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Team:NEAU-Harbin/link1.html
From 2014.igem.org
Agrobacterium-mediated transformation method
Agrobacterium-mediated fungal transformation (AMT) is a potential tool for performing targeted and random mutagenesis .This method is commonly used for plant-cell transformation and is recently widely applied to various fungi. Through AMT system, T-DNA can be transferred to a wide variety of fungi with high efficiency.
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Structure of A. tumefaciens' plasmid
• The gram-negative bacterium A. tumefaciens is a plant pathogen, which causes crown gall tumors.A. tumefaciens induces this tumorous growth by transferring a part of its DNA (T-DNA) which is located on its 200-kbp tumor-inducing (Ti) plasmid to the host. After integration into the host genome, genes that are naturally located on this T-DNA and encode enzymes for the production of plant growth regulators are expressed.
• And another segment, the virulence region, which is composed of a large number of vir genes, is necessary for the tumorigenicity. Proteins encoded by the virulence region are involved in the formation, transport and possibly also integration of the T-DNA. And the T-region of the Ti plasmid is surrounded by a 24-bp border repeat, which is the cis-acting signal for the DNA delivery system to plant cells. Otherwise, all the sequences of the natural T-DNA can be deleted and replaced by other DNA sequences without a negative effect. So the binary vector system is used, in which the T-DNA and the virulence region are placed on two separate plasmids.
• mechanism
• Phenolic compounds such as acetosyringone are used to induce the vir genes that encode the T-DNA transfer machinery of A. Tumefaciens. VirA, an inner membrane protein, senses acetosyringone and responds by autophosphorylation.
• The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides.
• The activated VirG, which has DNA-binding properties, then acts as a transcriptional activator of itself and other virulence genes after VirA transfers phosphoryl group to it.
• For the generation of a single-stranded DNA copy of the T-DNA,the virC and virD operons are needed.VirC1 can bind the25-bp “overdrive” sequence and thereby
• Stimulates T-strand production. The VirD2 protein, assisted by VirD1, stays covalently attached to the 5’ end of the T-strand.
• The next step in T-DNA transfer is piloting the T-strand through the bacterial membrane and cell wall. The VirB proteins form a transport pore and a structure on the surface, and the virulence proteins VirE2, VirE3, and VirF are also exported .VirE2 is a single-stranded DNA-binding protein and is thought to coat the T-strand in the host to protect it against nucleases and to keep the T-strand in an unfolded state to facilitate transport. Once inside the nucleus, the T-DNA stably integrates into the genome.
• advantages
• It has been shown to have several advantages over conventional transformation methods. AMT generates a high percentage of transformants with a single-copy integrated DNA, which facilitates the isolation of tagged genes, and the T-DNA is an efficient substrate for homologous recombination. Above all, AMT is well suited to perform insertional mutagenesis in fungi.
