Edible Mushroom Transgenic Service

Since 1978, when the genetic transformation of fungi entered a new stage, the genetic transformation of edible mushrooms has only taken off, and in 1991 it was first reported that researchers used PEG method to introduce exogenous DNA into the protoplasm of mushroom recipient bacteria to obtain transformants. Commonly used genetic transformation methods include PEG method, particle bombardment method, Agrobacterium-mediated method, etc.

• PEG (polyethylene glycol) method. The main principle is that PEG can cause the formation of molecular bridges between cell membranes or between DNA and membranes, which can promote intercellular contact and adhesion, or interfere with intercellular recognition by causing surface charge disorder, and facilitate intercellular fusion or entry of exogenous DNA.
• The electroexcitation method. The electroexcitation method is the use of high-voltage electrical pulses to form reversible instantaneous channels in the protoplast membrane by electroporation, thereby facilitating the uptake of exogenous DNA. This method is simpler and more convenient than PEG method and requires less DNA.
• Particle bombardment method. Particle bombardment method, also known as microprojectile bombardment, the main principle is to encapsulate the exogenous DNA on the surface of tiny gold or tungsten particles, and then under high air pressure, the particles are shot into the recipient cells or tissues at high speed. The exogenous DNA on the particles is then brought into the cell and integrated into the plant chromosome, thus achieving gene transformation.
• Restriction enzyme-mediated DNA integration (REMI). The main principle is that restriction endonucleases penetrate the cell and nuclear membranes, and in vivo, at specific enzymatic sites the resulting chromosomal DNA ends are then ligated to the linearized plasmid DNA cut by the restriction endonuclease in the presence of the host cell enzyme system.
• Agrobacterium-mediated transformation method. Agrobacterium tumefaciens is a Gram-negative soil bacterium that infects the injured parts of dicotyledonous plants and forms crown burrs. During crown burr formation, Agrobacterium tumefaciens remains only in the interstitial space of plant cells, and the T-DNA on its cyclic plasmid Ti is processed, sheared, replicated in the bacteria, transferred into plant cells, and randomly inserted into the plant genome. This system is a naturally effective genetic engineering system.

What We Offer

For the problems that may be encountered, such as low recombination activity, severe methylation, and inadequate expression under the regulation of heterologous promoters after the donor DNA enters the recipient bacteria, we have established a stable Agrobacterium-mediated transformation system for edible mushroom through Lifeasible's continuous search for new transformation methods and gene expression elements.

The promoters we currently use in edible fungus genetic transformation are ras promoter and gpd promoter. ras promoter has a strong role in regulating exogenous gene expression and can regulate the expression of GUS gene in Pleurotus ostreatus. gpd gene promoter has a stronger ability to regulate exogenous gene expression than ras promoter.

The genetic transformation screening markers we used for edible fungi can be grouped into four categories, nutrient-deficient markers, antibiotic resistance screening markers, herbicide/fungicide resistance screening markers, and metabolite resistance screening markers.

• Nutrient-deficient markers. The nutrient-deficient marker is obtained by transferring the wild-type allele into the corresponding nutrient-deficient strain and screening for prototrophic growth colonies on basic media to obtain transformants. It has the advantage of guiding the integration of the vector plasmid into the homologous part of the chromosome and has a very low transformation background, making it easy to select.
• Antibiotic resistance screening marker. After the expression vector is transferred into the recipient, the antibiotic resistance genes on the vector (e.g., kanf^R, Hyg^R, PhI^R, Ble^R) are integrated with the recipient genome to confer the corresponding resistance to the recombinant. Thus, the recombinants can be screened by adding the corresponding antibiotics to the culture medium. This marker can overcome the disadvantage of using nutrient-deficient phenotypes as screening markers.
• Herbicide/fungicide resistance screening marker. This marker can be used as a screening marker for some edible bacteria that are not sensitive to antibiotics.
• Metabolite resistance screening marker. This marker can overcome the disadvantage that some edible bacteria are not sensitive to antibiotics or antibiotics and herbicides are toxic to edible bacteria when used as screening markers by transferring exogenous functional genes into the receptor and getting expressed in the receptor.