What factors will affect the expression of the target gene in E.coli?
Recommended answer (1) Copy number of foreign gene
(2) Expression efficiency of foreign genes
① promoter strength
Effective ② Sex of ribosome binding site
③ Distance between SD sequence and start codon ATG
④ Codon
⑶ Expressed the stability of the product.
(4) Cell metabolic load
5] Cultivation conditions of engineering bacteria
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The vector was established, and the target gene was expressed and secreted outside the roots and root cells of plants. Please indicate the number of steps: 842 reward points: 20 | solve the problem.
Time: February 2009-14 09: 13 | Questioner: 370 629 225
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The ultimate goal of plant genetic transformation is not to establish plant expression vectors of specific foreign genes and transfer them to recipient plants. Ideal transgenic plants usually need high-level expression of exogenous genes in a specific place and at a specific time, and people expect phenotypic traits. However, in the development process of nearly twenty years, the expression efficiency is often low, the expression products are unstable, and even the genes are inactivated or silenced, which makes the exogenous genes of transgenic plants unable to be put into practical application in recipient plants. In addition, the safety of transgenic plants has attracted the attention of many countries, such as pollen transmission of transgenic plants, and antibiotic selection marker genes may make some antibiotics useless in clinic. The emergence of this high-tech plant genetic engineering is puzzling the above problems in an unprecedented period. In order to solve these problems, in recent years, people have planted transgenic technology, and the exploration and improvement, improvement and optimization of extensive plant expression vectors is one of the most important contents. The research progress of this paper is summarized.
The selection and transformation of 1 promoter with foreign gene expression level is often an important reason why transgenic plants are not ideal. Promoters play a key role in gene expression. Therefore, selecting the appropriate plant promoter to improve its activity is the first consideration to improve the expression of foreign genes.
In this plant expression vector, the widely used promoters are constitutive promoters, such as CaMV35S promoter used by most dicotyledonous plants, ubiquitin promoter used by monocotyledonous plants and Actinl promoter used by rice. Exogenous genes are under the control of promoters, expressed in these elements, and expressed in all parts and all development stages of transgenic plants. However, the expression of foreign genes in recipient plants is persistent and effective, which not only causes waste, but also often causes morphological changes of plants and affects their growth and development. In order to effectively play the role of plants and reduce the adverse effects of plants, more and more attention has been paid to the research and application of exogenous genes that specifically express promoters. 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 are destroyed, light-induced specific promoters of chemical-induced specific promoters, heat-shock-induced specific promoters, etc. The cloning of specific promoters laid a foundation for the expression of foreign genes in plants. For example, the Swiss CIBA- GEP PR-IA promoter controls the expression of Bt toxin gene in transgenic tobacco, and the promoter salicylic acid and its derivatives induce alcohol spraying, which is cheap and pollution-free, and it is obviously a very effective method to induce pests with resistant gene expression to occur again this season.
In plant transgenic research, satisfactory results are often not obtained, especially in the aspects of specific expression and induced expression using natural promoters, and the expression level is mostly unsatisfactory. It will be a very important way to transform existing promoters and construct composite promoters. For example, the promoter of octopine synthase gene and mannopine synthase gene in the transcription activation region of Ni. The results of GUS expression showed that the activity of the modified promoter was significantly higher than that of the 35S promoter. Ray Wu et al. increased the activity of the newly expressed promoter by nearly 10 times by manipulating the rice Actinl intron combination of the inducible PI-II gene promoter (patent). In the research of plant genetic engineering, these artificial syntheses play an important role.
Constructing vectors to improve the translation efficiency of foreign genes, usually gene modification, mainly considers three aspects:
2. 1 Improve the translation efficiency of 5'-3'- untranslated sequences
Many experiments have found that the normal expression of 5'-3'- untranslated sequence (UTR) of eukaryotic genes is very necessary, and the stability and translation level of mRNA in this region are often significantly reduced. For example, the upstream of the translation initiation site of tobacco mosaic virus (TMV) 126 kda protein gene consists of a 68bp nucleotide ω element, which provides a new ribosome binding site and improves the translation activity of Gus gene by dozens of times. There are many vectors with ω translation enhancer sequences at the 5'- end of foreign genes. Ingelbrecht described the 3'- terminal sequences of various genes that have been studied, and found that the 3'- terminal sequence of octopine synthase gene increased the instantaneous expression of NPTII gene by more than 20 times. In addition, the gene expression enhancement efficiency of different genes is different in promoting gene expression. For example, the 3'- terminal sequence of RBCs is more than 60 times that of 3'- terminal chalcone synthase gene.
