Submitted by: Kirti

Ph.D.

Advances in Genetic Engineering (MBB 602)New Promoter

and Selection

system for transgenics

PromotersAn obvious requirement for any genes that are to be expressed as transgenes in plants is that they are expressed correctly.

the major determinant of gene expression (level, location and time) is the region upstream of the coding region, termed Promoter.

A promoter sequence is the site to which RNA polymerase first binds during the initiation of transcription.

Affinity of RNA polymerase to the promoter sequences is several order of magnitude higher than that for other DNA sequences.

This is an important consideration as many of the genes (reporter genes,

selectable genes) that are to be expressed in plants are bacterial in origin.

They therefore have to be supplied with a promoter that will drive their expression in plants.

(Slater A, Plant Biotechnology)

In order to produce a transgenic plant, an isolated promoter is inserted into a vector and linked to a heterologous DNA sequence.

Any gene that are to be expressed in the transformed plants have to possess a promoter that functions in plants.

Types of Promoters used to regulate gene expression

1. Constitutive promoters are commonly used type of promoter. They are capable of expressing linked DNA sequences in all tissues of a plant throughout

normal development.

Monocot promoters- Plant ubiquitin promoter (Ubi), Rice actin 1 promoter (Act-1), Maize alcoh dehydrogenase 1 promoter (Adh-1).

In addition to promoters obtained from plant genes, there are also promoters of bacterial and viral origin which have been used to constitutively express novel sequences in plant tissues.

Examples of such promoters from bacteria include the octopine synthase (ocs) promoter, the nopaline synthase (nos) promoter and others derived from native Ti plasmids.

The 35S and 19S promoters of cauliflower mosaic virus are commonly used examples of viral promoters. Plant pathogen/Dicot promoters- Opine promoters, CaMV 35S promoter.

Promoters from the nopaline synthase (nos), octopine synthase

(ocs) and mannopine synthase (mas) genes have been isolated and inserted into transformation vectors upstream of foreign genes to control the expression of those genes.

Used mainly for transformation of dicotyledonous (dicot) plants.

Nos, ocs, mas are compact promoters less than 400bp long.

Nos is the weakest promoter in this group while ocs and mas are stronger in the order given.

The nos promoter is highest in older leaves, stem and flowers of

tobacco and is inducible by wound, auxin (Kim et al., 1994).

The CaMV 35S promoter (most widely used)

The 35S promoter is a very strong constitutive promoter, causing high levels of gene expression in dicot plants. it is less effective in monocots, especially in cereals. the differences in behavior are due to differences in quality and/or quantity of regulatory factors.

Part of the domain A of the CaMV 35S promoter, which contains the TATA box and extends from the -90 position to the transcription start site +1, is used as a "minimal promoter."

Apart from the TATA box, which is the binding site for RNA polymerase II, the region contains a least three CAAT-like boxes. These sequences potentiate the activity of upstream sequences and influence the efficiency of the promoter activity.

Minimal promoter does not drive the expression of a gene by itself. 

Additional sequences, such as an enhancer, are required.

The subdomain B of the 35S promoter harbors an enhancer element that increases promoter activity.

Cassava vein mosaic virus (CsVMV)

Because of its constitutive properties, the CsVMV promoter can be used in plant biotechnology as an alternative to the widely used 35S CaMV promoter.

34S promoter from figwort mosaic virus (FMV)

The sugarcane bacilliform badnavirus (ScBV) promoter

The carnation etched ring virus promoter

Maize alcohol dehydrogenase promoter (Adh)

In maize, there are two proteins identified that allow them to thrive under transient anaerobic conditions: alcohol dehydrogenase I (ADH-I) and II (ADH-II).

Adh-I gene promoter has been widely used.

In the 5' untranslated regions of the maize Adh genes, anaerobic regulatory elements (AREs) and an intron (of the Adh-1 gene) are important for driving gene expression in monocots.

The regulatory elements responsible for the anaerobic response of the genes are within a 247 bp (-140 and -99 of the maize Adh-1 promoter) upstream.

