What factors affect expression of desired genes in E. coli? Number of views: 904 times Tags: | Question time: 2009-4-28 17:30 | Asked by: lys2126
What factors will affect the expression of target genes in E. coli?
Recommended answers (1) Copy number of foreign genes
(2) Expression efficiency of foreign genes
①Promoter strength
< p>Effective ②The gender of the ribosome binding site③The SD sequence and the ATG distance between the start codon
④The codons
⑶The stability of the expressed product
(4) Cell metabolic load
⑸Engineering bacteria culture conditions
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Establish a vector to express the target gene Secreted outside plant roots and root cells. Please indicate the number of steps: 842 Reward points: 20 | Solving the problem
Time: 2009-2-14 09:13 | Questioner: 370 629 225
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Create a plant expression vector for a specific foreign gene and transfer it to the recipient plant, without the ultimate goal of genetic transformation of the plant. The ideal transgenic plant usually requires high levels of expression of the foreign gene at a specific location and at a specific time for the desired phenotypic trait. However, in the past two decades of development, the expression efficiency of transgenic plants has often been low, the expression products are unstable, and even gene inactivation or silencing and other undesirable phenomena have prevented the practical application of foreign genes in transgenic plants in recipient plants. In addition, the safety issues of genetically modified plants have attracted the attention of many countries. For example, the pollen transmission of genetically modified plants and antibiotic selection marker genes may make certain antibiotics clinically useless. The emergence of this high-tech plant genetic engineering comes at an unprecedented time plagued by the above issues. In order to solve these problems, in recent years, people have cultivated transgenic technology, explored and improved a wide range of plant expression vectors, and improvement and optimization are one of the most important contents. This article has reviewed the progress made.
Exogenous gene expression level 1 promoter selection and transformation is often an important reason for unsatisfactory transgenic plants. Promoters play a key role in determining gene expression, therefore, selecting a suitable plant promoter to enhance its activity is the first consideration to enhance the expression of exogenous genes.
In this plant expression vector, the widely used promoter is a constitutive promoter. For example, most dicotyledonous transgenic plants use the CaMV35S promoter, and monocotyledonous transgenic plants are mainly used from Ubiquitin promoter from maize, and Actinl promoter from rice. The exogenous gene turns under the control of the expression promoter of these components and is expressed in all parts of the transgenic plant and at all developmental stages. However, the continued and effective expression of foreign genes in the recipient plant not only causes waste, but also often causes morphological changes in the plant, affecting the growth and development of the plant. In order to effectively function in plants, and at the same time to reduce adverse effects on the plant, the research and application of specific expression promoters for foreign genes are receiving increasing attention. It has been found that specific promoters, including organ-specific promoters, induce specific promoters. For example, seed-specific promoters, fruit-specific promoters, mesophyll cell-specific promoters, root-specific promoters, damage-specific promoters, chemically induced specific promoters, light-induced specific promoters, Heat shock induces specific promoters. The cloning of specific promoters lays the foundation for the expression of specific foreign genes in plants. For example, the Swiss CIBA-GEIGY PR-IA promoter controls the expression of Bt toxin protein gene in transgenic tobacco. The promoter induces spraying of salicylic acid and its derivatives, which are cheap, non-polluting chemicals and induce the expression of resistance genes to pests. Happening again this season, it's obviously a very effective approach.
In plant transgenic research, satisfactory results are often not achieved, especially when using natural promoters for specific expression and induced expression, and the expression levels are mostly not ideal. It will be a very important way to transform the existing startup subsystem to build a composite startup.
For example, the promoter of the octopine synthase gene promoter and the mannoline synthase gene promoter in the transcription activation region of Ni, etc., the results of GUS expression show that the modified promoter activity is more significant than that of the 35S promoter improve. Wu Rui et al. used a combination of the rice Actinl intron to manipulate the inducible PI-II gene promoter, and a new expressed promoter activity increased nearly 10 times (patented). In plant genetic engineering research, the initiation of these artificial formations plays an important role.
