Plant gene direct transfer method
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Microinjection first frees plant cells out of protoplasts. A special instrument is used to make a small bell mouth that can accommodate a single protoplast, and the other end is connected to a micro tube, which is connected with a weak suction device to form a negative pressure. Then, with the aid of an inverted microscope and a micromanipulator, a single protoplast is sucked in a petri dish in a petri dish, and another microtubule having a diameter of about 0.5 to 1 μm is inserted into the protoplast; A micro-syringe is attached to the other end of the tube, and DNA of the target gene is injected into the recipient cells through the microtubes. This method requires strict operating techniques and must be performed in a special sterile micromanipulation chamber with low injection efficiency, which can cause damage to the recipient cells. Therefore, it has been rarely used, but it is still widely used in animal cell experiments.
Electroporation studies have shown that protoplasts can loosen the molecular structure of the plasma membrane under the impact of short-time high-voltage direct current pulses to form temporary pores with a diameter of 3 to 4 nm, but have little effect on protoplast viability. . At this time, the exogenous DNA present in the solution surrounding the protoplasts can enter the protoplasts through this small pore to achieve direct gene transfer. This method is more efficient and easy to use for gene transfer and has now been adopted by most researchers.
Microbeam laser drilling method This method is similar to electroporation. Protoplasts can form small holes with an area of ​​about 0.25 μm2 on the surface of the plasma membrane under short-time microbeam laser irradiation, allowing DNA molecules to enter cells. However, compared with the electroporation method, the instrument used in this method is complicated and requires high operating technology and is difficult to grasp. Improper handling can cause greater damage to the cells. If the formed pores cannot be recovered, the protoplasm content will cause cell death. At present, only a few qualified units have adopted it.
The particle gun is a specialized high-pressure gun for gene transfer. It can inject metal particles such as tungsten with an average diameter of about 4 μm into cells. If the DNA of the target gene is attached to the surface of metal particles, it can be realized. Direct gene transfer. The advantage of this method is that the metal particles can penetrate the cell wall, so they can be directly manipulated with cells or tissues without first dissociating the protoplasts, providing convenience for gene transfer. For example, according to reports from abroad in 1992, the University of Florida and the Monsanto Company cooperated to introduce the Bar gene, which is an anti-broad spectrum herbicide Basta, into a wheat embryogenic callus to obtain a transgenic wheat. China has also carried out research in this area and has obtained some research results. Recently, a new type of gene gun, which uses high-pressure microbeam liquid injection instead of metal particles injection, has been developed abroad, and has a better effect.
The direct transfer method of genes has expanded the scope of plant gene transfer, especially for cereals that are difficult to cultivate in protoplasts, and this has received much attention. The problem in the direct transfer of plant genes is that the foreign DNA will not be degraded by the nucleases in the cells after entering the cells, and whether it can be integrated into the plant chromosomes and be replicated, transcribed and translated together with the plant cells. In order to solve these problems, some researchers have combined the target genes with the reconstructed Agrobacterium plasmids to form recombinant DNA molecules, and then transferred them to plant cells using a direct transfer method, obtaining better results.