Within this 40 bp segment, there are two essential regions, each of around 15 bp, required for expression under low oxygen conditions.

The Adh-1 promoter has been used in cereals such as rice, oat and barley and superior transformation occurs when the Adh-I promoter is in conjunction with the intron.

Dicots such as tobacco, to drive the expression of genes of interest, it provides very low levels of expression.

The promoter construct has been tested in transient assays in various monocot crops, driving a very high level of gene expression compared to the 35S CaMV promoter.

Plant Actin promoters

The promoter of the rice Act-1 gene has been used as a strong constitutive promoter drives the expression of genes of interest in monocots.

The Act-1 gene from rice has a short 5' non-coding exon, separated by a 447 bp intron (intron 1) from the first coding exon.

The presence of the first intron of the gene has proved to be fundamental for the efficient gene expression from the Act-1 promoter. .

Other Constitutive Promoters

Promoters that has been successfully employed in monocot plants to achieve strong expression of the marker gene:

The Ubiquitin (from Maize) Ubi-1 and Ubi-2

pEmu

2.Tissue-specific promoters are those promoters that are capable of selectively expressing heterologous DNA sequences in certain plant tissues. Eg., anther specific promoter for induction of male sterility, LEA protein promoters for gene expression during embryonic development.

Fruit-specific promoter – promoter region from the ethylene regulated genes E4 and E8 and from

the fruit-specific polygalacturonase gene have been used to direct fruit specific expression of a heterologous DNA sequence in transgenic tomato plants.

It is preferable to use promoters from homologous or closely related

plant species to achieve efficient and reliable expression of transgenes in particular tissues.

Root specific promoters- have been of particular use in engineering resistance to nematodes and

improving plant tolerance to environmently stressfull conditions such as water, salt and heavy metals.

In an attempt to engineer resistance to the parasite in transgenic tomato plants, the root specific promoters (tob) was used to direct the expression of the sarcotoxin IA (Radi et al., 2006).

Result: sarcotoxin IA gene was selectively toxic to the parasite and non-toxic to the plant.

Seed Specific Promoters-

The majority of available seed specific promoters originate from seed storage proteins (SSPs) such as rice glutelin & globulin, soybean lectin & β-phaseolin, Brassica napin, Maize zein.

The genes that encode for the prolamin storage proteins are an ideal source for the isolation of seed specific promoters as these proteins are exclusively synthesized in the endosperm & are expressed at high levels during seed development in most cereals.

Tissue-specific promoters of interest in genetic engineering strategies include those associated with photosynthesis where expression is restricted to "green" tissues, and is considerably lower or absent in non-photosynthetic tissues like roots, mature flowers (excluding sepals) and mature fruit/nuts.

Transformation of tomato plants with the tomato RBCS3A [Rubisco small subunit] gene promoter fused to GUS resulted in leaf-specific expression compared to green fruits.

A minimal peach chlorophyll a/b-binding protein gene promoter (Cab19) was isolated and fused to an uidA (β-glucuronidase [GUS]) gene,the promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. (Bassett et al., 2007)

Tobacco plants were transformed by using a chimeric gene construction, in which a corn sucrose synthase-1 gene (Sh) promoter was used to direct expression of the beta-glucuronidase (Gus) reporter gene. Expression of Sh-Gus activity in these plants was found to be cell (phloem) type specifc.

3. Inducible promoters-

A very popular way to regulate the amount and the timing of protein expression is to use an inducible promoter.

An inducible promoter is not always active the way constitutive promoters are (e.g. viral promoters).

Their performance is not conditioned to endogenous factors but to environmental conditions and external stimuli that can be artificially controlled.

Some inducible promoters are activated by physical means such as the heat shock promoter. Others are activated by chemical such as Tetracycline (Tet).

Chemically-regulated promoters-

Promoters whose transcriptional activity is regulated by the presence or absence of alcohol, tetracycline, steroids, metal and other compounds.

