It is not the ultimate goal of plant genetic transformation to construct specific foreign genes in plant expression vectors and transfer them into recipient plants. Ideal transgenic plants usually need high-level expression of foreign genes in specific parts and within a specific time to produce ideal phenotypic traits. However, the development history of nearly 20 years shows that foreign genes often appear in recipient plants, such as low expression efficiency, unstable expression products and even gene inactivation or silencing, which leads to the failure of transgenic plants to be put into practical application. In addition, the safety of transgenic plants has attracted people's attention in many countries. For example, transgenes may spread with pollen, and antibiotic screening marker genes may invalidate some clinical antibiotics. The emergence of the above problems makes the high-tech plant genetic engineering in an unprecedented difficult period. In order to solve these problems, in recent years, people have explored and improved plant transgenic technology in many aspects, and the improvement and optimization of plant expression vector is one of the most important contents. This paper summarizes the progress that has been made.
Selection and transformation of 1 promoter
Insufficient expression of foreign genes is often an important reason for not getting ideal transgenic plants. Because promoters play a key role in determining gene expression, the first thing to consider is to select suitable plant promoters and improve their activity.
At present, the promoters widely used in plant expression vectors are constitutive promoters. For example, most dicotyledonous transgenic plants use CaMV35S promoter, while monocotyledonous transgenic plants mainly use ubiquitin promoter from maize and Actinl promoter from rice. Under the control of these constitutive expression promoters, foreign genes will be expressed in all parts and all development stages of transgenic plants. However, the sustained and efficient expression of exogenous genes in recipient plants not only causes waste, but also often causes morphological changes of plants and affects their growth and development. In order to make foreign genes play an effective role in plants and reduce the adverse effects on plants, people pay more and more attention to the research and application of specific expression promoters. The found specific promoters mainly include organ-specific promoters and induction-specific promoters. Such as seed-specific promoter, fruit-specific promoter, mesophyll cell-specific promoter, root-specific promoter, injury-induced promoter, chemical-induced promoter, light-induced promoter, heat-shock-induced promoter and so on. The cloning and application of these specific promoters laid a foundation for the specific expression of foreign genes in plants. For example, Swiss company CIBA- GEKKI uses PR-IA promoter to control the expression of Bt toxin gene in transgenic tobacco. Because 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 by spraying cheap and pollution-free chemicals in the season when pests recur.
In the research of plant transgene, it is often impossible to obtain satisfactory results by using natural promoters, especially when specific expression and induced expression are carried out, and the expression level is mostly unsatisfactory. It will be a very important way to transform existing promoters and construct composite promoters. For example, Ni et al. combined the transcriptional activation region of octopine synthase gene promoter with mannopine synthase gene promoter, and the GUS expression results showed that the activity of the modified promoter was significantly higher than that of the 35S promoter. Ray Wu et al. combined the inducible PI-II gene promoter with rice Actinl gene intron 1, and the expression activity of the new promoter was increased by nearly 10 times (patent). These artificial promoters have played an important role in plant genetic engineering research.
2. Improve translation efficiency
In order to enhance the translation efficiency of foreign genes, it is generally necessary to modify genes when constructing vectors, mainly considering three aspects:
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, and the deletion of this fragment often leads to a significant decline in mRNA stability and translation level. For example, there is an ω element composed of 68bp nucleotide upstream of the translation initiation site of tobacco mosaic virus (TMV) 126 kda protein gene, which provides a new binding site for ribosomes and can improve the translation activity of Gus gene several times. At present, many vectors add ω translation enhancement sequences to the 5'- end of foreign genes. Ingelbrecht and others studied the 3'- terminal sequences of various genes and found that the 3'- terminal sequence of octopine synthase gene can increase the instantaneous expression of NPTII gene by more than 20 times. In addition, the 3'- terminal sequences of different genes have different efficiencies in promoting gene expression. For example, the 3'- terminal sequence of RBC promotes gene expression 60 times higher than that of chalcone synthase gene.
2.2 Optimize the sequence around the initial password
Although the start codon is ubiquitous in biology, genes from different biological sources have their own special sequences around the start codon. For example, the typical feature of plant initiation codon peripheral sequence is AACCAUGC, animal initiation codon peripheral sequence is CACCAUG, and prokaryotic initiation codon peripheral sequence is very different from them. Cosac studied in detail the influence of site-directed mutation of the base around the start codon ATG on transcription and translation, and concluded that in eukaryotes, the transcription and translation efficiency is the highest when the sequence around the start codon is ACCATGG, especially the A in -3 position is very important for translation efficiency. This sequence was later called Cosac sequence, and was used to construct the expression vector. For example, there is a bacterial chitinase gene whose initial coding sequence is UUUAUGG. When it was transformed into ACCAUGG, its expression in tobacco increased by 8 times. Therefore, when constructing the expression vector with non-plant genes, it should be modified according to the characteristics of the sequence around the plant initiation codon.
