Lecture 12

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Plant transformation technologies: recent
developments
There are two major challenges in the field of plant transformation:
1. Stabilizing gene expression or Avoiding Gene Silencing
2. Multigene engineering or gene stacking
Issue:
1. Most of the transgenic plants produced by conventional
methods succumb to gene silencing.
2. Most of the methods are suitable for transfer of one or two
genes.
Many traits are governed by multiple genes.
Therefore, multi-gene engineering is needed to move into
future biotechnology.
Approaches for stabilizing gene expression:
These are based on:
1. Designing transformation vector judiciously.
2. Controlling transgene integration process.
3. Suppress host silencing machinery
Approaches for multigene engineering or gene stacking:
1.
2.
3.
4.
Make a large transformation vector.
Integrate one by one into a predetermined genomic position.
Plastids
Minichromosomes
Influence of the transformation system on gene expression
Agrobacterium-mediated
1. Gives simpler patterns of integration.
2. Inserts genes in ‘expressed areas’ of the genome.
However,
1. Not efficient for site-specific integration
2. Still several complex integration loci are generated
Particle bombardment
1. Gives more complex integration patterns
2. No site preference.
3. Non-polar transfer.
4. Efficient for site-specific integration
5. Simpler vector system.
Strategies
Tissue culture negatively influences transgene-expression stability
Therefore,
• Limit tissue culture phase.
• Limit exposure to phytohormone containing media.
• Use embryogenesis, if possible, rather than organogenesis.
Modifying locus structure after transformation: Multi-copy
lines can be included in breeding program after converting them
to single-copy lines
Conversion of complex locus to single copy was shown by
different methods:
1. Transposition of a single-copy into a new location.
2. Deletion of extra copies by Cre-lox to recover single-copy
Issue:
Solution:
Silencing may occur in homozygous
plants even if their hemizygous parents
were stable.
Monitor homozygous plants over a
few generations to ensure transgene
stability.
Issue:
Plasmid backbone integration is
not only undesirable, it may also
cause expression instability. Its
integration
may
induce
methylation due to the mismatch
with genomic isochore.
Integration of this vector can be
selected based on NPT expression
(kanamycin resistance), and cells
containing backbone can be
eliminated based on tms2 expression
(sensitive to indole acetamide)
Solution:
Apply backbone de-selection strategy
by using negative selection markers:
codA, tms2 (see below). This works
well with T-DNA vectors but not with
particle bombardment vectors.
RB- GOI- NPT-LB
Integration site and base composition
Plant genomes are mosaics of compositionally homogenous DNA segments with
defined GC content, termed isochores. Because the GC content of genes of
different origins, insertion of foreign DNA into an isochore may mark this region
for inactivation and methylation. In this respect, modification of transgene
sequences should not be limited to optimization of the codon usage to that of the
host species but, ideally, should be broadened to make sure that all sequences
match isochore composition. Since plant genomes consist of mosaics of isochores,
matching base composition means site-specificity. This is particularly true of
alloploid plants, because they contain two or more genomes. E.g.. Tobacco
(allotetraploid) consists of two parental genome.
Hypomethylated vs hypermethylated regions of the genome.
Site-specificity can be obtained by gene targeting employing Cre-lox system. Or
the transgene can be insulated with MARS to protect from surrounding
problems.
Construct design suitable for transgene expression stability:
1. Promoter choice: use plant promoters and avoid using viral
promoters.
2. Avoid inverted repeat structures or inverted transcription units
(see below)
(Promoter X-Gene 1-nos3’:: nos3’-Gene 2-Promoter X or Y)
3. May use MARS to stabilize gene expression
Matrix attachment regions (MARs) are operationally defined as DNA elements
that bind specifically to the nuclear matrix in vitro. It is possible, although
unproven, that they also mediate binding of chromatin (DNA + histones) to the
nuclear matrix in vivo, and alter the topology of the genome in interphase nuclei.
When MARs are positioned on either side of a transgene their presence
usually results in higher and more stable expression in transgenic plants or cell
lines, most likely by minimizing gene silencing. Our review explores current data
and presents several plausible models to explain MAR effects on transgene
expression. (From: Plant Mol Biol. 2000 Jun;43(2-3):361-76)
Expression of site-specifically integrated GUS gene in tobacco
Issue: variable position and structure of the locus cause variation in
transgene expression between individual transformants containing
identical gene construct
Approach:
Inserted GUS gene site-specifically using Cre-lox system.
