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In plant transgenic operations, in addition to the use of antibiotic resistance and herbicide resistance and other selection genes to exclude non-transformed cells to retain transformed cells, and the use of reporter genes such as Gus and GFP to show the success of transgenes, more importantly, the identification of positive at the molecular level The transformant clarifies the copy number and transcription and expression of the gene of interest in the transgenic plant. This article gives an overview of the commonly used methods for detection and identification of transgenic plants, and briefly introduces the new methods developed in the near future.
1.1.1 Conventional PCR
Because the amount of DNA required for PCR detection is small, the purity requirement is not high, no isotope is needed, the experiment is safe, the operation is simple, the detection is sensitive, the efficiency is high, and the cost is low, which makes it an indispensable method for transgenic detection today, and is widely used. application. However, PCR detection is prone to false positive results. Unreasonable primer design, cross-contamination of target sequences or amplification products, rearrangement and variation after exogenous DNA insertion will cause detection errors. Therefore, the results of conventional PCR are usually only used as the basis for the primary selection of transgenic plants. It is necessary to optimize the PCR technology and further verify the plants that are positive by PCR (real-time quantitative PCR).
1.1.2.1 Multiplex PCR
1.1.2.2 Falling PCR
1.1.2.3 rpPCR
1.1.2.4 Reverse PCR
1.1.2.5 Real-time quantitative PCR
1.2 Southern hybridization
2 Detection and identification of foreign genes in transformed plants
2.1 Northern hybridization
2.2 RT-PCR
Samia Djennane et al. introduced the tobacco nitrate reductase gene Nia2 into potato by Agrobacterium tumefaciens. After RT-PCR analysis, Nia2 gene was expressed in transgenic potato.
Since RT-PCR is performed at the level of total RNA or mRNA, RNA degradation and DNA contamination must be noted during the assay, and strict controls should be placed to prevent false results.
3 Detection and identification of exogenous gene expression in transgenic plants
3.1 ELISA test
The use of ELISA to detect foreign gene expression proteins is convenient, sensitive, specific, high degree of commercialization of reagents, low cost, wide application range, and easy to read test results. However, there are also problems such as excessive background and lack of standardization.
3.2 western hybridization
4 other techniques for detection of transgenic plants
4.1 Gene chip technology
Compared with conventional technologies, biochip technology is characterized by high parallelism, diversity, miniaturization and automation.
4.2 Test strip technology
4.3 In situ hybridization
4.4 Other methods
In summary, there are many methods for detecting transgenic plants. PCR can detect whether the target gene is integrated on the chromosome of the recipient cell, but the PCR detection is sensitive, susceptible to DNA contamination, and it is difficult to detect multiple sites. The most reliable method for detecting the integration of foreign genes on plant chromosomes is Southern hybridization and in situ hybridization. Southern hybridization can detect the copy number and insertion mode of foreign gene insertion. It is a more accurate analysis and is the authoritative method for identifying foreign genes in transgenic plants. In situ hybridization is a place to detect the presence of foreign genes. Integrate the chromosome of the foreign gene and the location of the foreign gene on the chromosome. At the transcriptional level of the foreign gene, Northern hybridization and RT-PCR can be used for detection. Northern hybridization is an important method to study the expression of foreign genes in transgenic plants, but it is more cumbersome, and RT-PCR is simpler and more sensitive than operation, especially in single copy. If the foreign gene encodes a protein, the expression of the protein in the transgenic plant can be detected by ELISA and Western hybridization. Western blotting was used to detect the expression of foreign genes, and ELISA was used for quantitative detection. The two were often used in combination.
From the detection technology of transgenic plants, new technologies are constantly emerging, and various technologies are combined with each other to complement each other and develop in the direction of high efficiency, convenience, safety and automation.
Molecular detection and identification methods for transgenic plants
With the continuous development of molecular biology and plant genetic engineering, more and more breeders have begun to use transgenic technology to obtain new germplasm and new varieties that are difficult to obtain by conventional breeding techniques. The biggest advantage of plant transgenic technology is that it can break the original reproductive isolation between natural species, promote the exchange of genes among different species, greatly enrich the types of variation, increase genetic diversity, and provide abundant breeding resources for the cultivation of new varieties of plants. . Through the study of gene function, the target gene can be screened, and the directional improvement of plant traits can be achieved. Therefore, since the first acquisition of genetically modified plants in 1983, the technology 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 successfully commercialized in more than 20 countries including the United States, Canada and China. produce. Genetically modified crop varieties are increasingly showing great potential in agricultural production.
