PLANT GENETIC ENGINEERING
IB6043
INTRODUCTION
Plant genetic engineering relies on multiple approaches to develop transgenic plants. The tools that facilitate the engineering of gene constructs and transfection of plants have been derived from multiple sources, which include both viruses and bacteria. The Cauliflower Mosaic Virus (CaMV)
and Tobacco Mosaic Virus (TMV) have both contributed to the development of vectors and promoter sequences for the design of vectors. The soil pathogen Agrobacterium tumefaciens and the Ti plasmid have both been exploited for the development of techniques for the efficient transfection of plants.
LEARNING OBJECTIVES
• To introduce the learner to the fundamental concepts of plant genetic engineering.
• To introduce the terminology associated with plant genetic engineering.
• To present the tools available to the genetic engineer.
• To introduce the user to techniques of plant genetic engineering.
LEARNING OUTCOMES
Upon completion of this module, the learner should deminstrate the ability to:
1. Design gene constructs for application to the engineering of plants.
2. Design experimental procedures for the genetic transformation
and selection of plants.
SECTION 1:
FUNDAMENTALS
INTRODUCTION
• Plants have been bred selectively by humankind for millenia.
• Most of the crops which we consume today have their origins in South America.
• These were developed as a result of selective breeding of their wild types.
• Genetic engineering involves the application of specific techniques and methods for the development of novel varieties.
• Legal challenges.
• Technical difficulties
• Consumer preference
• Debate on the morality of GM Crops.
Credit:Nicolle Rager Fuller, National Science Foundation
Agrobacterium tumefaciens
• Introduced approximately 30 years ago.
• Allows the direct transfer of one or just a few genes, between either closely or distantly related organisms
• Crop improvement can be achieved in a shorter time compared to conventional breeding
• Allows plants to be modified by removing or switching off specific genes.
• Natures genetic engineers involved in Horizontal Gene transfer (HGT) :
Agrobacterium tumefaciens.
CURRENT STATUS OF GMO CROPS
SOURCE: https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp
NUCLEAR, PLASTID & MITOCHONDRIAL DNA
GENOMIC DNA (CHROMOSOMES), PLASTID DNA (cpDNA 150 Kb) and MITOCHONDRIAL DNA (mtDNA)
GENETIC ENGINEERING OF PLANTS
• The trait.
• The association of the trait with specific genes.
• Chracterization of the complete gene(s) including regulatory regions.
• Design of the gene constructs.
• Selection of the vector.
• Transfection of the host plant.
• Selection of transformants.
• Establishment of genetically modified population.
ENGINEERING TRAITS
• Salinity tolerance.
• Pesticide resistance.
• Hairy root cultures to produce secondary metabolites.
• Yield.
• Efficient use of fertilizer.
• Nitrogen fixation.
• Pest resistance.
TYPICAL PLANT GENES
A typical plant gene consists of the regulatory and structural genes.
Regulatory genes are usually located at the 5′ upstream of a gene, with its own promoter, enhancer, or silencer region.
Structural genes begin with a catabolite activator protein (cap) site,
followed by a leader sequence, start codon, exons, introns, terminator,
and a polyadenylation site (poly-A tail). These elements are responsible
for DNA transcription.
THE CAULIFLOWER MOSAIC VIRUS
The 35S Promoter and its application to Plant GE
This Photo by Unknown author is licensed under CC BY-SA-NC.
Genetic
Engineering with CaMV
Cauliflower Mosaic Virus (CaMV) is a dsDNA pararetrovirus 9 (Gr. VII) which infects plants of the family
Brassicaceae . The dsDNA genome (Approx. 8 Kb) is segmented as a result of replication by reverse
transcription. The 35s RNA promoter sequence enables constitutive
expression in all plant tissues. The virus is Episomal and not
Endogenous. Infects Dicots but not Moncots.
Genome organization and expression strategy of CaMV.
Squires J et al. Plant Physiol. 2011;155:1908-1919
ORF Protein (ID) Function
1 Movement Intracellular Transport
2 Aphid transmission Adhesion to aphid stylet
3 DNA binding DNA replication
4 Capsid Viral coat protein
5 Reverse transcriptase,
Protease, RNase H Reverse transcription 6 Translational activator /
Inclusion Body Protein
Protein translation / Packaging of Virus
7 Unknown Unknown
RNA Designation Function
1 16 S Encodes the gene VI product.
2 35 S Polycistronic RNA involved in the expression of genes I to V.
Template for the reverse
transcription of CaMV genome
into DNA.
CaMV receptor in the Aphid stylet.
National Academy of Sciences et al. PNAS 2007;104:17899-17900
©2007 by National Academy of Sciences
Viral genome is segmented in the virus particle.
After entry into the host plant, the DNA fragments are ligated and circularized.