Constructing vectors to improve the efficiency of translation of foreign genes, usually genetic modification, mainly considers three aspects:
2.1 Improving translation efficiency 5'-?3' - non-translation Sequence
Many experiments have found that the 5'-3'-untranslated sequence (UTR) of eukaryotic genes is very necessary for the normal expression of the gene, and the absence of this region often results in the mRNA The stability and translation level are significantly reduced. For example, upstream of the translation start site of the 126 kDa protein gene of tobacco mosaic virus (TMV) is a 68 bp nucleotide omega element, which provides a new ribosome-binding site to increase the translation activity of the Gus gene. Dozens of times. There are many vectors that add an omega translation enhancer sequence to the 5'-end of the foreign gene. Ingelbrecht studied the 3'-terminal sequences of various genes and found that the 3'-terminal sequence of the octopine synthase gene increased the transient expression of the NPTII gene by more than 20-fold. In addition, the efficiency of gene expression enhancement of different genes is different in the promotion effect on gene expression, for example, the 3'-end sequence of rbcS 3'-end sequence of the 3'-end sequence of the chalcone synthase gene 60 times the sequence.
2.2 Optimize the sequence surrounding the start codon
Although start codons are common in the biosphere, genes from different biological sources have their own special external starts. codon sequence. For example, the typical feature of the sequence around the start codon of plants is AACCAUGC, while the sequence around the start codon of animals is CACCAUG, which is quite different from that of prokaryotes. Kozak studied in detail the effects of ATG-adjacent base-directed mutagenesis on transcription and translation before the start codon, and concluded that around ACCATGG in eukaryotes, the most effective sequence for the transcription and translation start codon, especially It is -3-digit A's entry efficiency is very important. The purchased sequence construct, called the Kozak sequence, is applied to the expression vector. For example, a bacterial chitinase gene, and the sequence surrounding the original start codon UUUAUGG to ACCAUGG, increased expression levels 8-fold in tobacco. Therefore, the use of expression vectors for gene constructs of non-plant origin should be based on the characteristics of the sequence surrounding the plant start codon to be transformed.
2.3 The coding region of the gene is for transformation
If the foreign gene is from a prokaryotic organism, the difference in the expression mechanism of these genes in plants is often very low Expression levels, for example, of the wild-type insecticidal protein gene from Bacillus thuringiensis expressed in plants, are very low, and studies have found that this is due to differences in the stability of prokaryotic and plant gene mRNAs. Monsanto Perlak premised on the genetic transformation of toxic proteins, insecticidal proteins without changing the amino acid sequence, plant-preferred codon usage, GC content, and deletion of elements in the original sequence that affect the stability of the mRNA, resulting in toxic proteins Expression in transgenic plants increased 30- to 100-fold in insects.
3
Eliminating the position effect after removing the foreign gene from the recipient plant, it is often very different expression levels in different transgenic plants. This is primarily a result of the different insertion sites of the foreign gene in the genome of the recipient plant. This is called the "position effect." In order to eliminate position effects, foreign genes are integrated into plant genome expression vectors in the transcriptionally active region. Current construction strategies usually take into account the nuclear matrix binding region and the application of site-directed integration technology.
The nuclear matrix binding region (matrix association region, MAR) is a DNA sequence that binds to specific portions of the nuclear matrix of eukaryotic chromatin.
Generally, MAR sequences are located in the circular structure of transcriptionally active DNA and their function is to enable division, thereby allowing each transcription unit to maintain relative independence from the influence of the surrounding chromatin. This study shows that placing the MAR on both sides of the MAR-containing MAR construct required for genetic transformation into a plant expression vector can significantly increase the expression level of the target gene and reduce differences in the target gene between transgenic plants. The difference in expression levels reduces the location of the effect. For example, Allen et al. studied the expression of heterologous MAR (from yeast) and homologous MAR (from tobacco) Gus genes in tobacco and found that the yeast MAR increased the average level of transgene expression by 12-fold, and the tobacco native MAR increased the expression of the transgene by 12-fold. The average level of transgene expression increased 60-fold. MAR's chicken lysozyme gene can also play the same role.
