It is not the ultimate goal of plant genetic transformation to construct a specific foreign gene in a plant expression vector and transfer it into a recipient plant. Ideal transgenic plants often need high-level expression of foreign genes in specific parts and within a specific time to produce desirable phenotypic traits. However, the development history of nearly twenty 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 make some clinical antibiotics ineffective. The emergence of the above problems makes the high-tech plant genetic engineering in an unprecedented troubled 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 obtaining 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 of transgenic plants and at all developmental stages. However, the sustained and efficient expression of exogenous genes in recipient plants not only causes waste, but also often causes morphological changes of plants, which affects the growth and development of plants. 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. For example, seed-specific promoter, fruit-specific promoter, mesophyll cell-specific promoter, root-specific promoter, damage-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, CIBA-GEIGY Company of Switzerland 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 way to induce the expression of insect-resistant gene in the season of pest recurrence by spraying cheap and pollution-free chemicals.
In plant transgenic research, using natural promoters often can't achieve satisfactory results, especially in specific expression and induced expression, the expression level is mostly not ideal. It will be a very important way to transform the existing promoters and construct compound promoters. For example, Ni et al. combined the transcription 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 intron 1 of rice Actinl gene, and the expression activity of the new promoter was improved by nearly 1 times (patent). These artificial promoters have played an important role in plant genetic engineering research.
2 Enhancing translation efficiency
In order to enhance the translation efficiency of foreign genes, genes are generally modified when constructing vectors, mainly considering three aspects:
2.1 Adding 5'-3'-untranslated sequence
mRNA experiments have found that the 5'-3'-untranslated sequence (UTR) of eukaryotic genes is very necessary for the normal expression of genes. For example, there is an ω element composed of 68bp nucleotides upstream of the translation initiation site of the 126kDa protein gene of tobacco mosaic virus (TMV), which provides a new binding site for ribosomes and can improve the translation activity of Gus gene by dozens of times. At present, many vectors have added ω translation enhancement sequences to the 5'-end of foreign genes. Ingelbrecht and others have studied the 3'-terminal sequences of various genes and found that the 3'-terminal sequence of octopine synthase gene can improve the instantaneous expression of NPTII gene by more than 2 times. In addition, the 3'-terminal sequence of different genes has different efficiency in promoting gene expression. For example, the 3'-terminal sequence of RBC has 6 times higher promotion effect on gene expression than the 3'-terminal sequence of chalcone synthase gene.
2.2 optimizing the sequence around the initial codon
although the initial codon is universal in the biological world, genes from different biological sources have their own special sequence around the initial 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 quite different from them. Kozak studied in detail the effects on transcription and translation caused by site-directed mutation of the base around the start codon ATG, 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 Kozak sequence, and was applied to the construction of expression vector. For example, there is a bacterial chitinase gene, whose original initial code sequence is UUUAUGG. When it is modified to ACCAUGG, its expression level in tobacco is 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 Transform the coding region of genes
If the foreign genes are 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 the wild-type insecticidal protein gene from Bacillus thuringiensis in plants is very low, which is found to be due to the differences between prokaryotes and plant genes, resulting in the decrease of mRNA stability. Perlak et al. 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. As a result, the expression of toxic protein in transgenic plants increased by 3 ~ 1 times, and obvious insect-resistant effect was obtained.
3 Eliminating the positional effect
When the foreign gene is transferred into the recipient plant, its expression level in different transgenic plants is 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 application of nuclear matrix binding region and site-directed integration technology is usually considered in the current expression vector construction strategy.
the 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 transcription DNA, and its function is to create a segmentation effect, so that each transcription unit remains relatively independent and is free from the influence of surrounding chromatin. Relevant research shows that putting MAR on both sides of the target gene to construct a plant expression vector containing MAR-gene-MAR structure for genetic transformation can obviously 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 an average of 12 times, while MAR of tobacco itself can increase the expression level of transgene by an average of 6 times. MAR derived from chicken lysozyme gene can also play the same role.
another feasible way is to adopt site-specific 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 at a specific site of the chromosome through homologous recombination. In practice, the DNA fragment of the transcriptional active region of chromosome should be separated first, 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, and the 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 exogenous genes, positional effect and insecurity caused by the diffusion of nuclear genes with pollen, a new genetic transformation technology-chloroplast transformation, which has emerged in recent years, is increasingly recognized and valued by people for its advantages and development prospects. Up to now, chloroplast transformation has been achieved in five plants, tobacco, rice, Arabidopsis, potato and rape (published by Hou Bingkai, et al.), which makes this transformation technology become a new growth point in plant genetic engineering.
At present, the complete sequence of chloroplast genome of many plants has been determined, which lays a foundation for the site-directed integration of foreign genes into chloroplast genome through homologous recombination mechanism. At present, the constructed chloroplast expression vectors basically belong to site-directed integration vectors. The constructed chloroplast expression vectors basically belong to fixed-point case vectors. 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 fragment on chloroplast genome. In chloroplast transformation for crop improvement, it is required that 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 have been selected 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 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 this vector is confirmed, this work will undoubtedly provide a good idea for constructing a convenient and practical new chloroplast expression vector.
due to the high copy of chloroplast genome, foreign genes integrated into chloroplast genome at fixed points are often expressed with high efficiency. for example, McBride et al. transferred Bt CryIA(c) toxin gene into tobacco chloroplast for the first time, and the expression amount of Bt toxin protein was as high as 3% ~ 5% of total leaf protein, while the usual nuclear transformation technology could only reach .1% ~ .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 2 ~ 3 times higher than that of nuclear transformation. Transgenic tobacco can not only resist sensitive insects, but also kill those insects with high resistance. 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 3 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.
5 Application of localization signal
The main purpose of the above 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 if some foreign genes are connected with appropriate localization signal sequences, the foreign proteins can be transported to specific parts of cells, such as chloroplasts, endoplasmic reticulum, vacuoles, etc., which can obviously improve the stability and accumulation of foreign proteins. This is because certain 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 to 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 1 ~ 2 times higher than that of the control. Recently, Ye Liang, Song Yanru, etc. also linked the transport peptide sequence of rbcS subunit to PHB synthesis related genes, trying to make the gene expression products accumulate 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 endoplasmic reticulum localization sequence (the coding sequence of tetrapeptide KDEL) with the foreign protein gene, and found that the content of foreign protein in transgenic plants was significantly improved. Obviously, the localization signal plays a positive role in promoting protein accumulation, but whether the same localization signal is suitable for all proteins needs to be further determined.
the application of intron 6 in enhancing gene expression
the role of intron in enhancing gene expression was first discovered by Callis et al. in transgenic maize, the first intron8,intron9 1) of maize alcohol dehydrogenase gene (Adhl) significantly enhanced the expression of foreign genes, and other introns (such as intron 8 and intron 9) of the gene also enhanced the expression of foreign genes to some extent. Later, Vasil et al. also found that the first intron of fructose synthase gene in maize could increase the expression level of CAT by 1 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 it may be internal