I’ll post something for you first. I hope it will be helpful to you.
Constructing a specific foreign gene into a plant expression vector and transferring it into the recipient plant is not The ultimate goal of plant genetic transformation. Ideal transgenic plants often require high-level expression of exogenous genes in specific parts and within a specific time to produce the desired phenotypic traits. However, the development history of the past two decades shows that exogenous genes Undesirable phenomena such as low expression efficiency, unstable expression products, and even gene inactivation or silencing often occur in recipient plants, making transgenic plants unable to be put into practical use. In addition, the safety issues of transgenic plants have attracted people's attention in many countries. , for example, transgenes may spread with pollen, antibiotic screening marker genes may make some clinical antibiotics ineffective, etc. The emergence of the above problems has put the high technology of plant genetic engineering in an unprecedented period of trouble. Targeted at To address these issues, people have explored and improved plant transgenic technology in many aspects in recent years. The improvement and optimization of plant expression vectors is one of the most important aspects. This article reviews the progress that has been made.
1 Selection and transformation of promoters
Insufficient expression of exogenous genes is often an important reason why ideal transgenic plants cannot be obtained. Since promoters play a key role in determining gene expression, selection Suitable plant promoters and improving their activity are the first issues to consider when enhancing the expression of exogenous genes.
The promoters currently widely used in plant expression vectors are constitutive promoters, for example, the vast majority Dicotyledonous transgenic plants all use the CaMV35S promoter, and monocotyledonous transgenic plants mainly use the Ubiquitin promoter from corn and the Actinl promoter from rice. Under the control of these constitutive expression promoters, foreign genes are expressed in all parts of the transgenic plants. and will be expressed at all developmental stages. However, the continuous and efficient expression of foreign genes in recipient plants not only causes waste, but also often causes changes in plant morphology and affects the growth and development of plants. In order to make foreign genes express in plants Effectively play a role in the body while reducing adverse effects on plants. At present, people are paying more and more attention to the research and application of specific expression promoters. The specific promoters that have been discovered mainly include organ-specific promoters and induction-specific promoters. For example, seed-specific promoter, fruit-specific promoter, mesophyll cell-specific promoter, root-specific promoter, damage-induced specific promoter, chemical-induced specific promoter, light-induced specific promoter, Heat shock-induced specific promoters, etc. The cloning and application of these specific promoters lay the foundation for the specific expression of foreign genes in plants. For example, the Swiss CIBA-GEIGY company uses the PR-IA promoter to control transgenic tobacco For the expression of Bt toxin protein gene, since the promoter can be induced by salicylic acid and its derivatives, it is obviously a very effective method to induce the expression of insect-resistant genes in the season when insect pests occur again by spraying cheap and non-toxic chemicals. approach.
In plant transgenic research, the use of natural promoters often fails to achieve satisfactory results, especially when performing specific expression and induced expression, the expression levels are mostly not ideal. For existing promoters It will be a very important way to transform the promoter and construct a composite promoter. For example, Ni et al. combined the transcription activation region of the octopine synthase gene promoter and the mannoline synthase gene promoter to form a composite promoter. The GUS expression results Said: The activity of the modified promoter is significantly higher than that of the 35S promoter. Wu Rui and others combined the operation-inducible PI-II gene promoter with the rice Actinl gene intron 1, and the expression activity of the new promoter was improved by nearly 10%. 10 times (patented). In plant genetic engineering research, these artificial promoters play an important role.
2 Enhance translation efficiency
In order to enhance the translation efficiency of foreign genes , when constructing vectors, genes generally need to be modified, mainly considering three aspects:
<p> 2.1 Add 5'-3'-untranslated sequence
Many experiments have found that the 5'-3'-untranslated sequence (UTR) of eukaryotic genes is very necessary for the normal expression of genes. , the deletion of this segment often leads to a significant decrease in the stability and translation level of mRNA. For example, upstream of the translation start site of the 126kDa protein gene of Tobacco Mosaic Virus (TMV), there is an Ω element composed of 68bp nucleotides , this element provides a new binding site for ribosomes, which can increase the translation activity of the Gus gene dozens of times. At present, many vectors have added Ω translation enhancement sequences to the 5'-end of foreign genes. Ingelbrecht et al. The 3'-end sequences of various genes have been studied and found that the 3'-end sequence of the octopine synthase gene can increase the instantaneous expression of the NPTII gene by more than 20 times. In addition, the 3'-end sequences of different genes enhance gene The efficiency of expression is different. For example, the rbcS 3'-end sequence promotes gene expression 60 times more than the 3'-end sequence of the chalcone synthase gene.