2.3 coding region of transformed gene.
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 level of wild-type insecticidal protein gene from Bacillus thuringiensis in plants is very low, which is found to be due to the difference between prokaryotic and plant genes, resulting in the decline of mRNA stability. Perlak and others of Monsanto Company in the United States modified the insecticidal protein gene without changing the amino acid sequence of the toxic protein, selected the codon preferred by plants, increased the GC content, and removed the elements that affected the mRNA stability under the original sequence. The results showed that the expression of toxin protein in transgenic plants was increased by 30 ~ 100 times, and obvious insect-resistant effect was obtained.
3 Eliminate position effect
When foreign genes are transferred into recipient plants, their expression levels in different transgenic plants are often very different. This is mainly due to the different insertion sites of foreign genes in the genome of recipient plants. This is the so-called "position effect". In order to eliminate the position effect and integrate all foreign genes into the transcriptional active region of plant genome, the nuclear matrix binding region and site-directed integration technology are usually considered in the current expression vector construction strategy.
Matrix association region (MAR) is a DNA sequence that exists in the chromatin of eukaryotic cells and specifically binds to the nuclear matrix. It is generally believed that MAR sequence is located at the boundary of the circular structure of active transcribed DNA, and its function is to create a segmentation effect, so that each transcription unit remains relatively independent and is not affected by the surrounding chromatin. Related research shows that putting MAR on both sides of the target gene and constructing plant expression vectors with MAR-gene-MAR structure for genetic transformation can significantly improve the expression level of the target gene, reduce the difference of the expression level of the target gene among different transgenic plants and reduce the positional effect. For example, Allen et al. studied the effects of heterologous MAR (from yeast) and homologous MAR (from tobacco) on the expression of Gus gene in tobacco, and found that MAR of yeast can increase the expression level of transgene by 12 times on average, while MAR of tobacco itself can increase the expression level of transgene by 60 times on average. MAR derived from chicken lysozyme gene can also play the same role.
Another feasible method is to adopt site-oriented integration technology. The main principle of this technology is that when the transformation vector contains DNA fragments homologous to the host chromosome, foreign genes can be integrated into specific sites of the chromosome through homologous recombination. In practical application, it is necessary to isolate the DNA fragment of the transcriptional active region of chromosome first, and then construct the plant expression vector. In microbial gene manipulation, homologous recombination and site-directed integration has become a conventional technology, and site-directed integration of foreign genes has been successful in animals, but in plants, except chloroplast expression vectors, there are few reports of successful nuclear transformation.
4. Construction of chloroplast expression vector
In order to overcome the problems of low expression efficiency of foreign genes, positional effect and insecurity caused by the diffusion of nuclear genes with pollen, a new genetic transformation technology, chloroplast transformation, has been increasingly recognized and valued by people for its advantages and development prospects. Up to now, chloroplast transformation has been achieved in tobacco, rice, Arabidopsis, potato and rape (published by Hou Bingkai, etc.), which makes this transformation technology a new growth point in plant genetic engineering.
At present, the complete sequences of chloroplast genomes of many plants have been determined, which lays a foundation for the site-specific integration of foreign genes into chloroplast genomes through homologous recombination mechanism. The chloroplast expression vectors constructed at present are basically site-directed integration vectors. The constructed chloroplast expression vector basically belongs to the site-specific case vector. When constructing chloroplast expression vector, a segment of chloroplast DNA sequence is usually connected to both sides of foreign gene expression cassette, which is called homologous recombination fragment or targeting fragment. When the vector is introduced into chloroplast, it is possible to integrate foreign genes into specific sites of chloroplast genome by homologous recombination of these two fragments with the same fragments in chloroplast genome. In chloroplast transformation for crop improvement, after homologous recombination, the insertion of foreign genes will not cause the loss of the original sequence of chloroplast genes, nor will it destroy the function of the original genes at the insertion point. To meet this requirement, two adjacent genes were selected as homologous recombination fragments, such as rbcL/accD, 16 strnv/rpsl2rps7, psba/trnk, rps7/ndhb. When homologous recombination occurs, the foreign gene is inserted into the spacer of two adjacent genes at a fixed point, which ensures 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 to construct a universal vector. Because the DNA sequences of trnA and trnI are highly conserved in higher plants, the author thinks that this vector can be used for chloroplast transformation in many different plants. If the universality of the vector is confirmed, this work will undoubtedly provide a good idea for constructing a convenient and practical new chloroplast expression vector.