Alcohol-regulated

alcR gene placed under the control of a strong constitutive promoter such as CaMV 35S, and a modified alcA (alcohol dehydrogenase I (Adh-I) encoded by the alcA gene) promoter linked to a gene of interest or target gene. 

In the presence of an inducer (e.g., an alcohol), the AlcR (transcriptional activator protein) binds to the specific sequences of the modified alcA promoter and the target gene is expressed.

Tetracycline-regulatedPromoter repressing system- a CaMV 35S promoter is modified by introducing a tet operator sequence upstream and downstream of the TATA box. In the absence of tetracycline, overexpressed TetR binds to the tet operator and prevents gene expression.In the presence of tetracycline, TetR no longer binds the operator and gene expression is turned on.

In tobacco, the expression of the tetracycline-inducible CaMV promoter could be modulated up to 500-fold. This inducible promoter has also worked in tomato and potato.

The system presents some problems: For tetracycline to work as an inducer, it must be supplied continuously to the medium due in part to the short-half life of the antibiotic.

In addition,TetR must be in high concentration to be effective as a repressor as it has to compete with at least forty proteins that assemble around the TATA box.

For some plants, such as Arabidopsis, high concentrations of the repressor are toxic and alter the photosynthetic physiology of the plant.

Tet as a promoter activating system-

TetR is fused to the acidic activation sequence of the herpes simplex virus protein 16 (VP16), forming a tetracycline transactivator (tTA) fusion protein which has the DNA binding specificity of TetR and the promoter activating function of VP16.

In the absence of tetracycline, tTA binds to tet operator sequences placed upstream of a TATA box in a target promoter and activates transcription. 

When tetracycline is provided, it forms a complex with tTA and releases the operator, thus, turning off gene transcription.

The system has worked in tobacco and in Arabidopsis.

Pathogenesis-related-

Pathogenesis-related (PR) proteins are a heterogeneous group of proteins induced in plants by pathogen infection and exogenous chemicals.

PR proteins take part in the systemic acquired resistance (SAR) that develops in a resistant plant upon infection with a pathogen.

Promoter sequences from diverse PR proteins have been isolated from plants such as Arabidopsis and maize.

Chemicals such as salicylic acid, ethylene, thiamine, etc have been identified as inducers of PR proteins.

One of the best studied promoters is the PR-1a promoter from tobacco.

The expression of the beta-glucuronidase (gus) gene, (when driven by the PR-1a promoter), increased 5-10 fold after 1-3 days of induction with salicylic acid.

The PR-1a promoter has also been used to induce the expression of Bacillus thuringienesis delta-endotoxin in transgenic plants.

Drawback: PR gene promoters are induce by common environmental stimuli such

as UV-B, ozone, and also oxidative stress

this feature complicate the control of gene expression by PR promoters in non-laboratory conditions

Physically-regulated promoters –

Promoters induced by environmental factors such as water or salt stress, temperature, illumination and wounding have potential for use in the development of plants resistant to various stress conditions.

These promoters contain regulatory elements that respond to such environmental stimuli.

Temperature-regulated promoters (soybean heat shock (hs) gene promoter linked to the chloramphenicol acetyl transferase (CAT) coding sequence and construct introduced in tobacco and thermoregulated expression of CAT activity examined in leaf extracts).

Light-regulated promoters

In plants, light-regulated promoters are critical in regulating plant growth and development through the modulation of expression of light-responsive genes.

Light-responsive elements from genes such as the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS) gene, the chlorophyl a/b binding protein, and the chalcone synthase have been widely studied.

4. Synthetic promoters- Among the elements of promoter are the TATA box, the transcription start site and the CCAAT consensus sequence, which are required for accurate transcription.

From the sequences of these elements in diverse organisms, it is possible to synthesize consensus sequences that may work across different organisms and are not necessarily derived from a particular organism.

For example: the synthetic promoters from Pioneer Hi-Bred's inventions contain: a TATA motif; a GC-rich region (at least 64% GC); and a transcription start site.