2.2 Optimize the start codon surroundings Sequence
Although the start codon is universal in the biological world, genes from different biological sources each have their own special sequence surrounding the start codon. For example, the typical sequence surrounding the plant start codon The characteristic is AACCAUGC, the sequence surrounding the start codon of animals is CACCAUG, and the sequence of prokaryotes is quite different from the two. Kozak has studied in detail the impact of site-directed mutations of the bases surrounding the start codon ATG on transcription and translation, and It is concluded that in eukaryotes, the transcription and translation efficiency is highest when the sequence surrounding the start codon is ACCATGG, especially the A at position -3 is very important for translation efficiency. This sequence is called the Kozak sequence by later generations and is used in In the construction of expression vectors. For example, there is a bacterial chitinase gene. The original start codon surrounding sequence is UUUAUGG. When it is modified to ACCAUGG, its expression level in tobacco increases by 8 times. Therefore, using non- When constructing expression vectors for genes derived from plants, they should be modified according to the characteristics of the sequences surrounding the plant start codon.
2.3 Modify the gene coding region
If the foreign gene is Coming from prokaryotes, due to differences in expression mechanisms, these genes are often expressed at very low levels in plants. For example, the expression level of wild-type insecticidal protein genes from Bacillus thuringiensis in plants is very low. Studies have found that this is due to The difference between prokaryotic genes and plant genes has caused a decrease in the stability of mRNA. Perlak and others from Monsanto Company in the United States modified the insecticidal protein gene without changing the amino acid sequence of the poisonous protein, using plant-preferred codons and increasing GC content, removing elements that affect the stability of mRNA in the original sequence. As a result, the expression of toxic proteins in transgenic plants increased by 30 to 100 times, and obvious anti-insect effects were obtained.
3 Eliminate position effects
When a foreign gene is transplanted into a recipient plant, its expression level in different transgenic plants often varies greatly. This is mainly due to the insertion site of the foreign gene in the genome of the recipient plant. Caused by differences. This is the so-called "position effect". In order to eliminate the position effect and enable foreign genes to be integrated into the transcriptionally active regions of the plant genome, the nuclear matrix binding region and the nuclear matrix binding region are usually considered in current expression vector construction strategies. Application of site-specific integration technology.
The matrix association region (MAR) is a DNA sequence present in the chromatin of eukaryotic cells that specifically binds to the nuclear matrix. It is generally believed that the MAR sequence is located The function of the boundary of the actively transcribed DNA circular structure is to create a partitioning effect so that each transcription unit remains relatively independent from the influence of surrounding chromatin. Relevant studies have shown that placing MAR at the target gene On both sides, a plant expression vector containing the MAR-gene-MAR structure is constructed for genetic transformation, which can significantly improve the target gene.
The expression level can reduce the difference in the expression level of the target gene between different transgenic plants and reduce the position effect. For example, Allen et al. studied the effect of heterologous MAR (from yeast) and homologous MAR (from tobacco) on the expression of Gus gene in tobacco. It was found that the MAR of yeast can increase the expression level of the transgene by an average of 12 times, while the MAR of tobacco itself can increase the expression level of the transgene by an average of 60 times. The use of MAR derived from the chicken lysozyme gene can also have the same effect.
Another feasible approach is to use site-directed integration technology. The main principle of this technology is that when the transformation vector contains DNA fragments homologous to the host chromosome, the foreign gene can be site-directed integrated through homologous recombination. Specific parts of the chromosome. In actual operation, the DNA fragment of the transcriptionally active region of the chromosome must first be isolated, and then the plant expression vector should be constructed. In the genetic manipulation of microorganisms, homologous recombination site-directed integration has become a routine technology. Site-directed integration has been successful, but in plants, except for chloroplast expression vectors that can achieve site-directed integration, there are few successful reports in nuclear transformation.