Genetic
Engineering with CaMV
The CaMV genomic DNA is an infective principle which can be engineered as a tool to deliver a gene of upto 900 bp into plant genomes, this is achieved by replacement of genes II and VII which are non-essential genes with the target gene. Addition of DNA to the 8 Kb
genome results in the loss of the ability of the virus to package itself.
Genetic Engineering with CaMV
Replacement of the insect transmission factor (Gene II) with the target gene results in an infective DNA molecule which can be physically transferred into plant tissue.
Agrobacterium tumefaciens
Agrobacterium tumefaciens
• Soil bacterium
• Natures genetic engineer
• Horizontal gene transfer
• Wild types induce hairy roots and plant tumors.
• Contain a Ti (Tumor Inducing) plasmid.
• Removal of the genes for virulence and replacement with the gene or genes of interest.
• Integrates within the host genome via genetic recombination.
Unknown (VIB-picture), CC BY-SA 3.0 <http://creativecommons.org/licenses/by-sa/3.0/>, via Wikimedia Commons
Marc Van Montagu Jeff Schell
Agrobacterium tumefaciens
Two key advances made Agrobacterium transformation the method of choice for plant transformation.
• Removal of all the T-DNA genes does not impede
the Agrobacterium ability to transfer DNA but does prevent the formation of tumors. This allowed scientists to produce "disarmed strains."
• The two main components for an Agrobacterium plasmid, the T-DNA and the virulence (vir) region can reside on separate plasmids . These
components form the basis of modern Ti plasmid vectors, termed binary Ti
vectors.
T-DNA
E
E Cloned in
Escherichia coli to achieve high copy number
Transfected into A. tumefaciens
Transfected into host plant
T-DNA
• T-DNA Plasmid ranges from 10,000 to 30,000 bases in size.
• Virulence genes (35) for translocation of the plasmid from the bacterium into the plant host.
• T-DNA has been engineered to develop the pCAMBIA vectors which contain a reporter gene and selectable markers.
• PCAMBIA vectors can be cloned in E. coli to increase copy number.
BINARY VECTORS
• A binary vector consists of two plasmids: a disarmed Ti plasmid
carrying the T-DNA region and a helper plasmid containing the
virulence genes.
SIZE OF THE INSERT
• Inserts with a size of less than 15,000 bp can be cloned in E. coli and transfected into the plant host.
• Inserts with a size in excess of 15,000 bp can become unstable in E.
coli.
PLANT PROMOTERS
The promoters can be categorized into three main groups:
Constitutive promoters are active at most of the developmental stages, and they directly participate in maintaining moderate and constant
level of gene expression.
Tissue-specific promoters provide restricted gene expression to certain tissues or gene expression involves in developmental-specific stages.
Gene expressions associated with the inducible promoters are greatly
affected by environmental stimuli, which allow for the regulation of
genes through external factors.
Promoter Source Activity
CaMV35S Cauliflower mosaic virus Constitutive
Ubiquitin RUBQ1, RUBQ2 and rubi3 Rice Constitutive
Ubiquitin Gmubi3 Soybean Constitutive
SCR, SRK Brassica rapa Pollen and stigma specific
Exo70C2 Arabidopsis Pollen and root specific
LMW Glu, HMW Glu-1D1 Wheat Seed specific
Expansin PcExp2 Sour cherry Ripened fruits
Potato class I patatin Potato Tuber/storage organ specific
NtHSP3A Tobacco Stress inducible
PLANT PROMOTER DATABASE
https://www.hsls.pitt.edu/obrc/index.php?page=URL1099585635
SECTION 2:
DESIGN OF GENE
CONSTRUCTS
This Photo by Unknown author is licensed under CC BY-SA.
Genetic Engineering Vectors
Plant transformation vectors are routinely used to genetically engineer
plant variants, these include gene knockouts for scientific research as well as genetically modified plants for commercial agriculture. The CaMV 35S promoter region is one of the most important components of the vector as its constitutive expression facilitates screening using reporter genes.
pCAMBIA Vectors
The pCAMBIA series of vectors are one of the most widely used vectors for plant transformation, they are engineered for integration into the plant
genome using the T-Border sites from the tumor inducing plasmid (Ti) derived from Agrobacterium sp flank the target DNA which needs to be integrated into the plant genome.
pCAMBIA Vectors
The vector is constructed based on the following criteria:
1.
Ability to replicate and be selected in bacteria.2.
Ability to integrate into the plant genome.3.
A verifiable reporter gene and plant selection marker.Target gene(s) can be inserted into the multiple cloning site, once inserted the genes will integrate into the host plant
genome and be expressed.
Following transformation into the plant cells, the genes will integrate randomly into the plant genome. This is one of the major challenges associated with Ti integration sites.
Protocol for GE using pCAMBIA
• Linearization of pCAMBIA vector
• Insertion of gene cassette.