Because of the high copy of chloroplast genome, foreign genes integrated into chloroplast genome can often be expressed efficiently. For example, McBride et al. first transferred Bt CryIA(c) toxin gene into tobacco chloroplast, and the expression of Bt toxin protein was as high as 3% ~ 5% of total leaf protein, while the usual nuclear transformation technology could only reach 0.00 1% ~ 0.6%. Recently, Kota et al. transferred Bt Cry2Aa2 protein gene into tobacco chloroplast, and also found that the expression of toxic protein in tobacco leaves was very high, accounting for 2% ~ 3% of soluble protein, which was 20 ~ 30 times higher than that of nuclear transformation. Transgenic tobacco can not only resist sensitive insects, but also kill those highly resistant insects. Staub et al. recently reported that the expression of human growth hormone gene transferred into tobacco chloroplast was as high as 7% of total protein in leaves, which was 300 times higher than that of nuclear transformation. These experiments fully show that the construction and transformation of chloroplast expression vector is one of the important ways to realize efficient expression of foreign genes.
Application of positioning signal
The main purpose of these vector optimization strategies is to improve the transcription and translation efficiency of foreign genes. However, whether high-level expressed foreign proteins can exist stably in plant cells and how much they accumulate is another important issue to be considered in plant genetic transformation.
In recent years, it has been found that the stability and accumulation of foreign proteins can be significantly improved if some foreign genes are connected with appropriate localization signal sequences to produce and transport them to specific parts of cells, such as chloroplasts, endoplasmic reticulum and vacuoles. This is because some areas such as endoplasmic reticulum provide a relatively stable internal environment for some foreign proteins, which effectively prevents the degradation of foreign proteins. For example, Wong et al. linked the transport peptide sequence of Arabidopsis rbcS subunit with insecticidal protein gene, and found that insecticidal protein could be specifically accumulated in chloroplasts of transgenic tobacco, and the total accumulation of foreign proteins was 10 ~ 20 times higher than that of the control. Recently,, Song, etc. The transport peptide sequence of rbcS subunit was also linked to PHB synthesis-related genes, in an attempt to accumulate gene expression products in the plastids of transgenic rapeseed seeds, thus increasing the content of foreign proteins. In addition, Wandelt et al. and Schouten et al. linked the localization sequence of endoplasmic reticulum (coding sequence of tetrapeptide KDEL) with foreign protein gene, and found that the content of foreign protein in transgenic plants was significantly increased. Obviously, the positioning signal has 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.
Application of intron 6 in enhancing gene expression
The effect of intron on enhancing gene expression was first discovered in transgenic maize by Callis et al. The first intron (intron 1) of maize alcohol dehydrogenase gene (Adhl) significantly enhanced the expression of foreign genes, and other introns of this gene (such as intron 8 and intron 9) also enhanced the expression of foreign genes to some extent. Later, Vasil and others also found that the first intron of corn fructose synthase gene could increase the expression level of CAT by 10 times. The third intron of rice actin gene can also increase the expression level of reporter gene by 2 ~ 6 times. Up to now, the mechanism of intron enhancing gene expression is not clear, but it is generally believed that the existence of intron may enhance processing efficiency and mRNA stability. Many studies by Tanaka and others show that the enhancement of gene expression by introns mainly occurs in monocotyledons, but not in dicotyledons.
Because introns can enhance gene expression, Mcelroy and others deliberately kept the first intron of rice actin gene downstream of the gene promoter when constructing monocotyledonous plant expression vectors. Similarly, Christensen et al. placed the first intron of maize ubiquitin gene downstream of the promoter when constructing a vector to enhance the expression of foreign genes in monocots. However, some studies have pointed out that the promotion of gene expression by specific introns depends on many factors such as promoter strength, cell type, target gene sequence, and sometimes even depends on the position of introns on the vector. For example, intron 9 of maize Adhl gene is located at the 5' end of Gus gene, and the expression of Gus gene is not enhanced under the control of CaMV35S promoter. When the intron is located at the 3- terminal of Gus gene, the expression level of Gus gene is increased by about 3 times under the control of the same promoter. It can be seen that the mechanism of intron on gene expression may be very complicated, and there is still a lack of fixed model on how to construct efficient plant expression vectors by intron, which deserves further discussion.