The GC-rich region located between the TATA motif and the transcription start site in plant promoters acts as a very strong inducer of constitutive expression. It increases transcriptional activation efficiency.

Plant-expressible promoters contain a region of about 40% GC, while a 64% or greater GC content is characteristic of animal promoters. The maize ubiquitin 1 gene (Ubi-1) promoter, which produces high levels of activity in monocots, has a GC content of 64%.

The activity of monocot-derived promoters is higher in monocots than in dicots, necessitating the development of both monocot and dicot promoters.

Several gene promoters have been evaluated for their ability to drive the constitutively high expression of transgenes in monocots including ZmUbi1 from maize (Zea mays), and Act1, OsTubA1, OsCc1, RUBQ1 and 2, rubi3 and OsAct2 from rice (Oryza sativa).

The ZmUbi1 promoter is widely used in monocot crops due to its ability to direct high levels of gene expression in virtually all tissues.

This promoter is active in many cell types and drives strong expression in young roots and leaves, but these expression levels decrease markedly as these organs mature (Cornejo et al., 1993).

(Analysis of five novel putative constitutive gene promoters in transgenic rice plants, Park et al., Journal of Experimental Botany, 2010)

The OsCc1 and Act1 promoters are active in both the vegetative and reproductive tissues of transgenic rice plants (McElroy et al., 1990; Jang et al., 2002).

The activity profile of the OsAct2 promoter is similar to that of Act1 in leaves and roots of transgenic rice plants, but is slightly stronger (He et al., 2009).

The rice ubiquitin gene promoters, RUBQ1 and RUBQ2, have been shown to drive higher GUS expression in transgenic rice plants by 8–35-fold, respectively, when compared with the 35S promoter (Wang and Oard, 2003).

The rice polyubiquitin gene promoter, rubi3, has been tested in all tissues and at all growth stages of transgenic rice plants and found to drive a higher level of constitutive expression of reporter genes than the maize ZmUbi1 promoter (Lu et al., 2008).

The promoter R1G1B is active in the whole grain including the embryo, endosperm, and aleurone layer (in rice), represents a constitutive promoter. The APX and PGD1 promoters may provide useful alternatives that facilitate strong constitutive expression of a transgene in the whole rice plant.

Examples of Promoters Promoter origin expression Induced/

upre-gulated by

Heterologous system

reference

Glutelin (Gt3) Oryza sativa

Endosperm Flowering Nicotiana tabacum

Russell and Fromm (1997)

Chalcone synthase (CHS8)

Phaseolus vulgaris

Root epical meristem, petal epidermis, cotyledons, primary leaves

Stress Nicotiana tabacum

Schmid et al. (1990)

Patatin Solanum tuberosum

Tuber Sucrose Not determined

Grierson et al. (1994)

Heat shock protein 17.7 G4

Helianthus annus

Vegetative tissues

Heat shock Not determined

Coca et al. (1996)

Sc Sugarcane bacilliform badnavirus

Constitutive Not determined

Several monocot and dicot species

Schenk et al. (2001)

At IRRI, transformation of rice with a synthetic cry1A(b) gene under the

control of the maize PEP carboxylase promoter produced a line with high expression of the cry1A(b) gene and increased resistance to striped stem borer and yellow stem borer (Ghareyazie, 1996).

The maize PEP carboxylase promoter give expression in leaf blades and sheath but not in endosperm.

The rbcL (gene encoding the large subunit of ribulose bisphosphate carboxylase) and atpB (gene encoding ATPase subunit β) gene in maize are transcribed from a PEP (plastid-encoded plastid RNA polymerase promoter) (Silhavy, 1998).

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions (Narusaka et al., Plant J. , 2003).

A promoter element that is important for dehydration and ABA-responsive expression was first identified in lea gene rab16A of rice (Mundy et al., 1990).

Analysis of the promoter regions of other dehydration induced genes led to the discovery of dehydration responsive element (DRE) in Arabidopsis.

The same motif known as C-repeat (CRT) was also identified in promoters of genes that respond to the cold stress.