2.2 Optimize the sequence around the start codon
Although the initiation codon is common in the biosphere, genes from different biological sources have their own special external initiation codon sequences. For example, the typical feature of the sequence around the plant initiation codon is AACCAUGC, and the sequence around the animal initiation codon CACCAUG is very different from that in prokaryotes. Cosac made a detailed study on the function of adjacent site-directed mutation of ATG and the transcription and translation of the initial codon, and concluded that the transcription and translation of the most effective sequence around ACCATGG in eukaryotes, especially the efficiency of -3 A entry, is very important. The purchased sequence, called Cosac sequence, was constructed for expression vector. For example, the expression level of bacterial chitinase gene and the sequence UUUAUGG around the original start codon increased by 8 times in tobacco. Therefore, the expression vector using gene constructs from non-plant sources should be based on the characteristics of the sequence around the initiation codon of the plant to be transformed.
2.3 The coding region of the gene is transformation.
If foreign genes come from prokaryotes, the expression level of these genes in plants is often very low due to the differences in expression mechanisms. For example, the expression of insecticidal protein gene from Bacillus thuringiensis wild type in plants is very low. It was found that the difference of mRNA stability between prokaryotes and plants was due to the decrease. Under the premise of Perlak of Monsanto, the gene transformation of toxic protein and insecticidal protein with the same amino acid sequence, codon selection and GC content of plants, and the deletion of elements that affect the stability of mRNA in the original sequence, as a result, the expression of toxic protein in transgenic plants increased by 30 to 100 times.
three
After removing the positional effect and foreign genes, the recipient plants usually have very different expression levels in different transgenic plants. This is the result that foreign genes have different insertion sites in the genome of recipient plants. This is the so-called "position effect". In order to eliminate the positional effect, foreign genes are the expression vectors of plant genomes in the transcriptional active region. At present, the construction strategy usually considers the integration of nuclear matrix binding region and site-specific integration technology.
The nuclear matrix binding region (MAR) is a DNA sequence that binds to a specific part of the chromatin nuclear matrix of eukaryotic cells. Generally, MAR sequence is located in the circular structure of transcription-active DNA, and its function is to make the division function, so that each transcription unit can maintain a relatively independent boundary under the influence of surrounding chromatin. This study shows that the genetic transformation of the structure containing MAR gene on both sides of MAR into the gene construction required by plant expression vector can significantly improve the expression level of the target gene, reduce the difference of expression level between different transgenic plants and reduce the position of this effect. For example, Allen et al. studied the expression of heterologous MAR (from yeast) and homologous MAR (from tobacco) Gus gene in tobacco, and found that yeast MAR can increase the average level of transgenic expression by 12 times, and tobacco MAR itself can increase the average level of transgenic expression by 60 times. MAR's chicken lysozyme gene can also play the same role.
Another feasible method is to use site-oriented integration technology. The main principle of this technology is to transform the chromosome of the vector host and integrate the homologous region of a specific DNA fragment into the chromosome through homologous recombination of foreign genes. In fact, the plant expression vector was constructed when the first isolated DNA fragment of the active region of chromosome was transcribed. Targeted integration of homologous recombinant microorganisms has become a routine gene manipulation, and chloroplast expression vectors of exogenous genes in plants have also been successfully integrated in animals, but there are few successful cases of targeted exogenous integration by nuclear transformation methods.
4. Construction of chloroplast expression vector
Exogenous nuclear transformation is often to overcome gene expression. Because of unsafe sexual behavior problems, such as the position effect of nuclear gene pollen transmission, a new genetic transformation technology-chloroplast transformation, which appeared in recent years, is recognized and valued by people more and more because of its advantages and development prospects. So far, five chloroplasts have been published to transform tobacco, rice, Arabidopsis, potato and rape (Hou Bingkai et al. ), which makes this transformation technology a new growth point in plant genetic engineering.
After determination, the exogenous genes of various plant chloroplast genome sequences were integrated into the chloroplast genome at fixed points, which laid the foundation for homologous recombination mechanism. The chloroplast expression vectors constructed at present basically belong to the integration vectors of specific websites. The construction of chloroplast expression vector is basically an example vector. The expression cassette of universal foreign gene that generates chloroplast expression vector, the two sides of chloroplast DNA sequence in each connection period are called homologous recombination fragments, or localization fragments (directional fragments). When the vector is introduced into chloroplast, the two fragments have the same homologous recombination fragment in chloroplast genome, which is a specific place for the integration of foreign genes into chloroplast genome. For the purpose of crop improvement, chloroplast transformation needs homologous recombination, and the original sequence of chloroplast genome inserted with foreign genes will not be lost, nor will the insertion point of the original gene be destroyed. In order to meet this requirement, two homologous recombination fragments of adjacent genes have been selected, such as rbcL gene/accd 1 6 strnv/rpsl2ps7psba gene/variant1,rps7/ndhB. After homologous recombination, the designated foreign gene is inserted into two adjacent gene intervals to ensure that the original function of the gene is not affected. Recently, Daniel et al. constructed a * * * universal vector by using homologous recombination fragments of tobacco chloroplast genome tRNA and trnI. In higher plants, because the sequences of tRNA and trnI DNA are highly conserved, the authors suggest that this vector can be used for chloroplast transformation in various plants. If the universality of the vector is confirmed, then this work is undoubtedly a good idea to construct a new, convenient and practical chloroplast expression vector.