1 Integration of foreign genes and identification of their integrated copy number
1.1 PCR (polymerase chain reaction) detection
The rapid amplification of the target fragment by PCR is actually an enzymatic reaction using DNA polymerase in the presence of template DNA, primers and four deoxyribonucleotides, through three temperature-dependent steps (ie, denaturation). , annealing and extension) repeated cycles of completion. The specificity of the fragment of interest obtained by PCR amplification depends on the specificity of binding between the primer and the template DNA. A pair of primers are designed according to the sequence of the foreign gene, and the fragment of the foreign gene in the transformed plant genome can be specifically amplified by the PCR reaction, and the non-transformed plant is not amplified, thereby screening the plants which may be transformed.
1.1.2 Optimized PCR
The optimization of PCR technology aims to improve the specificity of the amplification product, to speculate on the copy number and integration of the target gene, thereby improving the detection efficiency. Commonly used are multiplex PCR (MPCR), dropdown PCR (TD-PCR), rpPCR, inverse PCR (inverse PCR, IPCR), real-time quantitative PCR and the like.
MPCR is a method in which PCR amplification is performed using multiple sets of different regions for multiple DNA templates or the same template in the same tube PCR reaction system. Compared with the common PCR method, the MPCR reaction is faster and more economical, and only one PCR reaction can detect multiple target genes. Since the MPCR technique is to add multiple pairs of primers to the same reaction tube and simultaneously detect multiple target sites, the requirements for the primers are high, and the mutual interference between the different primers should be minimized; Can not be too close, otherwise it is difficult to separate when gel electrophoresis, can not be distinguished.
TD-PCR is a PCR protocol that achieves optimal amplification of a gene of interest through a series of reaction cycles in which a series of annealing temperatures are gradually reduced in a reaction tube or a few reaction tubes. It compensates for the shortcomings caused by the reaction system and the imperfect cycle parameters through the system's own compensatory function. This strategy ensures that the originally formed primer template hybrid has the strongest specificity. Although the annealing temperature used in the last cycle will fall to a non-specific Tm value, the amplification product at this time has begun to geometrically expand, surpassing any non-specific PCR product in the remaining cycles, so that the PCR product remains Specific amplification is presented. RHDon et al believe that the first few cycles in the PCR process are very important for the purity of the amplified product, so the higher annealing temperature of the first few cycles will increase the specificity of primer-template binding, and the TD-PCR method can prevent non-specificity. Formation of the product. Since the strategy of TD-PCR is to avoid low Tm pairing in earlier cycles, hot start techniques must be employed in TD-PCR.
Because of the frequent rearrangement of foreign genes into the target genome, Southern hybridization does not clearly analyze the copy number and integration of the transgene. Kumar and Fladung invented the rpPCR method when studying the foreign gene integration behavior of transgenic poplars. This method uses different primers for pairing, genomic DNA amplification, and based on the amplification of different primer pairs and the size of the product to estimate the copy number, integration and copy integrity of the T-DNA. Compared with Southern hybridization, the method has the advantage of being able to more quickly and easily indicate whether the repeating unit is complete and can indicate the direction of the repeat. The downside of this approach is that it does not show an effect when multiple copies are integrated on different chromosomes.
IPCR is identical to ordinary PCR in that it has a DNA fragment of known sequence, and the primers are each complementary to the ends of the known fragment. The difference is that for the known fragment, the 3'-ends of the two primers of the common PCR are relative, and the IPCRs are opposite to each other. Thus, IPCR can amplify an unknown sequence flanking a known sequence fragment. According to this feature, the number of copies of the foreign gene integrated in the plant genome can be analyzed. When multi-copy and multi-site integration is performed, the amplified product exhibits multiple bands on the electropherogram, and only one band is obtained in single copy.
However, this technique requires a DNA template complexity of less than 109 bp, and if it is higher than this value, the desired effect cannot be obtained. In addition, the efficiency of self-joining (cyclization) is also a factor limiting the success of this technology.