Another feasible method is to use site-directed integration technology. The main principle of this technology is to transform the chromosome of the vector host, and the foreign gene will be integrated into the homologous site of a specific DNA fragment on the chromosome through homologous recombination. sexual area. In actual operation, when the DNA fragment of the transcriptionally active region of the chromosome is first isolated, a plant expression vector is then constructed. Targeted integration of homologous recombination in microorganisms has become a routine gene manipulation, and has successfully been used to successfully integrate foreign genes in animals and chloroplast expression vectors in plants, and to achieve targeted integration of exogenous genes in plants. There are still very few successful cases of the nuclear transformation method.
4 Construction of chloroplast expression vector
Exogenous nuclear transformation is often used to overcome gene expression due to issues of unsafe sex, such as the position effect of pollen dispersion of nuclear genes , a new low-efficiency genetic transformation technology that has only appeared in recent years - chloroplast transformation has been increasingly recognized and valued for its advantages and development prospects. So far, 5 species of inherited chloroplast transformation have been published in tobacco, rice, Arabidopsis, potato and rapeseed (Hou Bingkai et al.), making this transformation technology begin to become a new growth point in plant genetic engineering.
It has been tested that the homologous recombination mechanism has laid the foundation for the site-specific integration of foreign genes into the chloroplast genome of various plant chloroplast genome sequences. The current construction of chloroplast expression vectors are basically site-specific integration vectors. . Constructing a chloroplast expression vector is basically a point-to-example vector. Generating chloroplast expression vectors generally involves an expression cassette for a foreign gene, with each ligation period flanked by chloroplast DNA sequences, referred to as homologous recombination fragments, or positioning segments (directional fragments). When the vector is introduced into the chloroplast, the two fragments have the same fragment and homologous recombination occurs in the chloroplast genome. It is a specific location that can integrate the foreign gene into the chloroplast genome. The purpose of chloroplast transformation in crop improvement requires that after homologous recombination occurs, the insertion of the foreign gene into the chloroplast genome neither causes loss of the original sequence, nor does it destroy the function of the original gene at the insertion point. In order to meet this requirement, existing work has selected homologous recombination fragments of two adjacent genes, such as rbcL gene/ACCD 4, 16StrnV/rpsl2rps7, psbA gene/variant, rps7/ndhB,. After homologous recombination of the designated foreign gene occurs, it is inserted into the spacer region between two adjacent genes to ensure that the original function of the gene is not affected. Recently, Daniel and others used homologous recombination fragments of tRNA and trnI from the tobacco chloroplast genome to construct a unique vector (universal vector). Since the tRNA and trnI DNA sequences are highly conserved in higher plants, the authors suggest that such vectors can be used for chloroplast transformation in a variety of different plants. If the versatility of this vector is confirmed, then this work undoubtedly provides a good idea for constructing a new, convenient and practical chloroplast expression vector.
The expression of site-specific integration of foreign genes into the chloroplast genome is often due to the high copy number of the chloroplast genome. In the first example, the cryIA (c) toxin gene introduction of BT by McBride et al. The high efficiency of tobacco chloroplast Bt toxin protein expression is as high as 3% to 5% of the total protein in the leaf, whereas a typical nuclear transformation technique can only achieve from 0.001% to 0.6%.
Recently, Kota Kinabalu, who introduced the Bt Cry2Aa2 gene into tobacco chloroplasts, also found that the expression of toxic proteins in tobacco leaves, accounting for 2% to 3% of soluble proteins, is 20 to 30 times higher than that in nuclear-transformed, transgenic tobacco. Not only fights sensitive insects, but also kills those that have a high resistance to insects. Stobbe recently reported that the expression level of human growth hormone gene introduced into tobacco chloroplasts was as high as 7% of leaf protein, more than 300 times higher than that of nuclear transformation method. These experiments fully developed the construction and transformation of chloroplast expression vectors, which is an important way to achieve high-level expression of foreign genes.