The expression of foreign genes integrated into chloroplast genome is often due to the high copy number of chloroplast genome. In the first example, McBride et al. introduced the cryIA(c) toxin gene of BT into the leaves of tobacco chloroplast Bt toxin protein with an efficiency as high as 3% to 5%, while the typical nuclear transformation technology can only achieve the total protein expression of 0.00 1% to 0.6%. Recently, Bt Cry2Aa2 gene was introduced into tobacco chloroplast in Kota Kinna. It was also found that the expression of toxic proteins in tobacco leaves accounted for 2% ~ 3% of soluble proteins, which was 20 ~ 30 times higher than that of nuclear transformation. Transgenic tobacco not only resists sensitive insects, but also kills highly insect-resistant ones. Staub recently reported that the expression of human growth hormone gene introduced into tobacco chloroplast was as high as 7% of leaf protein, which was more than 300 times that of nuclear transformation method. These experiments fully construct and transform chloroplast expression vectors, which is an important way to realize high-level expression of foreign genes.
5 Positioning signal application program
The main purpose of the above vector optimization strategy is to improve the transcription and translation efficiency of foreign genes. However, whether foreign proteins can be stably expressed at a high level in plant cells, other important issues need to consider the accumulation of plant genetic transformation.
Recent studies have found that if a foreign gene is correctly located in the connected signal sequence, the foreign protein can be transported to specific sites in cells, such as chloroplasts, endoplasmic reticulum, vacuoles and so on. It can significantly improve the stability of the system and the accumulation of foreign proteins. Is this because of a specific area? The endoplasmic reticulum of some foreign proteins provides a relatively stable environment, which can effectively prevent the degradation of foreign proteins. For example, before the transport peptide sequence of Rubisco subunit of Arabidopsis thaliana by Wong et al. was linked to the insecticidal protein gene accumulated in the chloroplast of transgenic tobacco with special insecticidal protein, the total accumulation of foreign protein was 10 to 20 times higher than that of the control group. Recently, liangye, Wang Songru Yan's rbcS subunit transport peptide sequence was linked to the pre-PHB synthesis gene, trying to make the gene product accumulate in transgenic rapeseed germplasm, thus increasing the content of foreign protein. , Wandelt and Stern linked endoplasmic reticulum. It was found that the sequence of foreign protein gene (coding sequence of tetrapeptide KDEL) significantly increased the content of foreign protein in transgenic plants. Obviously, the positioning signal has played a positive role in promoting the accumulation of protein, but whether the same positioning signal is applicable to all protein needs to be further determined.
6 introns enhance gene expression introns enhance gene expression. Callis and others first discovered transgenic corn. The first intron (intron 1) of maize alcohol dehydrogenase gene (ADHL) significantly enhanced the expression of foreign genes, and other introns (such as intron 9) also promoted it to some extent. Later, vasil Lev also found the first intron, and the expression level of corn fructose synthase gene CAT increased by 10 times. The third intron, rice actin gene, can also be composed of 2? The expression level of the reporter gene increased 6 times. The mechanism of jujube intron enhancing gene expression is not clear, but it is generally believed that the existence of intron can improve processing efficiency and mRNA stability. Tanaka et al., studies have shown that the enhancement of gene expression by introns mainly occurs in monocotyledons and dicotyledons, but it is not obvious.
Because of the enhancement of intron, mcelroy constructed monocotyledonous plant expression vector, especially the first intron of rice actin gene maintained the expression of genes downstream of promoter. Similarly, the gene of the first intron of maize ubiquitin constructed by Christensen et al. was placed downstream of the promoter to improve the expression of foreign genes in monocotyledonous plants. However, it has been pointed out that the influence of promoter strength, cell type, target gene sequence and other factors on gene expression depends on the function of specific intron, and sometimes even depends on the position of intron in the vector. For example, maize ADHL gene 9 is located at the 5' end of Gus gene and is not enhanced under the control of CaMV35S promoter of GUS gene. The intron gus gene was put into the 3' end of intron, which increased about 3 times under the control of the expression level of gus gene of the same initiator. The gene expression mechanism of intron may be very complicated. It can be seen that how to establish efficient plant expression vectors of introns lacks a fixed model and deserves further study.
