Talk about genetically modified plants "that matter"
2023-01-07 22:06:10
Imagine that you have to transport a machine from the production plant to the factory that needs it, and let it work smoothly. How many steps do you have to take?
First of all, we must first produce this machine, then pack it, put it on the car and transport it to the destination factory, install the machine, and finally, after a series of debugging, the factory can use this new machine to produce new products.
Similar to this process, the production of genetically modified plants requires a series of steps as described above. In a typical transgenic plant production process, these steps are referred to as gene cloning, vector construction, transformation, recombination, and screening, respectively.
1. Gene cloning.
It is difficult for a woman to be a rice-free product. To produce a genetically modified plant, there must be a gene that needs to be transferred. The sources of these genes are diverse, and the most widely used BT protein genes and glyphosate-tolerant genes are derived from two bacteria. However, Towson and Crick's blessing, we know that all cell organisms share a class of genetic material: DNA. Therefore, genes derived from bacteria can also play a role in plants.
Through sequence alignment and analysis, coupled with today's rich genomic database, we can easily find and determine the sequence of the target gene. Then, using the most commonly used PCR methods, we can clone the gene fragments we need from the original biological genome.
2. Vector construction.
However, it is not enough to clone the DNA of this gene. We have to pack it. Why do you want to pack it? Because everyone knows that DNA molecules are long-lined, but the ends of linear DNA molecules are more susceptible to degradation. Therefore, we have linked this gene of interest to a specific plasmid molecule by enzyme to form a new plasmid molecule inserted into the target fragment. There are several advantages to this. As a result, the plasmid is circular and has no free ends, so it can stabilize linear DNA molecules. Second, the plasmid can be replicated in E. coli with the division of E. coli. A plasmid molecule containing the gene of interest is conveniently obtained in large quantities. Third, the plasmid also has elements that allow the target fragment to enter the plant and express more easily. So why is it easy to get into the plant? Look at the third point:
3. Conversion.
In this step, we have to ask for an important transporter: Agrobacterium. Agrobacterium is a type of soil bacteria. Before it was "good", it often infects plants, causing a large number of cells to invade the site, forming a small sputum, which we call "crown." Scientists have found that Agrobacterium produces a crown of plants because Agrobacterium can insert a piece of DNA it contains into the genome of a plant. This DNA contains enzymes encoding the enzymes required for the synthesis of auxin, as well as compounds that the synthetic bacteria like to "eat". Therefore, scientists have used bioengineering methods to transform the DNA of Agrobacterium, retaining the ability of Agrobacterium to insert its own fragments into plants, but removing the genes for the synthesis of auxin and nutrients, and adding some "loading sites". "It is convenient for us to "load".
This modified DNA is actually the specific plasmid molecule we mentioned above. After the target gene is packaged, it will be transferred to Agrobacterium, and then, with the Agrobacterium containing the packaged target, the plant can be infested.
Because plants have thick cell walls, people generally choose relatively young parts for Agrobacterium infection. For corn, pollen tubes are used to infect, while for soybeans, shoot tip regions are used to infect. . After infestation, the intensive transporter of Agrobacterium transfers the DNA fragments carrying the gene (but not only) to the plant and integrates it into the plant genome to complete the "transportation" and "installation" process.
Because plants have thick cell walls, people generally choose relatively young parts for Agrobacterium infection. For corn, pollen tubes are used to infect, while for soybeans, shoot tip regions are used to infect. . After infestation, the intensive transporter of Agrobacterium transfers the DNA fragments carrying the gene (but not only) to the plant and integrates it into the plant genome to complete the "transportation" and "installation" process.
In addition to Agrobacterium transformation, it can also be transformed by gene gun or electroporation.
4. Screening However, it is not a good thing to let Agrobacterium run for a while. We have to solve a few questions: Does Agrobacterium have shipped the goods? Is the function normal? Sometimes, we want to know more about the part of the "machine" installed on the "line"? This requires screening.
4. Screening However, it is not a good thing to let Agrobacterium run for a while. We have to solve a few questions: Does Agrobacterium have shipped the goods? Is the function normal? Sometimes, we want to know more about the part of the "machine" installed on the "line"? This requires screening.
The test "has sent the goods", which is biologically called transformant screening. Usually, on that particular plasmid, there is another gene, which is to detect whether the "goods" have actually been delivered. When a DNA fragment is inserted into a plant's genome, the gene can produce a signal that tells people that "the goods are in place." This gene is the reporter gene. A commonly used reporter gene is a gene encoding a compound that can break down antibiotics. When this gene enters the plant genome, the plant tissue is no longer afraid of the antibiotics added to the medium, so that the plants that can survive on the medium are all the "plants already in place" plant organization. This method is very convenient, so it is very commonly used in scientific research. Most of the first generation of GM crops also adopt this strategy.
However, antibiotics will cause some people's concerns about the proliferation of antibiotics. Although the resistance of antibiotics to antibiotics is difficult to spread (after all, it encodes a protein, and the DNA encoding it and itself is easily destroyed in the environment and the human digestive tract), but in order to dispel people's worries, scientists also A "non-antibiotic screening system" was developed. For example, the pmi screening system does not use a resistance gene but uses a phosphomannose isomerase gene as a reporter gene. The name of the enzyme, which is very vocal, allows plants to use mannose, which is usually not used in the medium, as a source of carbon, so that plant cells that are "not in place" will remain hungry and the DNA fragments will succeed. The inserted plant tissue is “white and fat†and is easy to distinguish. In addition, the resistance gene can be cut off after some antibiotic screening is completed by some means. The commonly used Cre/Loxp system can accurately excise the resistance gene after screening, and thus can also produce transgenic plants without resistance genes. Of course, with the development of large-scale sequencing technology, we can also use the sequencing gene to directly determine whether the target gene is inserted or not, which is even more worth worrying about (except for small money).
After confirming that the DNA fragments have been inserted into the plant genome, we can organize these plant tissues. Since the plant cells are versatile, we can obtain complete plants carrying the target genes. By definition, these plants can already be counted as “transgenic plantsâ€. However, things have not yet completely ended. These “transgenic plants†also need to pass a test that is very important, but often overlooked by laymen, that is, to determine whether the target gene is normally expressed. Since the nature of the gene of interest is very diverse, the means of detection are also diverse. For example, by thermal asymmetric interlaced PCR, we can determine the location of the target gene inserted into the plant genome, detect target gene expression levels by Real-time PCR and western hybridization, and even analyze plant genome size by transcriptome and metabolomics techniques. Expression and changes in products, etc. However, for a transgenic plant whose production target is commercial planting, the principle is that the trait of the target gene must be produced; at the same time, try not to produce other non-target traits; if non-target traits occur, then it must be none. Harmful. The finished product obtained through the above test is a true transgenic plant.
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