120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences (Narusaka et al., Plant J. , 2003).

To develop an efficient and reliable transformation approach, selectable marker is the prerequisite and extremely useful in enabling successful transformation.

Selectable markers are those which allow the selection of transformed cells, or tissue explants, by their ability to grow in the presence of an antibiotic or a herbicide.

Due to the very low efficiency and randomness of transgene integration into genome of host cells, a marker gene is always needed to distinguish the “true” transformed cells, tissues and regenerated shoots from non-transformed ones.

The genetic markers developed for use in plant cells in general have been derived from either bacterial or plant sources and can be divided into two types: selectable and screenable markers.   

The selectable functions on most general transformation vectors are prokaryotic antibiotic-resistance enzymes which have been engineered to be expressed constitutively in plant cells.

Enzymes affording protection against specific herbicides have also been used successfully as dominant marker genes.

The enzyme coding sequence is normally fused to promoters isolated from T-DNA or the CaMV genome at the 5’ end, and a polyadenylation signal, often from a T-DNA gene, at the 3’ end. (Plant Transformation and Genetic Markers, Rao K.S and Rohini V.K)

The most widely used selectable markers in Cereal transformation are the genes encoding Neomycin phosphotransferase (nptII), Hygromycin phosphotransferase (hpt), Phosphinothricin acetyltransferase (bar) (Cheng et al., 2004).

Neomycin phosphotransferase (nptII)

nptII is the most widely used selectable marker for plant transformation.

It was initially isolated from the transposon Tn5 from the bacterium strain Escherichia coli K12.

Neomycin phosphotransferase-II (NPT-II) is a small (25 kd) bacterial enzyme which catalyses the ortho-phosphorylation of a number of aminoglycoside antibiotics including neomycin and kanamycin.

The reaction involves transfer of the gamma-phosphate group of ATP to the antibiotic molecule, which detoxifies the antibiotic by preventing its interaction with the target site-the ribosome.

Plants such as maize, cotton, tobacco, Arabidopsis, flax, soybean and many others have been successfully transformed with the nptII gene.

In plants, it is normally used in concentrations ranging from 50 to 500 mg/l. It is very effective in inhibiting the growth of untransformed cells.

Kanamycin is ineffective as a selection marker for several legumes and gramineae. In rice, kanamycin interfere with the regeneration of transformed cells to green plants.

As an alternative, paromomycin can be used for selecting nptII-transformed rice cells.

Thus, the choice of the selective agent is important and based on the plant species to be transformed.

Monsanto is the owner of patents on the use of nptII as antibiotic resistance gene for plant transformation.

The bifunctional marker by the National Research Council of Canada has the nptII gene linked to a gus gene.

The hygromycin phosphotransferase (denoted hpt, hph or aphIV) gene was originally derived from Escherichia coli.

The gene codes for hygromycin phosphotransferase (HPT), which detoxifies the aminocyclitol antibiotic hygromycin B.

A large number of plants have been transformed with the hpt gene and hygromycin B has proved very effective in the selection of monocotyledonous plants.

Most plants exhibit higher sensitivity to hygromycin B than to kanamycin in case of cereals.

The sequence of the hpt gene has been modified for its use in plant transformation. Deletions and substitutions of amino acid residues close to the carboxy (C)-terminus of the enzyme have increased the level of resistance in certain plants, such as tobacco.

Bar gene as a selection marker

Members of the genus Streptomyces produces antibiotic bialaphos. It consists of a glutamic acid analogue moiety, called phosphinothricin

and two alanine residues.

Bialaphos is an inhibitor of the key enzyme in the nitrogen assimilation pathway, glutamine synthetase (GS).

It becomes active after removal of the alanine residues by intracellullar peptidases.

The remaining glufosinate compound inhibits GS and leads to accumulation of toxic levels of ammonia in plant cells.

A bar gene has also been isolated from Alcaligenes faecalis encodes a phosphinothricin acetyl transferase (PAT) enzyme, detoxify glufosinate by acetylation of the amino group.