5 Applications of Positioning Signals
The main purpose of the above vector optimization strategies is to improve the efficiency of transcription and translation of foreign genes. However, high-level expression of foreign proteins Whether it can be stabilized in plant cells and other important questions need to be taken into account in the accumulation of genetically transformed amounts in plants.
Recent research has found that if a certain foreign gene is connected to the correct positioning of the signal sequence, the foreign protein will be produced and transported to a specific site in the cell, for example: chloroplasts, endothelial cells, Plasma reticulum, vacuoles, etc., can significantly improve the stability of the system and the accumulation of foreign proteins. Is this because of a specific area? The environment within the endoplasmic reticulum of certain exogenous proteins provides a relatively stable environment that effectively prevents the degradation of exogenous proteins. For example, Wong et al. linked the Arabidopsis thaliana Rubisco large subunit transit peptide sequence to a specialized insecticidal protein gene before accumulating in the chloroplasts of transgenic tobacco. The total accumulation of foreign protein increased 10 to 20 times compared with the control group. . Recently, Ye Liang and Song Wang Yanru linked the transit peptide sequence of the rbcS subunit to the pre-PHB synthesis gene in an attempt to enable the accumulation of gene products in the mass of transgenic rapeseed seeds, thereby increasing the exogenous protein content. , Wandert and Stern connected the endoplasmic reticulum localization and found that the content of the foreign protein in the transgenic plants had been significantly improved by the sequence of the foreign protein gene (the coding sequence of the tetrapeptide KDEL). Clearly, the localization signal plays a positive role in promoting protein accumulation, but whether the same localization signal applies to all proteins needs further determination.
The intron-enhanced gene expression of 6 introns was originally discovered in transgenic corn by Callis et al., the first intron of the corn alcohol dehydrogenase gene (ADHL) ( Intron 1) The expression of foreign genes is significantly enhanced, and other intronic genes (such as intron9) also have a certain amount of promotion effect. Later, Vasillev also discovered that the first intron of the corn fructose synthase gene CAT increased the expression level 10-fold. The third intron, the rice actin gene, also increased the expression level of the reporter gene by 2 to 6-fold. The mechanism by which date introns enhance gene expression is unclear, but it is generally believed that the presence of introns may improve the efficiency of mRNA processing and the stability of the mRNA. Tanaka et al., some studies have shown that the enhancement effect of introns on gene expression mainly occurs in monocots and dicots but is not obvious.
McElroy constructed a monocot expression vector due to intron enhancement, specifically the first intron of the rice actin gene that maintains gene expression of genes downstream of the promoter. Similarly, Christensen et al. placed the gene of the first intron of the maize ubiquitin vector downstream of the promoter in the constructed vector to enhance the expression of foreign genes in monocots. However, studies have pointed out that the influence on gene expression depends on many factors such as promoter strength, the cell type, the target gene sequence, etc., depending on the role of the specific intron, and even sometimes depends on the position of the intron in the vector. . For example, the maize ADHL gene 9 is placed at the 5' end of the Gus gene, and the GUS gene is expressed under the control of the CaMV35S promoter without enhancement; when the 3' end of the intron is placed in the intron of the gus gene, Under the control of the same initiator, the expression level of the gus gene increased approximately 3 times. The mechanism of gene expression by introns may be very complex. It can be seen that there is a lack of a fixed model for how introns can establish efficient plant expression vectors and is worthy of further study.
Policies of polygene control
So far, most studies on the genetic transformation of recipient plants are based on the introduction of a single exogenous gene.
