"
Team:NEAU-Harbin/link1.html
From 2014.igem.org
| Line 153: | Line 153: | ||
<div id="neiyes"></div> | <div id="neiyes"></div> | ||
<div id="neiyez"> | <div id="neiyez"> | ||
| - | <p style="padding-left:20px">Agrobacterium-mediated transformation method | + | <p style="padding-left:20px">Agrobacterium-mediated transformation method</p> |
| - | + | <p style="padding-left:20px">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. </p> | |
<p style="text-align:center"></p> | <p style="text-align:center"></p> | ||
</div> | </div> | ||
<div id="neiyex"><img src="http://nd.cn.usa.wakelion.net/project/link1.jpg"></div> | <div id="neiyex"><img src="http://nd.cn.usa.wakelion.net/project/link1.jpg"></div> | ||
| - | <p style="padding-left:20px">（more）: | + | <p style="padding-left:20px">（more）: </p> |
| - | Structure of A. tumefaciens' plasmid | + | <p style="padding-left:20px">Structure of A. tumefaciens' plasmid </p> |
| - | • 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. | + | <p style="padding-left:20px">• 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.</p> |
| - | • 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. | + | <p style="padding-left:20px">• 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.</p> |
| - | • mechanism | + | <p style="padding-left:20px">• mechanism</p> |
| - | • 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. | + | <p style="padding-left:20px">• 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.</p> |
| - | • The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides. | + | <p style="padding-left:20px">• The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides.</p> |
| - | • 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. | + | <p style="padding-left:20px">• 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.</p> |
| - | • 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 | + | <p style="padding-left:20px">• 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</p> |
| - | • Stimulates T-strand production. The VirD2 protein, assisted by VirD1, stays covalently attached to the 5’ end of the T-strand. | + | <p style="padding-left:20px">• Stimulates T-strand production. The VirD2 protein, assisted by VirD1, stays covalently attached to the 5’ end of the T-strand.</p> |
| - | • 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. | + | <p style="padding-left:20px">• 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.</p> |
| - | • advantages | + | <p style="padding-left:20px">• advantages</p> |
| - | • 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. | + | <p style="padding-left:20px">• 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.</p> |
| - | • Reference | + | <p style="padding-left:20px">• Reference</p> |
| - | Caroline B. Michielse. Agrobacterium -mediated transformation as a tool for functional genomics in fungi</p> | + | <p style="padding-left:20px">Caroline B. Michielse. Agrobacterium -mediated transformation as a tool for functional genomics in fungi</p> |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 06:58, 8 October 2014
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.
（more）:
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.
• Reference
Caroline B. Michielse. Agrobacterium -mediated transformation as a tool for functional genomics in fungi