(for molecular strategy see lecture 10 slide 7).
Result:
All transgenic lines containing the site-specific integration of the GUS gene
express GUS gene at more or less at the same level. No gene silencing, if
viral promoter is not used.
Reference:
Day et al. (2000). Genes Dev. 14, 2869 2880.
Chawla et al. (2006) Plant Biotech J. 4: 209 – 218
Using Mutant Hosts To Avoid Gene Silencing: host is impaired in the
gene silencing pathway From: Plant J. 39(3):440-9, 2004
Basic and applied research involving transgenic plants often requires consistent high-level
expression of transgenes. However, high inter-transformant variability of transgene expression
caused by various phenomena, including gene silencing, is frequently observed. Here, we show
that stable, high-level transgene expression is obtained using Arabidopsis thaliana posttranscriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation
(T1) plants transformed with a β-glucuronidase (uidA or GUS) gene driven by the 35S
cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants
shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise,
when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under
control of p35S, all transformants showed a clear phenotype typified by serrated leaves,
whereas such phenotype was only observed in about one of five wild type transformants. p35Sdriven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked
contrast to the variable expression patterns observed in wild type T2 populations. We further
show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme
gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants,
reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild
type background. MAR-based plant transformation vectors used in a PTGS mutant background
might be of high value for efficient high-throughput screening of transgene-based phenotypes
as well as for obtaining extremely high transgene expression in plants.
From: Plant J. 39(3):440-9, 2004
Schematic representation of T-DNA vectors pp35S-uidA, ppCASuidA, ppOMA1-uidA and
pMAR-p35S-uidA.
Not to scale. uidA: β-glucuronidase coding region; pat: phosphinothricin acetyltransferase
coding region; pNOS: nopaline synthase promoter; p35S: cauliflower mosaic virus 35S
promoter; pCAS: cassava vein mosaic virus promoter; pOMA1: hybrid octopine and
mannopine synthase promoter; tOCS: octopine synthase terminator; tNOS: nopaline synthase
terminator; tg7:terminator of gene 7 of Agrobacterium tumefaciens; chiMAR: chicken
lysozyme MAR; RB and LB: right and left T-DNA border, respectively.
From: Plant J. 39(3):440-9, 2004
Engineered minichromosomes in plants
Genetic engineering for complex or combined traits requires the simultaneous
expression of multiple genes, and has been considered as the bottleneck for
the next generation of genetic engineering in plants. Minichromosome
technology provides one solution to the stable expression and maintenance of
multiple transgenes in one genome. For example, minichromosomes can be
used as a platform for efficient stacking of multiple genes for insect, bacterial
and fungal resistances together with herbicide tolerance and crop quality
traits. All the transgenes would reside on an independent minichromosome,
not linked to any endogenous genes; thus linkage drag can be avoided.
Engineered minichromosomes can be easily constructed by a telomeremediated chromosomal truncation strategy. This approach does not rely
on the cloning of centromere sequences, which are species specific,and
bypasses any complications of epigenetic components for centromere
specification. Thus, this technique can be easily extended to all plant species.
The engineered minichromosome technology can also be used in combination
with site-specific recombination systems to facilitate the stacking of multiple
transgenes.
From: Current Opinion in Biotechnology 2007, 18:425–431
Production of minichromosomes:
1.
2.
Radiation induced chromosomal breakage
From B chromosomes by a breakage fusion-bridge (BFB) cycle initiated in a
specialized translocation between the B chromosome and the short arm of
chromosome 9
From: Current Opinion in Biotechnology 2007, 18:425–431
A chromosomes: Normal set of chromosomes
B chromosome: super-numeray chromosomes (mostly inert)
Yu W, Han F, Gao Z, Vega JM, Birchler JA: Construction and behavior of
engineered minichromosomes in maize. Proc Natl Acad Sci U S A 2007,
104:8924-8929.
This paper describes the targeting of minichromosomes with genes for applications in
plant genetic engineering. It provides proof of concept that engineered
minichromosomes can be used as platforms for the second generation technology of
‘output trait’ GM crops. Also demonstrated that maize supernumerary B
chromosomes can support foreign gene expression and can be modified as vectors for
genetic engineering.
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