Agrobacterium reforming process, the key technology of plant genetic engineering
Product Manager：Harrison Michael
Plant genetic transformation technology, also called plant transgenic technology, is a key technique in modern biotechnology. It can improve plant genetic characteristics by introducing foreign genes into the plant genome. This article focuses on agrobacterium mediated method the efficiency and stability of plant genetic transformation technology.
I. Introduction to Agrobacterium-mediated transformation
1. Advantages of Agrobacterium-mediated method
The agrobacterium-mediated method has significant advantages over other transformation methods such as gene gunshots:
1. High transformation frequency: Agrobacterium tumefaciens can efficiently introduce foreign DNA into plant cells.
2. DNA into larger pieces, you can import large fragment DNA contains more than one gene.
3. Low copy number: import the exogenous gene copy number is usually low, help stabilize the genetic and expression.
4. Mendelian inheritance: In most cases, the transformed gene expression follows the Mendelian inheritance law.
5. Wide host range: The host range of Agrobacterium has expanded from plants to prokaryotes, fungi and even human cells.
2. The principle of agrobacterium mediated method
Agrobacterium tumefaciens is a Gram-negative bacterium whose T-DNA from the Ti plasmid can be transferred into plant cells by conjugation. Through molecular cloning, the tumorigenic gene in T-DNA can be replaced by the target gene of interest, making Agrobacterium tumeto an effective gene transfer tool. Below is a introduction to the technology:
1. Agrobacterium tumefaciens and Ti plasmids
Agrobacterium tumefaciens is a soil bacterium that can infect plants and cause tumor formation. This ability is attributed to the Ti plasmid it carries. The Ti plasmid is a large plasmid that contains a region called T-DNA (transfer DNA) that can be transferred into plant cells by Agrobacterium tumefaciens.
2. Structure of T-DNA
T-DNA is the part of the Ti plasmid that contains the genes used by Agrobacterium to infect plants. In transgenic technology, T-DNA is modified to contain foreign genes that scientists want to transfer into plants.
3. Integration of exogenous genes
When agrobacterium tumefaciens infects plants, T-DNA is cleaved out and integrated into the plant genome within the plant cells. This process is achieved through the DNA repair machinery of plant cells.
4. Selection of markers and reporter genes
In T-DNA, scientists often add selection marker genes and reporter genes. Selection marker genes are used to screen transgenic plants containing T-DNA, while reporter genes, such as GUS genes, are used to test for integration and expression of T-DNA.
5. Agrobacterium-mediated infection process
Preparation phase: First, the scientists cloned the foreign gene into a modified Ti plasmid, which no longer contains the tumor-causing gene, but contains the target gene.
Transformation phase: Next, the modified Ti plasmid was introduced into Agrobacterium tumefaciens.
Infection stage: Agrobacterium tumefaciens carry the modified Ti plasmid and deliver T-DNA to plant cells through wounds or contact with plants under specific conditions.
Integration and expression of phase: T - DNA integration within the plant cells, and begin to express exogenous genes.
3. Biological mechanisms of the transformation process
Agrobacterium-mediated plant genetic transformation is a complex biological process involving the following key steps:
1. Plant tissue injury: phenolic compounds are released to induce agrobacterium recognition and attachment.
2. Signal transduction: Agrobacterium VirA and VirG proteins respond to plant signals to activate the Vir gene region.
3. T-DNA replication: VirD1/D2 protein complexes replicate T-DNA to form T-strands.
4. T-DNA transfer: The T-DNA complex enters the recipient cell under the action of Vir protein.
5. Nuclear localization: The T-DNA complex enters the nucleus through nuclear pores.
6. Integration: T-DNA targets the integration site under the interaction of parasitic chromatin.
7. Expression: After integration of T-DNA, the gene expression regulatory sequence within it causes the gene to be expressed in plant cells.
II. Experimental operation of Agrobacterium transformation method
1. Culture of Agrobacterium
Cultivation of agrobacterium is the initial step in transformation experiments and is usually performed in LB medium.
1. LB culture medium preparation: 10 g/L 5 g/L peptone and yeast extract, 10 g/L sodium chloride dissolved in water, adjust the pH to 7.0 after sterilization.
2. Inoculation: Agrobacterium strains containing Ti plasmids were inoculated into LB medium.
3. Culture: The bacteria were cultured in a constant temperature shaker at 37 ° C until they grew to the logarithmic development phase.
2. Preparation of receptive cells of Agrobacterium Tumefaciens
In order to improve the conversion efficiency, it is necessary to prepare agrobacterium into receptive cells.
1. Collect bacteria: Collect the cultured Agrobacterium by centrifugation and remove the supernatant.
2. Wash: Resuspension of bacteria with a buffer containing 50 mM glucose, 10 mM MgSO4 and 50 mM CaCl2.
3. Prepare the receptive state: Put the bacteria on ice to make it a state that is easy to accept foreign DNA.
3. Transformation of foreign DNA
The target genes of plasmid DNA mixed with competent agrobacterium cells, promote the absorption of DNA by heat shock treat ment.
1. Mixed DNA and cells: Purified plasmid DNA was mixed with competent cells.
2. Heat shock, heat shock under 42℃ 90 seconds, and then quickly transferred to the ice to cool.
3. Recovery: The cells were incubated at 28℃ for 1-2 hours to restore cell viability.
4. Transformation of plant cells
After transformation of agrobacterium and plant cell culture, and realize the transfer of exogenous gene.
1. Preparation plant explant: choose the suitable plant tissue, such as leaves, stem segments or embryoid, and disinfection treatment.
2. Co-culture: Plant explants were co-cultured with transformed Agrobacterium tumefaciens cells. The co-medium is usually MS medium and its main components include:
o Ammonium nitrate 1900 mg/L
o Potassium nitrate 1650 mg/L
o Potassium dihydrogen phosphate 170 mg/L
o Magnesium sulfate 375 mg/L
o Ferric sulfate is 27.8 mg/L
o Trace element and vitamin mixture
o Sucrose 30 g/L
o AGAR powder 7 g/L (for preparation of solid medium)
3. Screening: Plant cells containing exogenous genes were screened using screening medium containing antibiotics. The screening medium is MS medium supplemented with antibiotics such as:
o Rifampicin 50-100 µg/mL
o K+ 50 µg/mL
4. Regeneration: will be transferred to regeneration medium after the screening, promote the proliferation and differentiation of plant cells.
5. Evaluation of conversion efficiency
The integration and expression of the exogenous genes were identified by molecular biological methods such as PCR and Southern blot, and the transformation efficiency was evaluated.
Conclusion
Agrobacterium transformation is an efficient and stable plant genetic transformation technology, which is widely used in plant gene function research and genetic improvement. Precise control of experimental conditions and optimization of operation steps can significantly improve the transformation efficiency and success rate. With the continuous progress of molecular biology technology, agrobacterium transformation will play a more important role in plant biotechnology. Aladdin helps plant research by providing comprehensive biochemical reagents with high quality and high purity reagents to help life science.
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