Issues on the use of antibiotic resistance genes

There are concerns that antibiotic selectable marker genes might unexpectedly recombine with pathogenic bacteria in the environment or with naturally occurring bacteria in the gastrointestinal tract of mammals who consume genetically modified food

contributing to the growing public health risk associated with antibiotic resistance for infections that cannot be treated with traditional antibiotics and considered as a hindrance in their commercialization.

The presence of antibiotic resistance genes in foods might produce harmful effects- 1. consumption of these genetically modified foods might reduce the effectiveness of antibiotics to fight bacterial diseases; antibiotic resistance genes produce enzymes that degrade antibiotics. 2. antibiotic resistance genes might be transferred to human or animal pathogens, making them resistant to antibiotics.

(POTENTIAL ADVERSE HEALTH EFFECTS OF GENETICALLY MODIFIED CROPS, Anita Bakshi, Journal of Toxicology and Environmental Health, Part B, 6:211–225, 2003)

A genetically engineered Bt corn variety from Novartis includes an ampicillin resistance gene (Cannon, 1996). Ampicillin is an antibiotic that is used to treat a variety of bacterial infections in humans and animals. A number of European countries, including Britain, have refused to allow the Novartis Bt corn to be grown.

(POTENTIAL ADVERSE HEALTH EFFECTS OF GENETICALLY MODIFIED CROPS, Anita Bakshi, Journal of Toxicology and Environmental Health, Part B, 6:211–225, 2003)

Safety issue- GE crops fed to animals today (e.g. Syngenta’s insect-resistant GE maize, Bt176) contain antibiotic resistance genes. The survival of GE DNA in the gut of animals raises the possibility of horizontal gene transfer of GE DNA to gut bacteria. Precaution clearly demands that any use of antibiotic resistance genes in GE crops be prohibited. The phasing out of antibiotic resistance genes is required by the EU and FAO/WHO. (Greenpeace briefing, sept. 2005)

Alternative methods to antibiotic resistance marker genes

Two general strategies have been pursued to avoid the use of antibiotic resistance genes :

1. Elimination of the selectable marker gene in the resultant

transgenic organism.

2. Use of a non−toxic compound that favors or promotes the regeneration and growth of transformed cells expressing a transgene product that acts on the compound.

In the first strategy, the methods currently employed are:

Co-transformation- A simple approach is to co-transform plant cells with two separate pieces of T-DNA, one with a selective marker gene and the other with genes of interest and to select marker free progeny from the co-transformants (Hohn et al., 2001).

Unlinked integrations of the two T-DNAs lead to the segregation of the marker gene from the gene of the interest in the T1 generation.

Co-trasformation can be performed using either two strains or a single strain of A.tumefaciens.

Hohn et al. (2001) suggested that the elimination of marker gene by co-transformation may be especially useful when using Agrobacterium-mediated transformation.

Though simple and effective, this method carries several unavoidable limitations:

It is time consuming and compatible only for sexually propagated fertile plants.

The tight linkage between co-integrated DNAs may limit the efficiency of co-transformation.

Both may integrate in the same loci and only a proportion of plants carrying the selectable marker will also carry the desired gene at an unlinked site (Scutt et al., 2002).

This method may not be suitable for species with very low transformation efficiency.

(An update on the progress towards the development of marker-free transgenic plants, Botanical Studies (2010) 51: 277-292)

Site−specific recombination systems- In site specific recombination, DNA strand exchange takes place between segments possessing only a limited degree of sequence homology (Coates et al., 2005).

Three site specific recombination systems are well known and described for the elimination of selection marker genes.

These are the Cre/lox site specific recombination system (Dale and Ow, 1991), the FLP/FRT recombination system from Saccharomyces cerevisiae (Landy, 1989; Kilby et

al., 1995; Lyznik et al., 1996), and the R/ RS recombination system from Zygosaccharomyces rouxii (Onouchi et al., 1995; Sugita et al., 2000).

