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Download Lecture 7 Manipulation of gene expression and secretion of foreign
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Lecture 7 Manipulation of gene expression and secretion of foreign protein Manipulation of gene expression The transformed plants were assayed for the production of the foreign protein and studied physiologically to assess how the presence of additional proteins affects the whole plant. Earlier experiments utilized promoters that were expressed constitutively in a range of plant cells. More recently, many additional plant promoters have been isolated and characterized and used to express foreign proteins in specific cells at certain times during the growth and development of the plant. For example, instead of strong constitutive 35S promoter from cauliflower mosaic virus, which is expressed in all plant tissues, tissue specific promoters (promoters active only in specific tissues such as roots or flowers) were used to control the expression of foreign genes. Isolation and Use of Different Promoters Specialized vectors, called promoter tagging (labeling) vectors, have been used to isolate plant promoters from several plant species. This approach relies on the Agrobacterium mediated Ti plasmid transformation system. Briefly, a promoter less reporter gene is placed next to the right border of the Ti plasmid vector. After transfer of the T-DNA into a plant chromosome the reporter gene from the vector is situated adjacent to the plant DNA (Fig 1A). If the T-DNA is inserted at the promoter region of a functional gene, transcription of the reporter gene occurs. For example, with the neomycin phosphotransferase (npt) gene as a reporter, its expression is detected by selecting kanamycin-resistant transformants. However, with this method it is difficult to identify (tag) a promoter that is active only during a certain developmental stage or that is induced by a specific environmental factor. To overcome this problem, a two-gene selectable marker system was devised. In this case, a hygromycin resistance gene was placed under the control of a constitutive promoter next to a promoter less reporter gene within the T-DNA (Fig. 1B). After hygromycin-resistant transformants are selected, the transformants can be checked by an enzyme assay under different conditions for expression of the reporter gene. With this strategy 5- 30% of the transformed plant cells have the reporter genes under the control of an active promoter. Figure 1. Using a promoter less reporter gene to isolate a plant promoter A. A promoter less neomycin phosphotransferase (npt) reporter gene is placed down stream from a right border (RB) sequence of the T-DNA. After transfer of the T-DNA into the plant chromosomal DNA, if the TDNA is inserted near the promoter (P) region of a functional plant gene that is oriented in the same direction as npt ,transcription occurs. The expression of npt is detected by selecting kanamycin transformants. B. To ensure that transformed cells are selected, a hygromycin resistance (Hygr) gene, under the control of a constitutive promoter (P), is placed downstream from the promoter less reporter gene within the T-DNA. Both npt and Hygr genes are equipped with transcription terminator TT regions. The cauliflower mosaic virus 35S promoter is frequently used as a strong promoter in plant systems, although the level of expression of a foreign protein under the control of this promoter is often lower than desired. To address this problem, it is necessary to test different promoter—gene constructs in plants to see if more effective promoters can be found. In addition to the promoter, several other elements may enhance foreign gene expression. These include enhancer sequences that are found from one to hundred nucleotides upstream of the promoter sequence, introns that may stabilize messenger RNA (mRNA), and transcription terminator sequences. In one series of experiments, DNA constructs that contained all or some of the following elements were tested: the 35S promoter, the nopaline synthase gene transcription terminator, from one to seven tandemnly repeated enhancer elements, and a DNA sequence from tobacco mosaic virus called omega that increases gene expression at the translational level. The most active construct contained seven enhancer elements and directed much higher level of foreign gene expression in both transgenic tobacco and rice plants than when the 355 promoter alone was used. These promoter constructs directed a wide range of foreign gene expression in transgenic plants. This variation is probably due to the site within the plant genome where the T-DNA is inserted, nevertheless, this work shows that it is possible to engineer promoters that are much stronger than the naturally occurring 35S promoter. With this approach, it should be possible to engineer promoters that are tissue specific, developmentally regulated, and strong. Targeted Alterations in Plant DNA In bacteria, and to a lesser extent in animal cells, it is relatively straight for ward to alter the genomic DNA of an organism by homologous recombination between the native form of the target DNA, in the genome, and a modified form of the target DNA usually on a plasmid vector. Using similar techniques, the targeted alteration of plant cell genes occurs quite infrequently. However, based on results with animal cells, researchers have used RNA/DNA chimeric molecules—actually; the RNA is 2’-O- methyl RNA—to introduce stable changes into the genomic DNA of plant cells. These chimeric oligonucleotides are designed to have one or more bases that do not pair with the endogenous plant DNA sequence. Following the delivery of chimeric oligonucleotide into a plant cell by