VirC
Function: VirC1 binds to the enhancer driver sequence outside the T-DNA terminal sequence, enhancing the tumorigenicity of Agrobacterium (by increasing the ability of the VirD operon to nick at a specific site within the T-DNA terminal sequence). The function of VirC2 is currently unknown.
VirD gene family (1-4)
They encode four different protein molecules with molecular masses of approximately 16.2, 47.4, 21.3 and 5.8 kDa, all of which are related to T-DNA processing.
VirD1: encodes a DNA relaxase.
VirD2: ① It has the ability to cleave the N-terminal end of T-DNA; ② It contains a nuclear localization signal. After VirD2 covalently binds to T-DNA, it can guide the complex toward the plant cell nucleus.
VirD3: Its function is still unclear. VirD3 proteins have little homology among different Agrobacterium species.
VirD4: The N-terminus carries a signal peptide sequence that guides the protein to be transported to the cell membrane.
VirE
The encoded VirE2 protein is a single-stranded DNA binding protein.
Function: VirE2 covalently binds to the 5' end of processed T-DNA molecules to form a T-complex, which protects them from degradation by nucleases.
VirF
Possible function: Assists in the transfer of T-DNA into host plant cells
VirH
Function: It may detoxify certain bactericidal or antibacterial compounds produced by plants, thereby preventing the growth of Agrobacterium from being inhibited by these substances.
Tzs
Encodes a cytokinin isopentenyltransferase related to zeatin, which can secrete zeatin outside the cell (zeatin can promote dedifferentiation and cell division of plant tissues at the site of Agrobacterium infection)
Necessary conditions for T-DNA integration into the plant nuclear genome.
(1) T-DNA border sequence
(2) Vir region genes
(3) Genes on the chromosome of Agrobacterium
12. The working principle of the cointegration vector system.
(1) The two plasmids of Agrobacterium and Escherichia coli have a homologous region
(2) After the two plasmids are joined, homologous recombination is performed to form a large co-integrated plasmid
(3) The foreign gene to be introduced into the plant is cloned and manipulated in Escherichia coli. After homologous recombination with the Agrobacterium Ti plasmid, the foreign gene is located between the two T-DNA border sequences.
(4) E. coli plasmids cannot replicate autonomously in Agrobacterium, so they cannot exist stably in Agrobacterium without recombination.
13. The working principle of the binary vector system.
(1) Consists of helper plasmid and shuttle plasmid
(2) Helper plasmid: contains the Vir segment necessary for T-DNA transfer, providing toxic gene function; contains the Agrobacterium replication initiator; and has no T-DNA border sequence.
(3) Shuttle plasmid: has 1-2 T-DNA border repeat sequences; broad host range replication and mobility functions; bacterial selection markers, such as kanamycin resistance genes; recently constructed shuttle vectors carry the cosI replication initiator (from
pBR322), so plasmids can be prepared in large quantities; encode plant selection markers, expression signals, T-DNA border sequences; have multiple cloning sites for foreign gene subcloning
(4) Neither plasmid has the ability to induce tumors when present alone
(5) After conjugation, the two plasmids can coexist autonomously in the same Agrobacterium cell under selection pressure. When Agrobacterium infects a wounded plant, the Vir gene on the Ti plasmid interacts in trans with the right border sequence on the shuttle plasmid, thereby transferring the T-DNA into the plant cell.
14. Compare the characteristics of the cointegration vector system and the binary vector system.
Cointegration Vector System:
Binary vector system: (1) It consists of two mutually compatible plasmids that can coexist autonomously in the same Agrobacterium cell (basic characteristics)
(2) No need for in vivo recombination
(3) Only one plasmid needs to be transferred into bacteria
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