Transformation of plant cells by Agrobacterium involves a series of complex reactions, including the attachment of Agrobacterium to plant cells, the release of signal molecules from plant cells, the induction of expression of Vir region genes, the transfer of transfer DNA (T-DNA ) and its integration and expression on the plant nuclear genome, and the acquisition of a complete transgenic plant after cell and tissue culture.
Ti plasmid is a circular double-stranded DNA molecule outside the chromosome of Agrobacterium tumefaciens, about 200 kb, including vir region, con region, ori region and T-DNA region. Among them, the vir region, the toxicity site on the chromosome of A. tumefaciens and the border sequence of T-DNA are necessary for T-DNA transfer.
After Agrobacterium attaches to the plant wound tissue, through the interaction between the two, Agrobacterium forms fiber filaments to fix it on the plant cell wall, and at the same time induces the injured plant tissue to release some phenolic and other compounds. As a receptor protein, VirA is induced by the secretions of damaged plant cells, and after autophosphorylation, it further activates the VirG protein. The latter is a DNA transcription activator. After activation, it can specifically bind to a sequence called Vir box upstream of the promoter region of other Vir genes to initiate the transcription of these genes. Among them, the VirD gene product cuts T-DNA to produce a T-DNA single strand (T-strand), which combines with VirD2 and VirE2 to form a T-strand protein complex and then traverses bacterial cell membranes, cell walls, plant cell walls, cell membranes and nuclear membrane. The T-DNA that enters the human cell nucleus is randomly integrated into the plant chromosome in the form of single or multiple copies. Studies have shown that T-DNA is preferentially integrated into transcriptionally active regions, and the integration frequency of T-DNA is also higher in homologous regions of T-DNA and highly repetitive regions of DNA. The T-DNA integrated into the plant genome also suffers from deletions, duplications, and transboundary phenomena to a certain extent.
Different plants are affected by factors such as genotype, developmental status, and difficulty in tissue culture, so Agrobacterium-mediated transformation adopts different methods accordingly. Currently, the commonly used Agrobacterium-mediated transformation method is the leaf disk method. In this method, the transformation receptors have been extended to organs, tissues, cells, and protoplasts. Agrobacterium-mediated transformation of foreign genes is easy to operate and has high transformation efficiency. Gene transfer is a naturally occurring behavior. The number of copies of foreign genes integrated into the recipient genome is small, the degree of gene rearrangement is low, and the inheritance of transgenic traits in future generations is relatively stable.
1. Preparation of bacterial solution
Pick a single colony of Agrobacterium after 2 d of subculture and place it in liquid LB medium supplemented with 50 mg/L Kan. The culture was carried out with shaking at 200 r/min, and the culture conditions were 28°C and darkness. After 16-20 h, place the bacterial solution in a centrifuge tube, centrifuge at 5,000 g for 5 min and collect the bacteria. The collected bacteria were diluted to a suitable concentration with liquid LB medium supplemented with 50 mg/L Kan or liquid medium for tobacco leaf callus induction.
2. Preparation of tobacco leaf disc explants
Take young tobacco leaves (tissue culture seedlings or greenhouse field seedlings). If the materials are taken from the field or greenhouse, wash them with tap water and soak them in 2% sodium hypochlorite solution for 3-5 min. Rinse 3 times with sterile water, punch into small discs with a 5mm hole punch, and store in sterile water for later use.
3. Infection and co-culture
The tobacco leaf disc explants were soaked in the liquid LB medium of Agrobacterium LBA4404 strain containing plasmid pBI121 and 50-100 mg/L Kan, and in the liquid medium of Agrobacterium LBA4404 strain without plasmid pBI121. Among them, plasmid pBI121 carries the selectable marker gene or the target gene. Soak for 4-5 min, take it out, absorb the bacterial liquid with filter paper, and then inoculate it into callus induction medium for co-culture. The culture conditions are 24-28°C and cultured in the dark.
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4. Selective culture
After co-cultivation for 2-3 days, the tobacco leaf discs were soaked in liquid callus induction medium supplemented with 300-500 mg/L Carb. After 30-60 min, inoculate them in the selection medium for selective culture until complete regenerated plants are obtained. The culture conditions are as follows: the callus induction stage is 24-28°C, dark culture; the adventitious shoot and root regeneration stage is 24-28°C, 13-16 h/d light, and the light intensity is 40-50 µmol/(m2·s).
5. Detection of transgenic plants
a. Histochemical method. GUS gene expression was detected in calli and leaves according to the histochemical method. GUS detection of Kan-resistant callus: Cut the obtained Kan-resistant callus or leaves into small pieces, add them to the prepared X-gluc substrate solution, and incubate at 37°C overnight. Fix with FAA solution for more than 1 h, and decolorize with 70%, 90%, and 100% ethanol in sequence, and visually observe whether there is GUS gene expression on the surface and inside the callus.
b. PCR identification. The genomic DNA of tobacco to-be-transgenic plants was extracted as a template. Design primers based on the selection marker gene or target gene sequence, and conduct PCR amplification detection using the total DNA of non-transgenic plants (negative control), empty strain transformed plants (negative control) and vector plasmid DNA (positive control) as controls to preliminarily identify whether the foreign gene has been integrated into tobacco genomic DNA.
c. Southern blot analysis. The genomic DNA of tobacco to-be-transgenic plants was extracted and double-digested with restriction endonucleases. The processed DNA was electrophoresed on a 0.7%-0.9% agarose gel, and probe labeling, DNA transfer, membrane treatment, and hybridization operations were performed. Further identify whether the foreign gene is integrated into the genomic DNA of tobacco and the copy number of the foreign gene (control selection is the same as above).
d. RT-PCR. Use the Trizol kit to extract the total RNA of tobacco to-be-transgenic plants, and use the reverse transcription PCR kit to perform RT reaction and PCR reaction on the total RNA according to the prescribed procedures. Further identify the expression of exogenous genes at the RNA level (control selection is the same as above).
e. Northern blot analysis. Extract and purify the total RNA or mRNA of tobacco to-be-transgenic plants, conduct 1.2% (W/V) formaldehyde-denaturing agarose gel electrophoresis, and perform probe labeling, RNA transfer, membrane treatment, and hybridization operations. Further identify the expression of foreign genes in plant cells (control selection is the same as above).
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