Polygenic control policy
Up to now, most studies have been carried out on genetic transformation of recipient plants by introducing a single exogenous gene. But it is powerful enough, sometimes because of the expression of a single gene or a single mechanism of action, the ideal transgenic plants are not obtained. If two or more genes have synergistic effects on plants, at the same time, better transformation results will be obtained than a single gene. This strategy has been applied to cultivate transgenic plants with disease resistance, insect resistance and stress resistance. For example, according to the different mechanisms of insect spectrum and insect-resistant gene function, two vectors with complementary functions are constructed, and two insect-resistant genes are introduced into plants in some way. Wang Wei, together with other lectin genes and protease inhibitor genes, was simultaneously transferred into transformed plants that already contained bivalent insect-resistant genes in cotton. Barton Bt insecticidal protein gene and scorpion toxin gene were introduced into tobacco, which greatly improved the insect resistance and the ability to prevent pests from producing drug resistance (patent). With strong disease resistance, we constructed a plant expression vector containing β- 1, 3- glucanase bivalent gene and chitinase gene in Lanyan laboratory and introduced it into rape and cotton. The results showed that transgenic plants had obvious resistance. Recently, Feng Daorong and Li Baojian 2? Three antifungal genes and hpt genes are even on the vector, whether the insect-resistant gene and bar gene are connected to another vector, and the results of introducing the gene gun into rice together show that 70% of R's offspring contain introduced foreign genes (6? 7), and the trend of integration of one or two genomic sites of introduced foreign genes.
Generally speaking, it is impossible to introduce foreign DNA fragments above 25KB into plant cells. Function-related genes, such as quantitative trait loci in plant disease resistance genes, mainly exist in the form of gene clusters. If some large DNA fragments larger than 100KB, such as a series of foreign genes in natural plant chromosome gene clusters or non-phase chains, are introduced into the same point in the plant genome, the phenomenon of being controlled by multiple genes may appear. Excellent traits may produce broad-spectrum insect resistance and disease resistance, and a new metabolic pathway can be obtained to produce new biomolecules in recipient cells. In addition, large fragments of gene clusters or synchronous insertion of gene clusters can also overcome the position effect caused by transgene to some extent and reduce the occurrence of undesirable phenomena such as gene silencing. Recently, Hamilton and Alfred developed a new generation of vector system, cloned large DNA fragments, and directly transformed plants BIBAC and TAC with the help of Agrobacterium. These two operators should not only speed up the map cloning of genes, but also control multiple genes, so that variety improvement will have potential applications. At present, the application research in multi-gene transformation has just begun. On BIBAC and TAC carriers
8 Selection of marker genes for use and deletion
Selection of Marker Genes A marker gene selected by a transformable cell (or individual) from a large number of untransformed cells in genetic transformation. They can usually make the produced transgenic cells resistant to selector products. Therefore, the transgenic cells added with this selection in the normal growth medium show the sensitivity of this selection agent because of lack of resistance rather than growth, development and differentiation. In the vector, the connecting side of marker gene is constructed and selected, and both of them have their own gene regulatory sequences (such as promoter, terminator, etc.). There are two main types of selectable marker genes: antibiotic resistance genes and herbicide resistance genes. The former will produce antibiotic resistance, while the latter will produce drug-resistant herbicides. The antibiotic resistance genes used include NPTII gene (hygromycin resistance), Gent gene (neomycin phosphotransferase, kanamycin resistance) and HPT gene (gentamicin resistant ADM). Herbicide resistance genes include EPSP gene (producing 5- pyruvate shikimic acid -3- phosphate synthase, glyphosate), GOX gene (degrading glyphosate by glyphosate oxidase) and bar gene (producing PPT acetyltransferase, anti-bisphosphamide or glufosinate).
The above 1, 2, 3, 5 and 6 are all remarkable, especially because you can choose PB I 12 1 as the extracellular secretion skeleton carrier, and then you can change the genotype on it.
The cloning procedure is relatively simple.
1, the first restriction site, to obtain the desired gene, and it is a good vector for uploading changes.
2 plasmid transformation of Escherichia coli DH5α amplification
Transforming plant cells with well-amplified plasmids
4. Collect extracellular culture medium to detect protein expression and secretion. whether or not
As for the carrier of transformation, just say a few words. After the answer, if you really do a good job, you can start your own company.