4 Constructing chloroplast expression vectors
In order to overcome the problems of low expression efficiency of exogenous genes, position effect and unsafety caused by the diffusion of nuclear genes with pollen that often occur in nuclear transformation, an emerging genetic transformation technology has emerged in recent years - chloroplast transformation , is increasingly being recognized and valued for its superiority and development prospects. So far, it has been achieved in five species of plants: tobacco, rice, Arabidopsis thaliana, potato and rapeseed (published by Hou Bingkai et al., et al.) Chloroplast transformation has made this transformation technology a new growth point in plant genetic engineering.
Since the complete chloroplast genome sequences of many plants have been determined, this provides the opportunity for foreign genes to be transformed through homologous recombination. Mechanistic site-specific integration into the chloroplast genome has laid the foundation. The chloroplast expression vectors constructed so far are basically site-specific integration vectors. The constructed chloroplast expression vectors are basically site-specific instance vectors. When constructing chloroplast expression vectors, they are generally based on exogenous gene expression cassettes. A segment of chloroplast DNA sequence is connected to both sides, which is called a homologous recombination fragment or targeting fragment. When the vector is introduced into the chloroplast, homologous recombination occurs between these two fragments and the same fragment on the chloroplast genome. Integrate exogenous genes into specific sites of the chloroplast genome. In chloroplast transformation for the purpose of crop improvement, it is required that after homologous recombination occurs, the insertion of exogenous genes will neither cause the loss of the original sequence of the chloroplast genes nor destroy it. The function of the original gene at the insertion point. To meet this requirement, existing work has selected two adjacent genes as homologous recombination fragments, such as rbcL/accD, 16StrnV/rpsl2rps7, psbA/trnK, rps7/ndhB .When homologous recombination occurs, the foreign gene is inserted into the spacer region between two adjacent genes to ensure that the function of the original gene is not affected. Recently, Daniel et al. used tobacco chloroplast genes trnA and trnI as homologous recombination fragments , constructed a universal vector. Since the DNA sequences of trnA and trnI are highly conserved in higher plants, the author believes that this vector can be used for chloroplast transformation in a variety of different plants. If this vector is universal, If confirmed, this work undoubtedly provides a good idea for constructing a convenient and practical new chloroplast expression vector.
Due to the high copy nature of the chloroplast genome, foreign genes that are integrated into the chloroplast genome at specific sites are often High-efficiency expression will be obtained. For example, McBride et al. transferred the Bt CryIA(c) toxin gene into tobacco chloroplasts for the first time. The expression level of Bt toxin protein is as high as 3 to 5% of the total leaf protein, while the usual nuclear transformation technology can only achieve 0.001 to 0.001% of the total leaf protein. 0.6. Recently, Kota et al. transferred the Bt Cry2Aa2 protein gene into tobacco and into tobacco chloroplasts. They also found that the expression level of the toxic protein in tobacco leaves is very high, accounting for 2 to 3% of the soluble protein, which is higher than that in cells.
Nuclear transformation is 20 to 30 times higher. Transgenic tobacco can not only resist sensitive insects, but also kill 100% of those insects that have developed high resistance. Staub et al. recently reported that the human growth hormone gene was transferred into tobacco chloroplasts, and its expression The amount is as high as 7% of the total leaf protein, which is 300 times higher than that of nuclear transformation. These experiments fully demonstrate that the construction and transformation of chloroplast expression vectors is one of the important ways to achieve efficient expression of foreign genes.
5 Application of positioning signals
The main purpose of the above vector optimization strategies is to improve the transcription and translation efficiency of foreign genes. However, whether high-level expression of foreign proteins can stably exist and accumulate in plant cells The amount is another important issue that needs to be considered in plant genetic transformation.
