNA are produced allowing the plasmid to be passed on to the next generation during cell division. Most plasmids used as cloning vectors can replicate to high numbers in the host cells resulting in many copies of the gene(s) of interest. Other plasmids have origins of replication that allow them to replicate in more than one type of bacteria (e.g. Escherichia coli and Agrobacterium tumefaciens.
- A selectable marker gene: This gene is required to distinguish cells containing the plasmid with the gene of interest from those that did not take up the plasmid. The most commonly used selectable marker genes are those that confer resistance to antibiotic or herbicide. Common eselectable markers used in plant transformation include the neomycin phosphotransferase II (NPTII) kanamycin resistance gene, for selection in kanamycin-containing media, phosphinothricin acetyltransferase gene, for selection in media containing phosphinothricin (PPT) or hygromycin phosphotransferase (HPH) gene, for selection in media containing hygromycin B. Those cells that do not have the selectable marker genes will not be able to grow in the presence of the antibiotic or herbicide targeted by the gene incorporated; only those cells that do have the plasmid with the marker gene will regenerate.
- A cloning site: This is a place within the plasmid where a piece of DNA (with a gene of interest) is inserted.
An example of a commonly used Ti plasmid is pBIN19 (Lee and Gelvin, 2008). pBIN19 carries two antibiotic resistance genes, one on the plasmid with a bacterial promoter to allow for selection of bacteria that have the plasmid. A second one is included within the T-DNA region driven by a plant promoter to allow for selection of transformed plant cells. It also carries Escherichia coli and A. tumefaciens origins of replication. pBIN19 is an example of a shuttle vector (i.e., it can be propagated in two different host species). pBIN19 can be manipulated in E. coli to rapidly make multiple copies of the gene of interest and carry out the necessary assembly steps to put together the transgene (desired gene, promoter, selectable marker and T-DNA borders in the right positions). It can then be transferred to A. tumefaciens, which is more difficult to work with, for use in plant transformation.
Agrobacterium tumefaciens has the natural ability to transfer DNA into a chromosome of the host plant. This bacterium often contains a plasmid known as Ti plasmid, which is a large, circular, double-stranded DNA that can replicate independent of the A. tumefaciens genome. During infection of the host plant, a portion of the Ti plasmid called T-DNA (for transferred DNA), which includes the gene of interest, is cut off and transferred into the host’s chromosome. Once in the plant cells, the T-DNA incorporates into the chromosome, resulting in genetically modified cells. The GM plant cells are then regenerated into whole plants containing the gene of interest, which will be passed on to future generations of the plant by the normal inheritance process.
Virus-based vectors also can be used as a tool for gene transfer to plant cells, for specialized purposes. For further information on virus-based vectors, see Chung et al. 2006.
- Bacterial Plasmids. California State University Northridge