But powerful enough, sometimes due to the expression of a single gene or a single mechanism of action has not yet been obtained? Ideal transgenic plants. If two or more genes have a synergistic effect on the plant at the same time, a more preferable result will be obtained than the conversion of a single gene. This strategy has been applied to the breeding of transgenic plants that are disease-resistant, insect-resistant, and stress-tolerant. For example, according to the insect spectrum and different mechanisms of action of insect-resistant genes, two vectors of functionally complementary genes can be constructed, and the two insect-resistant genes can be introduced into plants in a certain way. Wang Wei and other lectin genes and protease inhibitor genes were simultaneously transferred into cotton transformed plants that already contained bivalent insect resistance genes. When the Barton Bt insecticidal protein gene and scorpion toxin gene are introduced into tobacco, the insect resistance and ability are greatly improved to prevent pests from developing resistance (patented). With strong disease resistance, Lanyan's laboratory constructed plant expression vectors containing β-1,3-glucanase bivalent genes and chitinase genes, and introduced them into rapeseed and cotton. The results showed that the transgenic plants produced significant resistance. Recently, Fengdaoantong and Li Baojian even put 2 to 3 fungal disease resistance genes and hpt genes on vectors. Both the insect resistance gene and the bar gene were connected to another vector and introduced into rice using a gene gun. The results showed that 70% The progeny of R contain the introduced foreign genes (6 to 7), and the tendency of one or both of the introduced genomic loci of the multiple foreign genes to be integrated.
In general conventional conversion, it is not possible to introduce foreign DNA fragments exceeding 25KB into plant cells. Functionally related genes, such as quantitative trait loci in plant disease resistance genes, are mainly in the form of gene clusters. If some large fragments of DNA larger than 100KB, such as gene clusters in natural plant chromosomes or a series of non-linked foreign genes are introduced into the same point in the plant genome, it may appear to be controlled by multiple genes. Excellent traits may lead to broad-spectrum insect resistance and disease resistance, and a completely new metabolic pathway can be obtained in the receptor cells to produce new biomolecules. In addition, large fragments of gene clusters or synchronous insertion of gene clusters can also overcome the position effect caused by transgenes to a certain extent and reduce the occurrence of undesirable phenomena such as gene silencing. Recently, Hamilton and Alfred in the United States developed a new generation vector system to clone large DNA fragments and directly transform plants BIBAC and TAC with the help of Agrobacterium tumefaciens. These two operators will not only speed up map-based cloning of genes, but also have potential applications for multi-gene control and variety improvement. Currently, applied research in multigene transformation has just begun. On BIBAC and TAC vectors
8 Use and deletion of selectable marker genes
Cells (or individuals) that can be transformed during genetic transformation of selectable marker genes are screened from a large number of non-transformed cells out marker genes. They can often produce transgenic cells that have resistance to the product of a selection agent, so that the transgenic cells in normal growth media add such selection due to the lack of resistance of non-transgenic cells that exhibit sensitivity to this selection agent. rather than growth, development and differentiation. The vector is constructed to connect the side of the selectable marker gene to the target gene, both of which have their own gene regulatory sequences (such as promoter, terminator, etc.). There are two main categories of selectable marker genes: antibiotic resistance genes and herbicide resistance genes. The former can produce antibiotic resistance, and the latter can produce resistance to herbicides. The antibiotic resistance genes used include the NPTII gene (hygromycin resistance), and the Gent gene that produces hygromycin phosphotransferase, neomycin phosphotransferase, and kanamycin resistance), the HPT gene (that produces ( Herbicide resistance genes, including EPSP genes (produces 5-pyruvate-shikimate-3-phosphate synthase, glyphosate), GOX genes (glyphosate oxidase degrades glyphosate), bar Genes (producing PPT acetyltransferase, anti-Bialaphos or glufosinate)
The above 1, 2, 3, 5, 6, they are all remarkable, especially since you are going into cells. The exocrine backbone vector can be selected as PB I121, and then you can make changes in the above genotype.
The cloning steps behind it are relatively simple.
1. First restriction enzyme digestion. site to obtain the desired gene, plus a good vector for introducing changes.
2. Amplification of plasmid transformed into Escherichia coli DH5α
Amplification of plant cells transformed with good plasmid
4. Collection of extra-root medium to detect protein expression and secretion. Is it
As for the carrier transformation, a few simple steps will suffice. After answering, if we really make a good carrier, we can open our own company.