Research in recent years has found that if certain exogenous genes are connected to appropriate positioning signal sequences, the exogenous protein will be produced. Directed transport to specific parts of the cell, such as chloroplasts, endoplasmic reticulum, vacuoles, etc., can significantly improve the stability and accumulation of exogenous proteins. This is because specific areas such as the endoplasmic reticulum provide certain exogenous proteins with Create a relatively stable internal environment, effectively preventing the degradation of foreign proteins. For example, Wong et al. connected the transit peptide sequence of the Arabidopsis rbcS subunit before the insecticidal protein gene and found that the insecticidal protein can specifically accumulate in In the chloroplasts of transgenic tobacco, the total accumulation of foreign proteins increased 10 to 20 times compared with the control. Recently, Ye Liang, Song Yanru, and others also connected the transit peptide sequence of the rbcS subunit before the PHB synthesis-related genes in an attempt to express the gene. The product accumulates in the plastids of transgenic rapeseed seeds, thereby increasing the content of exogenous proteins. In addition, Wandelt et al. and Schouten et al. connected the endoplasmic reticulum positioning sequence (coding sequence for the tetrapeptide KDEL) to the exogenous protein gene and found that exogenous protein The protein content in transgenic plants has been significantly increased. Obviously, localization signals have a positive effect in promoting protein accumulation, but whether the same localization signal is applicable to all proteins remains to be determined.
Within 6 The application of introns in enhancing gene expression
The effect of introns in enhancing gene expression was first discovered by Callis et al. in transgenic corn. The first intron of the corn alcohol dehydrogenase gene (Adhl) The intron (intron 1) has a significant enhancement effect on the expression of foreign genes, and other introns of the gene (such as intron8, intron9) also have a certain enhancement effect. Later, Vasil et al. also discovered the first fructose synthase gene in corn. Introns can increase the expression level of CAT by 10 times. The third intron of the rice actin gene can also increase the expression level of the reporter gene by 2 to 6 times. So far, the mechanism by which introns enhance gene expression is unclear. It is clear, but it is generally believed that the presence of introns may enhance the processing efficiency and mRNA stability of mRNA. Multiple studies by Tanaka et al. have shown that the enhancement effect of introns on gene expression mainly occurs in monocots and in gemini. It is not obvious in leaf plants.
Because introns can enhance gene expression, Mcelroy et al. deliberately added the first intron of the rice actin gene when constructing a monocot expression vector. Retained downstream of the promoter of the gene. Similarly, Christensen et al. placed the first intron of the maize Ubiquitin gene downstream of the promoter when constructing the vector to enhance the expression of foreign genes in monocots. However, there are Studies have pointed out that the promotion effect of a specific intron on gene expression depends on various factors such as promoter strength, cell type, target gene sequence, etc., and sometimes even depends on the position of the intron on the vector. For example, the maize Adhl gene Intron 9 is placed at the 5' end of the Gus gene. Under the control of the CaMV35S promoter, the expression of the Gus gene is not enhanced. When the intron 9 is placed at the 3' end of the Gus gene, under the control of the same promoter, the expression of the Gus gene is not enhanced. However, the expression level increased by about 3 times. It can be seen that the mechanism of introns on gene expression may be very complicated. How to use introns to construct efficient plant expression vectors currently lacks a fixed model.
It is worthy of further discussion.
7 Multi-gene strategy
So far, most genetic transformation studies have transferred a single exogenous gene into the recipient plant. However, sometimes due to the expression of a single gene If the strength is not enough or the mechanism of action is single, ideal transgenic plants cannot yet be obtained. If two or more synergistic genes are transferred into plants at the same time, more ideal results will be obtained than single-gene transformation. This The strategy has been applied in cultivating disease-resistant, insect-resistant and other stress-resistant transgenic plants. For example, according to the different insect-resistant spectrum and action mechanism of the insect-resistant gene, two genes with complementary functions can be selected for vector construction, and through certain method to transfer two insect-resistant genes into one plant at the same time. Wang Wei et al. transferred the lectin gene and the protease inhibitor gene into cotton at the same time, and obtained transformed plants containing bivalent insect-resistant genes. Barton et al. transformed Bt The insecticidal protein gene and the scorpion toxin gene were simultaneously transferred into tobacco, which greatly improved its insect resistance and ability to prevent pests from developing resistance (patented). In terms of disease resistance, Lan Haiyan and others in our laboratory constructed a gene containing β-1, Bivalent plant expression vectors of 3-glucanase gene and chitinase gene were introduced into rapeseed and cotton. The results showed that the transgenic plants had obvious disease resistance. Recently, Feng Daorong, Li Baojian and others will 2 to 3 antifungal disease genes and the hpt gene were connected to one vector, and two insect resistance genes were connected to the bar gene on another vector. They were simultaneously introduced into the rice plants using a gene gun. The results showed that 70 The R. generation plants contain all the introduced foreign genes (6 to 7), and the introduced multiple foreign genes tend to be integrated at one or two sites in the genome.