Real-time quantitative PCR is a method in which a fluorescent group is added to a PCR reaction system, and the entire PCR process is monitored in real time by using fluorescence signal accumulation, and finally the unknown template is quantitatively analyzed by a standard curve. Its characteristics are: good specificity, real-time quantitative PCR technology to identify the template by specific hybridization of primers or probes, with high accuracy, low false positive; high sensitivity, using sensitive fluorescence detection system for fluorescent signals Real-time monitoring; linear relationship is good, because the intensity of the fluorescent signal is linear with the logarithm of the template amplification product, the initial template concentration is quantified by the detection of the fluorescent signal, the error is small; the operation is simple, the automation is high, real-time Quantitative PCR technology for the amplification and detection of PCR products in the case of closed tube is completed in the next step, no need to open the lid, cross-contamination and less chance of polluting the environment; no post-treatment, no hybridization, electrophoresis, photographing.
Southern hybridization utilizes the specific hybridization of labeled DNA and RNA probes with target DNA to analyze the integration of foreign genes on plant chromosomes (such as copy number, insertion method) and the stability of foreign genes in transgenic offspring. . Southern hybridization can eliminate the DNA contamination during the operation and eliminate the false positive signal caused by the residual plasmid in the transformation. It has high accuracy and specificity, and is the most reliable method for studying the integration of foreign genes in transgenic plants. It has been widely used in the detection of transgenic plants of various crops such as rice, wheat, corn, soybean, rapeseed and peach. However, the method is complicated, high in cost, and requires high experimental technical conditions, which limits its use.
The transcription level of the foreign gene in the transformed plant can be analyzed by hybridizing the total RNA and mRNA of the cell with the probe, which is called Northern hybridization, which is an important means to study the expression and regulation of the foreign gene in the transgenic plant. The Northern hybridization procedure is generally divided into three parts: preparative blotting and hybridization of extraction probes for total RNA of plant cells. Northern hybridization is closer to the performance of the target trait than Southern hybridization, so it is more practical. However, the sensitivity of Northern hybridization is limited, and the detection rate of low-abundance mRNA in cells is low. Therefore, in practice, the transcription level of foreign genes is detected by RT-PCR (reverse transcription PCR).
The principle of RT-PCR is to synthesize cDNA from the mRNA of the plant to be tested under the action of reverse transcriptase, and then use the cDNA as a template to amplify specific DNA. Therefore, RT-PCR can detect whether the gene of interest is expressed at the mRNA level. RT-PCR is very sensitive and can detect low-abundance mRNA. Especially when the foreign gene is integrated in a single copy, the mRNA is detected by RT-PCR.
Although the expression of foreign genes can be studied to a certain extent at the mRNA level, mRNA is specifically degraded in the cytoplasm, and the correlation between mRNA and expressed protein is not high (correlation coefficient is less than 0.5), gene expression Studies of intermediate product mRNA levels are not a substitute for studies of the final expression products of genes. The product of the expression of the foreign gene of the transgenic plant is generally a protein, and the ability of the foreign gene encoding protein to be normally expressed in the transgenic plant and exhibiting the proper function is the ultimate goal of plant gene transformation. The detection of foreign gene expression proteins mainly utilizes the principle of immunology, and ELISA and Western hybridization are classical methods for detecting proteins expressed by foreign genes.
ELISA is the abbreviation of enzyme-linked immunosorbent assays. The basis is the assimilation of antigens or antibodies and the enzymatic labeling of antigens or antibodies. The high specificity, sensitivity and efficient catalysis of antigen-antibody reactions. The characteristics are organically combined for the purpose of qualitative or quantitative determination. The ELISA has the direct method, the indirect method and the double-anti-sandwich method. The most commonly used method is the double-anti-sandwich method, which has the highest sensitivity. The general ELISA is a qualitative test, but if a standard curve of the known concentration of the transgenic component and the absorbance value is made, the content of the transgenic component of the sample can also be determined to achieve a semi-quantitative determination. The method has been applied to the detection of various transformed plants such as cotton, pepper, rice, tobacco, and tomato.
Western hybridization is a protein detection technology that combines protein electrophoresis, imprinting and immunoassay. The principle is to immobilize the target protein separated by polyacrylamide gel electrophoresis (SDS-PAGE) in situ on a solid phase membrane (such as nitrocellulose). Membrane), the membrane is incubated in a high concentration protein solution to block non-specific sites, and then hybridized with a specific antibody (primary antibody) on the blot, and then added to the primary antibody. The bound labeled secondary antibody is finally detected by the nature of the labeled compound on the secondary antibody. Based on the detection results, it is known whether the target protein is expressed, the concentration, and the approximate molecular weight. This method is highly specific and can be used for qualitative testing.
