How to construct a vector

The selection and transformation of 1 promoter 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 the C a M V 3 5 S promoter, while monocotyledonous transgenic plants mainly use the U b i q u i t i n promoter from corn and the A c t i n l 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, CBA-Geigey Company of Switzerland uses P R-I A promoter to control the expression of B t 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, N i et al. combined the transcriptional activation region of octopine synthase gene promoter with mannan synthase gene promoter, and the G U S expression results showed that the activity of the modified promoter was significantly higher than that of the 3 5 S promoter. Ray Wu et al. combined the inducible promoter of PI-III gene with intron 1 of rice A c t i n l gene, and the expression activity of the new promoter was improved by nearly 1 0 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 sequences. Many experiments have found that the 5 ′-3 ′-untranslated sequence (U T R) of eukaryotic gene is very necessary for the normal expression of the gene, and the deletion of this fragment often leads to the stability and translation level of m R N A. For example, there is an ω element composed of 6 8 b p nucleotides upstream of the translation initiation site of the 1 2 6 k D a protein gene of tobacco mosaic virus (T M V), which provides a ribosome. At present, many vectors add ω translation enhancement sequences to the 5' end of foreign genes. I n g e l b r e c h t et al. 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 N P T I I I I gene by more than 2 0 times. In addition, the 3'- terminal sequences of different genes have different efficiencies in promoting gene expression. For example, the 3'- terminal sequence of RBCs is 6 0 times higher than that of chalcone synthase gene to promote gene expression. 2.2 Optimizing the sequence around the start codon Although the start codon is universal in biology, genes from different biological sources have their own special sequences around the start codon. For example, the typical feature of the sequence around the plant initiation codon is A A C C A U G C, and the sequence around the animal initiation codon is A C A U G, which is very different from prokaryotes. K o z a k studied the influence of site-directed mutation of bases around the start codon A T G on transcription and translation in detail, and concluded that in eukaryotes, when the sequence around the start codon is C C A T G G, the transcription and translation efficiency is the highest, especially the position of AT-3 is very important for translation efficiency. This sequence was later called k0z a K sequence, and was used to construct expression vector. For example, there is a bacterial chitinase gene whose original code sequence is U U U A U G G, and when it is modified to A C C A U G G, 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 Modification of gene coding region If foreign genes come from prokaryotes, the expression level of these genes in plants is often very low due to 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 difference between prokaryotic and plant genes, which leads to the decline of the stability of m R N A. On the premise of not changing the amino acid sequence of the toxic protein, P e r l a k and others of the American mN SA NTO Company have modified the insecticidal protein gene, selected the codon preferred by plants, increased the content of G C, and removed the elements that affected the stability of m R N A under the original sequence. As a result, the expression of toxin protein in transgenic plants increased by 30 ~ 100 times, and obvious insect-resistant effect was obtained. 3 Eliminating positional effects 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. M A T R I X A S O C I A T I O N R E G I O N (M A R) is a D N A sequence, which exists in the chromatin of eukaryotic cells and specifically binds to the nuclear matrix. It is generally believed that the sequence of M A R is located at the boundary of the circular structure of active transcription D N A, and its function is to cause division, so that each transcription unit remains relatively independent and is not affected by surrounding chromatin. Related research shows that putting M A R on both sides of the target gene and constructing a plant expression vector with M A R-g e n e-M A R 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, l l e n et al. studied the effects of heterologous M A R (from yeast) and homologous M A R (from tobacco) on the expression of G u s gene in tobacco, and found that the M A R of yeast can increase the transgenic expression level by 65438 0.2 times on average, while the M A R of tobacco itself can increase the transgenic expression level by 6 0 times on average. Using M A R 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 D N A fragments homologous to the host chromosome, foreign genes can be integrated into specific parts of the chromosome through homologous recombination. In practice, firstly, the D N A fragment of the transcriptional active region of chromosome should be isolated, and then the plant expression vector should be constructed. 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, positional effect and insecurity caused by the diffusion of nuclear genes with pollen in nuclear transformation, a new genetic transformation technology-chloroplast transformation, which has emerged in recent years, 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 D N A sequence of chloroplast is usually connected to both sides of foreign gene expression cassette, which is called homologous recombination fragment or localization fragment (T a r g e t i n g f r a g m e n t). 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. In order to meet this requirement, two adjacent genes were selected as homologous recombination fragments, such as r b c L/a c c D,16TrNV/R PS L 2RPS 7, PB BA/T R NK, RP S 7/N D HB. 