Generally conventional Transformation cannot yet introduce exogenous DNA fragments larger than 25kb into plant cells. However, some functionally related genes, such as quantitative trait genes and disease resistance genes in plants, mostly exist in the form of "gene clusters". If some genes are Large fragments of DNA larger than 100kb, such as naturally occurring gene clusters in plant chromosomes or a series of non-linked exogenous genes introduced into the same site of the plant genome, may result in the emergence of excellent traits controlled by multiple genes or the production of a wide range of It can also give recipient cells a new metabolic pathway and produce new biomolecules with a wide range of insect resistance, disease resistance, etc. Not only that, the simultaneous insertion of large fragments of gene groups or gene clusters can also overcome the problem to a certain extent. The position effect caused by transgenes can reduce the occurrence of undesirable phenomena such as gene silencing. Recently, Hamilton in the United States and Liu Yaoguang in China have respectively developed a new generation of vector systems, which have the ability to clone large fragments of DNA and directly transfer it with the help of Agrobacterium. BIBAC and TAC for transforming plants. These two vectors can not only accelerate map-based cloning of genes, but also have potential application value for cultivar improvement that achieves multi-gene control. At present, there is no research on the use of BIBAC and TAC vectors in multi-gene transformation. Applied research has just begun.
8 Utilization and deletion of screening marker genes
Screening marker genes refer to genes that can make transformed cells (or individuals) distinguish themselves from numerous non-transformed cells during genetic transformation. Marker genes screened in cells. They can usually make transgenic cells produce products that are resistant to a certain selection agent, so that transgenic cells can grow normally on the medium added with this selection, while non-transgenic cells lack resistance. It shows sensitivity to this selective agent and cannot grow, develop and differentiate. When constructing the vector, the screening marker gene is connected next to the target gene, and each has its own gene regulatory sequence (such as promoter, terminator, etc.) . Currently, there are two main categories of commonly used screening marker genes: antibiotic resistance enzyme genes and herbicide resistance enzyme genes. The former can produce resistance to a certain antibiotic, and the latter can produce resistance to herbicides. They are the most commonly used. The antibiotic resistance enzyme genes include NPTII gene (produces neomycin phosphotransferase, resistance to kanamycin), HPT gene (produces hygromycin phosphotransferase, resistance to hygromycin), and Gent gene (produces resistance to gentamicin ), etc. Commonly used herbicide resistance genes include EPSP genes (produces 5-enolpyruvylshikimate-3-phosphate synthase, resistance to glyphosate), GOX genes (produces glyphosate oxidase, reduces
glyphosate), bar gene (produces PPT acetyltransferase, resists Bialaphos or glufosinate), etc.
Among the above, 1, 2, 3, 5, and 6 are worthy of note, especially 5 , because you want to secrete it outside the cell. You can choose PB I121 as the skeleton vector, and then you can change the genotype on it.
The next step is the cloning step, which is relatively simple.
1 First, obtain the target gene plus enzyme cutting site and connect it to the modified vector.
2 Transfer the plasmid to E. coli DH5a for amplification
3 Transfer the amplified plasmid to Enter the plant cells for expression
4. Collect the root extracellular culture medium to detect whether there is expression and secretion of the protein.
If you want to answer the question, please briefly explain the steps of transforming the vector. That’s fine. After all, if you really make a good carrier, you can start your own company.