7 polygene strategy
Up to now, most genetic transformation research is to transfer a single exogenous gene into recipient plants. However, sometimes due to the lack of a single gene expression intensity or a single mechanism of action, ideal transgenic plants cannot be obtained. If two or more synergistic genes are transferred into plants at the same time, better results will be obtained than single gene transformation. This strategy has been applied to cultivate transgenic plants resistant to pests and diseases. For example, according to the different insect-resistant spectrum and action mechanism of insect-resistant genes, two genes with complementary functions can be selected for vector construction, and the two insect-resistant genes can be simultaneously transferred into plants in a certain way. Wang Wei and others transferred lectin gene and protease inhibitor gene into cotton at the same time, and obtained transformed plants containing bivalent insect-resistant genes. Barton et al. transferred Bt insecticidal protein gene and scorpion toxin gene into tobacco at the same time, which greatly improved the insect resistance and the ability to prevent pests from producing resistance (patent). In terms of disease resistance, Lan Haiyan and others in our laboratory constructed a bivalent plant expression vector containing β- 1, 3- glucanase gene and chitinase gene, and introduced it into rape and cotton. The results showed that transgenic plants had obvious disease resistance. Recently, Feng Daorong, Li Baojian and others connected two or three antifungal genes and hpt genes to one vector, and two insect-resistant genes and bar genes to another vector, and introduced them into rice plants by gene gun. The results showed that 70% of the progeny plants of wild rice contained all introduced foreign genes (6 ~ 7), and the introduced foreign genes tended to be integrated at one or two loci in the genome.
Generally, conventional transformation cannot introduce foreign DNA fragments larger than 25kb into plant cells. 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 large DNA fragments larger than 100kb, such as naturally occurring gene clusters in plant chromosomes or a series of unrelated foreign genes, are introduced into the same site of plant genome, it will be possible to have excellent characters controlled by multiple genes or produce broad-spectrum insect resistance and disease resistance, and it will also give recipient cells a brand-new metabolic pathway and produce new biomolecules. Moreover, the synchronous insertion of large genome or gene cluster can also overcome the position effect brought by transgene to some extent and reduce the occurrence of undesirable phenomena such as gene silencing. Recently, Hamilton in the United States and Liu Yaoguang in China developed a new generation of vector systems, namely BIBAC and TAC, which cloned large fragments of DNA and directly transformed them into plants with the help of Agrobacterium. These two vectors can not only accelerate the map-based cloning of genes, but also have potential application value for variety improvement under the control of multiple genes. At present, the application of BIBAC and TAC vectors in multi-gene transformation has just begun.
8 Utilization and deletion of screening marker genes
Screening marker genes refer to marker genes that can enable transformed cells (or individuals) to be screened from many non-transformed cells in genetic transformation. They can usually make transgenic cells produce products that are resistant to a certain selector, so that transgenic cells can grow normally on the medium added with this selector, while non-transgenic cells are sensitive to this selector and cannot grow, develop and differentiate due to lack of resistance. When constructing the vector, the screening marker genes are connected to one side of the target gene, and each gene has its own gene regulatory sequence (such as promoter, terminator, etc.). At present, there are two kinds of commonly used screening marker genes: antibiotic resistance enzyme gene and herbicide resistance enzyme gene. The former can be resistant to some antibiotics, while the latter can be resistant to herbicides. The most commonly used antibiotic resistance enzyme genes are NPTII gene (producing neomycin phosphotransferase and resisting kanamycin), HPT gene (producing hygromycin phosphotransferase and resisting hygromycin) and Gent gene (resisting gentamicin). Commonly used herbicide-resistant genes include EPSP gene (producing 5- enolpyruvate shikimic acid -3- phosphate synthase and resisting glyphosate), GOX gene (producing glyphosate oxidase and degrading glyphosate), bar gene (producing PPT acetyltransferase and resisting bisphosphamide or glufosinate) and so on.
Above, 1, 2, 3, 5, 6 are all noteworthy, especially 5, because you want to secrete it outside the cell. The skeleton vector can be PB I 12 1, and then you can change the genotype on it.
The next step is cloning, which is relatively simple.
1 Firstly, the restriction site of the target gene was obtained and connected to the modified vector.
2. The plasmid was transferred to E.coli DH5a for amplification.
3. Transfer the amplified plasmid into plant cells for expression.
4. Collect extracellular culture medium of roots to detect protein expression and secretion.
As for the steps of transforming the carrier, if you answer the question, just talk about it briefly. After all, if you really make the carrier, you can start your own company.