Download Lecture 7 Manipulation of gene expression and secretion of foreign

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