The recombination sites are typically between 30 to 200 nucleotides in length and consist of two motifs with a partial inverted repeat symmetry.

The strategy involves the generation of plants that express the cre gene and crossing them with plants in which the selection marker gene is flanked by lox sites.

The marker gene is excised in the F1 generation and the cre gene is segregated away in the subsequent generations.

System-Cre-lox, Flp/Frt and RS/R. The gene construct contains the Lox/Flp/R recombinase sequences flanked by the selection marker gene while the gene of interest lies outside the recombinase sequences (a). When the transgenics containing the Cre/FRT/RS is crossed with the other transgenic containing the Cre, FLP or R recombinase gene sequences (b), the recombination process starts (c) and the marker genes are removed (d) from chromosome. Remaining Cre recombinase sequence in chromosome is segregated out during the advancement of the generation. SMG; Selection marker gene, GOI; Gene of interest. Symbol cycle in the figure represent the recombinase (Cre, FLP or R) proteins.

Transposition-

The strategy is to connect either the transgene or the selectable marker with transposable sequences in such a way that the two entities can be separated from each other in a controlled reaction after transformation and selection.

In the first one, the marker gene is placed on a mobile element which is lost after transposition (Gorbunova and Levy, 2000).

Marker free transgenic tobacco and aspen plants have been generated at low frequencies by inserting the able ipt gene into the transposable element Ac (Ebinuma et al., 1997).

Second, relocation of the desired gene away from the original transgene locus. The feasibility of this approach was demonstrated in tomato (Goldsbrough

et al., 1993; Yoder and Goldsbrough, 1994).

Advantage – transformation of re- calcitrant plants.Disadvantage- time consuming.

Current progress of biosafe selectable markers in plant transformation

Zhengyi Wei, Xingzhi Wang and Shaochen Xing Journal of Plant Breeding and Crop Science Vol. 4(1), pp. 1-8, 15 January, 2012

Once the transgenic plants are generated, selection markers become useless and are likely to have a negative impact on the metabolic activity of transgenic plants due to over-expression of marker gene-encoded proteins or enzymes.

A number of environmental safe and user-friendly selectable marker genes have been exploited in recent years and utilized to mitigate as a result of concerns for bio-security.

Selection strategies Positive selection- marker genes allow for the transformed cells to be

identified without causing lethal effect in the non-transformed counterparts.

Genes associated with hormone biosynthesis

The uidA gene -This gene is originally isolated from Escherichia coli, and encodes the β-glucuronidase enzyme (GUS).

It has been extensively used as a reporter gene in plant transformation in which a glucuronide derivative of the cytokinin benzyladenine (benzyladenine N-3-glucuronide) is utilized as the selectable reagent to inactive cytokinin.

Upon hydrolysis by GUS, active cytokinin is released which will stimulate the transformed cells to propagate.

Development of the non-transformed cells will be restrained due to the lack of GUS gene, thereby the selection of transformed versus non-transformed cells can be achieved.

The ipt gene- The isopentenyl transferase (IPT) is a key enzyme in cytokinin biosynthesis.

The ipt gene isolated from Agrobacteriun tumefaciens could be used as a marker for transformed cells without any additional selection reagent.

In this system, the ipt transgenic cells can be regenerated into plants on the medium without cytokinin because IPT enhances the de novo biosynthesis of cytokinin, non-transformed cells cannot survive due to the lack of the hormone. Overexpression of the ipt gene, a component of the T-DNA, leads to increased cytokinin relative to auxin, which triggers shoot regeneration.

This overproduction of shoots can result in a phenotype of a large number of shoots. This phenotype can be used as a marker (Ebinuma et al., 1997).

Saccharide Metabolism Pathway Related Genes The genes in this group encode particular enzymes in saccharide

metabolism pathway and those rare saccharides (mostly monosaccharides) cannot be used as carbon sources by plants under normal conditions.

The pmi gene The pmi gene from Escherichia coli encodes the phosphomannose

isomerase (PMI), which catalyzes the reaction to convert mannose-6-phosphate into fructose-6-phosphate.