Since Western hybridization detects the expression of the target gene at the translation level, it can directly express the influence of the introduction of the target gene on the plant, and to some extent reflects the success or failure of the transgene, so it has very important significance and is widely used. The method has been applied to the expression of tobacco, artemisia, sorghum, poplar and other related genes. The disadvantage of Western hybridization is that the operation is cumbersome and costly, and it is not suitable for batch testing.
Biochip technology originated from the hybridization of nucleic acid molecules. It was proposed in the 1980s and developed rapidly in the early 1990s. Biochip refers to bioinformatics molecules (such as oligonucleotides) that are immobilized on a solid support medium at high density. A microarray of gene fragments, cDNA fragments or polypeptides, proteins). Biochips can be divided into gene chips and protein chips. Both types of chips can be used for the detection and identification of transgenic plants, but the current application potential is the cDNA chip for the regulation of the expression of foreign genes in transgenic plants. The cDNA chip is capable of detecting any small difference in expression of the plant genome caused by integration of foreign genes and different integration of foreign genes. The mRNAs of different tested samples were labeled with different fluorescent substances, and the same amount of probes were mixed with the same array to obtain the difference of the expression intensity of the foreign genes, thereby realizing the comparative study of the regulation of the expression of the foreign genes. The current universal reporter gene, selectable marker gene, target gene, promoter and terminator specific fragment are immobilized on a slide to prepare a detection chip, and hybridized with the DNA extracted, amplified and labeled from the plant to be tested, and the hybridization signal is After the scanner scans, it can be analyzed and judged by computer software to effectively screen the transformed plants.
At present, due to the high cost, the promotion and application of this technology has been limited. At the same time, some false positive backgrounds have limited its application. It is believed that with the continuous development of life technology and the further development and utilization of computer processing software, biochips will surely become more and more applications.
Similar to the ELISA principle, the difference is that the nitrocellulose membrane replaces the polystyrene reaction plate as a solid phase carrier. The specific antibody is first adsorbed on the membrane, and the membrane is placed in a solution mixed with the sample. The protein diffuses with the liquid phase, encounters the antibody, reacts with the antigen-antibody, screens the positive result through the negative control, and gives the genetically modified component. The approximate range of content. The test strip method is a quick and simple qualitative detection method. The test strip is placed in the sample extract to be tested, and the test result can be obtained in 5-10 min. The test process does not require special instruments and skilled skills, and the economy is economical. Convenient, especially suitable for field and on-site inspection. However, test strip detection can only detect a specific single target protein.
In addition, recent literature reports on the new development of test strip detection technology, which can use the test strip technology to specifically detect a nucleic acid sequence in a sample, and realize the multiple nucleic acid sequences on the same test strip. Simultaneous detection. This undoubtedly expands the scope of application of this technology and contributes to the improvement of detection efficiency.
In situ hybridization is to determine the original position of the sample in the sample by hybridization. It is the main method for localization of the foreign gene on the chromosome and the expression of the foreign gene in the tissue. In situ hybridization of chromosomal DNA can be used to determine the integration of foreign genes on the chromosome, which is important for studying the genetic characteristics of foreign genes. Many experiments have shown that positional effects are important factors influencing the stability and expression of foreign genes. In situ hybridization of mRNA can visually observe the expression of exogenous mRNA and the difference in expression at different developmental stages. The tissue immunolocalization of foreign gene expression proteins can be used to determine the distribution of expressed proteins in transgenic plant tissues and cells, and become an important means to study the function of foreign genes and the stability of foreign proteins in transgenic plants. APSantos et al. reported the use of in situ hybridization to visualize the exogenous genes (including single-copy genes) and their transcripts of different tissues and species during the interphase, compared with genes in the mid-cell division. The expression of the source gene mainly occurs in the interphase of chromosome division, so it is more intuitive and more meaningful, which helps to accurately predict whether the foreign gene is expressed or not, and is expected to reduce the detection time of the foreign gene.
In recent years, some new methods for detecting foreign genes have been developed, such as mass spectrometry, chromatographic analysis, biosensors, near-infrared spectroscopy, and microfabricated devices, which are used in the detection of transgenic plants.