• Transformation of pCAMBIA-gene cassette into plant cells by electroporation.
• Screening for transformants by selection on Hygromycin B.
• Propagation of genetically modified plants.
Gene Stacking
Gene stacking involves the construction of a synthetic chromosome which when transformed into a plant, does not integrate into the plant genome but replicates independently of the existing host chromosomes. This
procedure is essential when more than one gene, or alternately, a cluster of genes involved in a biosynthetic pathway have to be engineered into a
plant.
A synthetic Chromosome with its independent origin of replication.
Each gene cassette comprises a regulatory region with a
promoter binding site, a ribosome binding site, a tissue specific signal tag and a 3’ regulatory region consisting of a terminator and a poly adenylation signal. The gene encoding the enzyme is
indicated in blue.
1. Acetyl CoA Thiolase 2. HMG CoA Synthase 3. HMG CoA Reductase 4. Mevalonate Kinase
5. Phosphomevalonate Kinase
6. Mevalonate pyrophosphate decarboxylase
A gene stack comprising six genes involved in the Isopenteny diphosphate biosynthetic pathway.
Risks associated with Genetic Modification
1.
Transgene escape into landraces.2.
Vertical gene transmission via seeds.3.
Horizontal gene transfer via vectors.4.
Accumulation of toxic gene products.5.
Development of herbicide tolerant varieties.6.
Evolutionary advantage over native crops.GENETIC ENGINEERING: CASE STUDY
Steps in Genetic Engineering
1. Identification of the desired ‘trait(s)’.
2. Characterization of the pathway.
3. Identifying genes involved in the pathway.
4. Gene isolation 5. The construct
6. The transformation and delivery system.
7. Transformation.
8. Screening and commercialization.
Step 1: The trait
Let us consider a hypothetical case in which a protein “DRR” is linked to drought tolerance in Oryza sativa. This protein is found only in wild type O. sativa variety WT-6.
DRR
Step 2: Pathway Characterization
Protein DRR is not the product of a single gene, rather, it is the end product of a pathway, the enzymes of which are encoded by the genes Drase1, Drbase1, Drcase4 and Drrase4.
DRa
DRb
DRc
DRd
DRR Drase1
Drbase1
Drcase4
Drrase4
Step 3: Gene / Gene Cluster
Each of these genes is encoded on a different chromosome in WT-6
1 3 7 8
Drase1
Drbase1
Drcase4
Drrase4
Step 4: Gene Isolation
Have you already learnt to isolate the genes? Yes!
Drase1
Drbase1
Drcase4
Drrase4
Step 5: The Construct
A gene construct: This is a simplified version to help you
understand the concept. Promoters can be tissue specific and this has several advantages.
Drase1 Drbase1 Drcase4 Drrase4
Promoter
Step 6: T & D System
We have to develop a suitable vector or transformation process in order to deliver our genetic construct to its intended target chromosome.
Inducible promoter Recombination site
Recombination site
Selectable Marker Reporter gene Construct
Step 7: Transformation
Transformation can be carried out using Biolistics, Agrobacterim containing Ti plasmid, CaMV or other viral mediated transformations.
Plant Tissue Culture System
Callus transforme d using Biolistics
Screening
Regeneration Shoot
regeneration
Root
regeneration
Grow-out and seed
harvesting
Step 8: Screening / Commercializing
Finally, we screen for transformants carrying our genetic construct and breed commercial lines based on the
molecular breeding strategy.
What can go wrong ?
When a ‘foreign’ or ‘engineered gene’ is introduced into a plant, the following situations can be encountered:
1. Gene does not integrate into the host genome / expresses transiently.
2. Gene integrates but is not expressed.
3. Gene is expressed only in the first generation.
What makes GE difficult?
1. Fate of RNAs.
2. Promoter functionality.
3. Loss of introduced gene(s) as a result of recombination.
4. Lethal introductions.
5. Interference in regulatory pathways.
Transgene Escape
Bacillus thuringiensis and the Monarch Butterfly
Cartagena Protocol on Biosafety
The Cartagena Protocol on Biosafety to the Convention on Biological Diversity is an international agreement which aims to ensure the safe handling, transport and use of living modified organisms (LMOs) resulting from modern biotechnology that may have adverse effects on biological diversity, taking also into account risks to human health. It was adopted on 29 January 2000 and entered into force on 11 September 2003.
Regulatory Guidelines
Genetic modification of plants or in that case any living organisms in
Malaysia is governed by the Biosafety Act 2007. The Malaysian government has implemented a new measure aimed at promoting biotechnology within the country, while complying with the standards set out by the Cartagena Protocol on Biosafety (CPB). The Malaysian Biosafety Act 2007 was
approved by the House of Representatives and entered into force on 1 December 2009.
Governing Body: Genetic Modification Advisory Committee (GMAC)
