Genetic engineering (DNA recombination technology) is the artificial "processing" of genetic material (DNA) of different organisms under in vitro conditions, and recombination according to people's wishes to change the traits and functions of the organism, and then through appropriate carriers. The recombinant DNA is transferred into an organism or a cell and expressed in an organism or in a cell to obtain a new biological function. Such plants obtained by genetic engineering techniques are generally referred to as "genetically engineered plants." Since the first acquisition of genetically modified plants in 1983, it has been favored by breeders and has flourished. More than 200 plants have been successfully transgenic in more than 30 families. More than 3,000 transgenic plants have been approved in the field for field trials, and have been successfully commercialized in more than 20 countries including the United States, Canada, and China. . Improved crop varieties through genetic engineering are increasingly showing great potential in future agricultural production.
There are many methods for transgenic, and the most commonly used are Agrobacterium-mediated genetic transformation and gene gun transformation. Either way, the transformed cells are only a minority compared to the non-transformed cells, and there is competition between the two, and the transformed cells as xenogeneic cells are weakly competitive. Therefore, transformed cells must be screened.
Under selective pressure conditions, the expression of the resistance gene in the transformant is advantageous for selecting a transformed clone from a large number of non-transformed cells. The selective reagents currently used mainly include antibiotics, herbicides, methotrexate, and amino acids.
1. Antibiotic resistance gene:
Npt, neomycin phosphotransferase gene, resistant to kanamycin, G418, paromomycin and neomycin;
aphIV, hygromycin phosphotransferase gene, resistant to hygromycin;
Spt, streptomycin phosphotransferase gene, resistant to streptomycin;
Cat, chloramphenicol acetyltransferase gene, causes chloramphenicol to lose antibiotic activity;
Aacc3 and aacc4, gentamicin 3-N-acetyltransferase gene, resistant to gentamicin;
Ble, a bleomycin resistance gene, resistant to bleomycin.
2. Herbicide resistance gene
Bar, encoding a phospholipid flavonoid acetyltransferase (PAT), acetylates the free amino group of PPT (phosphonated flavonoids) and poisons PPT. Herbicide resistant glufosinate and bialaphos;
Epsps, glyphosate resistance marker gene, glyphosate resistant;
Als, green huanglong resistance marker gene.
3. Color or luminescent reporter gene
(1) GUS Enzyme activity detection
GUS enables β-glucosidase to catalyze the cleavage of a series of glucosides, producing a series of chromophores or fluorescing substances. The GUS activity can be quantified and spatially localized by spectrophotometer, fluorometer or histochemical method. Analysis, detection method is simple and sensitive.
(2) Detection of luciferase activity
The luciferase-catalyzed substrate converts the 6-hydroxyquinoline analog to decarboxylate the substrate under the action of magnesium ions, ATP and oxygen to form an activated oxidized fluorescein, which is converted into a normal oxidative fluorescence after emitting photons. Prime.
(3) Green fluorescent protein detection
GFP produces green fluorescent protein that emits unique green fluorescence at wavelengths of 395 nm and 490 nm.
The traditional screening method is to use antibiotics and herbicides, but the effects of these substances on humans and the environment are not fully understood. Therefore, non-antibiotic screening systems have emerged.
The mannose positive selection system belongs to one of the non-antibiotic screening systems. The principle is that mannose cannot maintain the growth of various explants. When culturing plant cells to add mannose, it is only converted to mannose-6-phosphate, which cannot be further involved in metabolism and accumulates to toxic levels. Scientists cloned the gene encoding 6-mannose mannose isomerase as a selection gene, constructed an expression vector for genetically transformed plants, used mannose as a selection agent, and selected the gene-encoded 6-mannose mannose isomerase to extract 6-phosphate mannose. The sugar is converted to fructose 6-phosphate which is prone to glycolysis.
Compared with traditional genetic transformation methods, new screening methods including mannose selection facilitate the regeneration and growth of transgenic tissues, while killing non-transgenic tissues due to hunger, rather than killing them, called positive selection. The traditional antibiotic selection method or herbicide selection method to kill untransformed cells is called negative selection.
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