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, D 'ANIEL et al. used tobacco chloroplast genes t r n A and t r n I as homologous recombination fragments to construct a U NIV E R S A L E C T O R (U NIV E R S A L E C T O R). Because the D N A sequences of t r n A and t r n I 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, for the first time, the gene of M C B R Y I A (c) toxin was transferred into tobacco chloroplast, and the expression of B t 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, K o t a et al. transferred the gene of Bt CrY2A2 protein into tobacco chloroplast, and also found that the expression of toxin 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. Recently, STA AUB and others reported that the expression of human growth hormone gene in leaves was as high as 7% of total protein, which was 3 0 0 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 strategy 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, W o n g G and others linked the transporter sequence of Arabidopsis r b c S 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 Rb-C-S subunit was also linked with genes related to P H B synthesis, in an attempt to accumulate gene expression products in the plastids of transgenic rapeseed seeds, thus increasing the content of exogenous proteins. In addition, W a n d e l t et al. and S c h o u t e n et al. linked the localization sequence of endoplasmic reticulum (the coding sequence of tetrapeptide K D E L) with the 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. The application of introns in enhancing gene expression was first discovered in transgenic corn by Cal Llis et al. The first intron (i n t r o n 1) of maize alcohol dehydrogenase gene (A d h l) obviously enhanced the expression of foreign genes, and other introns of the gene (such as i n t r o n 8, i n t r o n 9) also enhanced the expression of foreign genes to some extent. Later, V a s i l and others also found that the first intron of fructose synthase gene in maize could increase the expression level of C A T by 1 0 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 the processing efficiency and stability of m R N A. Many studies by Tannaka et al. show that intron enhancing gene expression mainly occurs in monocotyledonous plants, but not in dicotyledonous plants. Because introns can enhance gene expression, M c e l r o y and others deliberately kept the first intron of rice actin gene downstream of the gene promoter when constructing monocotyledonous plant expression vectors. Similarly, C H R I S E N S E N et al. placed the first intron of maize UBI Q U I N gene downstream of the promoter to enhance the expression of foreign genes in monocotyledonous plants. 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 ADDHL gene is located at the 5' end of G u s gene, and the expression of G u s gene is not enhanced under the control of CAMV V35S promoter; When the intron is located at the 3- terminal of G u s gene, under the control of the same promoter, the expression of G u s gene is increased by about 3 times. 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 Multi-gene Strategy Up to now, most of the genetic transformation research is to transfer a single foreign 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. B a r t o n et al. transformed B t 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 h p t genes to one vector, and two insect-resistant genes and b a r genes to another vector, and introduced them into rice plants by gene gun. The results showed that 7 0% 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 can not introduce exogenous D N A fragments larger than 2 5 k b 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 fragments of D N A larger than 1 0 0 k b, such as gene clusters naturally existing in plant chromosomes or a series of unrelated foreign genes, are introduced into the same site of plant genome, it may have excellent characters controlled by multiple genes or produce broad-spectrum insect resistance and disease resistance, and it may 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, H a m i l t o n in the United States and Liu Yaoguang in China have developed a new generation of vector systems, namely, B I B A C and T A C, which cloned large fragments of D N A and directly transformed 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 research of B I B A C and T A C 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 a large number of 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 genes of antibiotic resistance enzymes are npt ii ii (producing neomycin phosphotransferase, resisting kanamycin), H P T (producing hygromycin phosphotransferase, resisting hygromycin) and G e n t T (resisting gentamicin). Commonly used herbicide-resistant genes include ESP gene (producing 5- enolpyruvate shikimic acid -3- phosphate synthase, resisting glyphosate), G O X gene (producing glyphosate oxidase, degrading glyphosate), b a r gene (producing PPP acetyltransferase, resisting B i a l a p h o s or g l u f o s i n a t e) 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 plasmodium B I 1 2 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 D5A for amplification 3. The amplified plasmid was transferred to plant cells for expression 4. The extracellular culture medium of roots was collected to detect whether protein was expressed and secreted. 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. Let me briefly talk about the steps: 1 Select the appropriate restriction site, mainly referring to the multi-cloning site of the vector you use. Generally, it is recommended to use double restriction, and there is no problem of direction verification; 2. Design primers to clone genes according to the restriction sites you choose; 3. The P-C-R products of the vector and the gene were digested with enzymes respectively, and then recovered. You can choose to cut the glue and recycle it, or you can use anhydrous ethanol to precipitate it; 4 ligase ligation 5 transformation 6 plasmid verification. P C R verification and enzyme digestion verification should be carried out. 7 sorting