Plant cells lacking this enzyme are not capable of surviving on the medium containing mannose as the main or sole carbon source.

In pmi/mannose selection system, only the transformed cells genetically modified to express E. coli manA gene can utilize mannose and develop further while the non-transformed cells have negligible growth without appropriate energy source (Reed et al., 2001).

The pmi gene is the most widely used alternative bio-safe selectable marker and has been successfully applied in transformation of many species such as wheat (Gadaleta et al., 2006) and sugarcane (Jain et al., 2007).

The xylA gene

Xylose isomerase (XI), encoded by xylA gene from Streptomyces rubiginosus catalyses the formation of D-xylulose from D-xylose.

Some plant species can utilize D-xylulose instead of D-xylose as carbon source.

Cells expressing xylA can grow and recover de novo plants while the wild-type cells are disabled when using xylose as the sole carbon source.

Successfully used in tobacco (Haldrup et al., 2001) and sunflower (Morawala and Rajyashri, 2007).

The atlD gene

The atlD from E. coli strain C encodes the arabitol dehydrogenase, which converts arabitol into xylulose metabolized by plant cells.

AMINO ACID METABOLISM ASSOCIATED GENES The AK and DHPS genes

AK gene encodes aspartate kinase (AK) and the DHPS encodes the dihydrodipicolinate synthase (DHPS)

two key enzymes in branched-chain amino acids (lysine, threonine, methionine and isoleucine) synthesis pathway from asparagine

In a wide range of plant species, the activity of these two enzymes is strongly feedback-inhibited by lysine.

The homologous enzymes from bacteria are less sensitive to lysine inhibition and can be used as selectable markers for plant transformation.

In AK/lysine and DHPS/lysine selection system, the metabolism of endogenous AK and DHPS is feedback-inhibited by milli-molar of external lysine in non-transgenic cells.

In contrast, the transformants can grow normally and regenerate de novo plants. These systems have been used in transformation of several plants successfully (Ufaz and Galili, 2008).

Negative selection

This strategy differs from positive selection in that the selection of transformed tissue is achieved at the cost of non-transformed tissue.

The badh gene

An enzyme in the plant biosynthetic pathway, betaine aldehyde dehydrogenase (BADH) converts the toxic chemical betaine aldehyde (BA) (Selection agent) to the non-toxic glycine betaine (GB), one of best-studied compatible solute protecting plants against various abiotic stresses (Chen and Murata, 2011).

Kumar et al. (2004) reported that the transformed chloroplast genome of carrot harboring spinach-derived badh gene showed highest lever of GB accumulation in transgenic plants conferred strong tolerance to salt stress.

DOG1

Gene isolated from yeast encoding 2-deoxyglucose-6-phosphate phosphatase (2-DOG-6-P) gives resistance to 2-deoxyglucose (2-DOG).

Hexokinase catalyzes the conversion of 2-DOG into 2-DOG-6-phosphate, which can cause inhibition of respiration, impair cell wall formation and protein glycosylation and eventually leading to the cell death.

2-DOG-6-phosphate phosphatase can detoxify 2-DOG-6-phosphate by converting it into 2-DOG through dephosphorylation reaction

In this selection system, only the transformed cells expressing DOGR1 gene can survive and develop into shoots.

When over-expressed in transgenic plants, it was used as a positive selection system for tobacco and potato plants (Kunze et al., 2001).

A new positive selection marker gene AtTPS1, encoding trehalose- 6-phophate synthase, has been developed (Leyman et al., 2008).

The selection agent is nontoxic and common sugar glucose. Wild-type non-transformed Arabidopsis thaliana plantlets germinated on glucose had small white cotyledons (stoppage of the photosynthetic mechanism by external sugar) while transgenic plants expressing AtTPS1 became insensitive to glucose.

The selectable marker gene which encodes trehalose-6-phophate synthase catalyzes the first reaction of the two-step trehalose synthesis.

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