microprojectile bombardment, it is thought that DNA repair enzymes recognize the mismatches between the targeted gene and the chimeric oligonucleotide. During the repair process, the altered DNA is incorporated into the plant genome. The changed chromosomal DNA can be readily detected phenotypically if the mutation that is created is dominantly or co dominantly expressed. This is because plants are diploid with two copies of each gene, and this procedure changes only one of those two copies. In addition to changing one or two bases in the sequence of a plant gene, this technique may also be used to modify plant DNA through the site-specific insertion or deletion of a single base. The use of RNA/DNA chimeric molecules to stably modify plant genomes is conceptually similar to conventional mutagenesis and selection procedures. An example of a chimeric oligonucleotide used to change the nucleotide sequence of plant DNA. The 2’-O-methyl RNA residues are shown in lower case letters, the DNA residues are shown in uppercase letters. Targeting foreign DNA into chloroplast genome While the vast majority of plant genes are found as part of the nuclear DNA, both the chloroplast and mitochondria contain genes that encode a number of important and unique functions. However, not all of the proteins that are present in these organelles are encoded by organellar DNA. Some chloroplast and mitochondrion proteins lie encoded in the nuclear DNA, synthesized in the cell’s cytoplasm and then, by a special mechanism, imported into the appropriate organelle. Accordingly there are two ways that specific foreign protein can be introduced into the chloroplast or mitchondrion. In one way, a fusion gene encoding the foreign protein and additional amino acids that direct the transport of the protein to the organelle can be inserted into the chromosomal DNA, and, after synthesis, the recombinant protein can be transported into the targeted organelle. In the other way the gene for the foreign protein can be inserted directly into chloroplast or mitochondrial DNA. Higher plants have approximately 50 to 100 chloroplast s per leaf cell, and each chloroplast has about 10 to 100 copies of the chloroplast DNA genome. Stable genetic transformation of chloroplasts in order to modify chloroplast functioning or to produce foreign proteins requires insertion of the foreign DNA into the chloroplast genome rather, than into the much larger chromosomal DNA. (Plant chromosomal DNA is generally around 10 to 100 times larger than chloroplast DNA.) Moreover, the foreign DNA needs to be present in approximately all the chloroplast DNA genomes per leaf cell. Initially, foreign DNA was introduced by microprojectile bombardment into the chloroplast genome on a plasmid vector with both the non selectable foreign DNA and selectable marker such as an antibiotic resistance gene, flanked by specific chloroplast DNA sequences –homologous recombination is the normal mode of DNA integration into the chloroplast genome. Figure2. Plasmid vector used for integrating tandem genes into the chloroplast genome Approaches for studying expression of foreign genes One of the most direct approaches for studying the expression of foreign genes in transformed plant tissue is to measure the abundance, or activity, of the gene products encoded by the transferred genes. Many promoters and putative gene regulatory sequences have been analysed by making fusions with reporter genes. These are usually the coding sequences of bacterial enzymes for which convenient and sensitive assays are available and whose activities are not normally found in higher plant tissues. Among the most common are octopine and nopaline synthase, CAT, NPT-II and GUS. In situations where the gene product under investigation has no enzymatic activity, gene expression may be screened by western blotting or other standard immunological methods. In addition to this, molecular analysis like polymerase chain reaction, Southern analysis and Northern analysis are carried out to check for the integration of the transgenes. Secretion of foreign proteins Transgenic plants have the potential to be exploited as bioreactors for production of vaccines, therapeutics, antibodies etc.But harvesting and purification of these compounds from the plant cells are very difficult. To overcome this problem, plants are engineered to secrete foreign proteins through the roots directly to a hydroponic culture medium by a process called “rhizosecretion”.Normally roots secrete large amounts of sugar and aminoacids, but relatively lower levels of proteins. Bacteria found in root zone of plants use root exudates as source of nutrients. Small organic molecules like sugar and amino acids are first secreted to the root intercellular space (apoplast) before they are exuded by the roots. Three different foreign proteins namely-xylanase from Clostridium thermocellum, GFP from the jelly fish Aequorea victoria and human placental secreted alkaline phosphatase were engineered to secrete through roots. These three proteins were directed to the root apoplast using three different direction signals. Each protein was efficiently exuded by the roots of transgenic tobacco plant, as long as the genetic construct contained a DNA fragment encoding a signal peptide, placed upstream of the gene whose protein was targeted for secretion. Both the 35S promoter which is expressed in all types of plant cells and mas2’ promoter, which is preferentially expressed in roots direct the synthesis of target protein in root tissue. With the 35S promoter the foreign protein can be also recovered from the guttation fluid (leaf exudates). Pmas2’ SP DNA Construct for GFP protein Pmas2’-mannopine synthase promoter SP –signal peptide gfp- gfp gene isolated from A.victoria gfp