Plant Biotechnology

Question 1

Give examples of traits which are selected for in plants and reasons why

Answer 1

Reduced branching - more resources available to fewer seeds (bigger seeds)

Reduced height - more resources available to seeds (bigger seeds)

Reduced seed shattering - reduced seed loss before harvest

Pest resistance - lower pesticide usage, less risk to human health

Herbicide tolerance - fewer treatments (improved weed management, reduced cost, lower energy input, reduced ploughing)

Question 2

Give two examples of architectural modifications that have driven agricultural improvement

Answer 2

1: Modification of Teosinte Branched 1 (TB1) gene 7,500 years ago. Produced domesticated maize from teosinte

2: Modification of the RHT (Reduced Height) gene 50 years ago - caused the green revolution

Question 3

Give a study to show the importance of minimising biotic and abiotic stress on plants

Answer 3

Bray et al. (2000) showed that on average, between 64-87% of potential crop yield is lost due to biotic and abiotic stress, abiotic stress has a significantly larger effect.

Question 4

Describe the TB1 mutant in maize

Answer 4

• During maize development, Mexican farmers found an unbrachnced plant in which the TB1 gene was over expressed
• Over expression is due to a promotor mutation which is present in all modern varieties of maize
• Loss of function of maize TB1 results in a highly branched growth habit - similar to teosinte, but more extreme (Hubbard, 2002)

Question 5

Describe the action of the TB1 gene

Answer 5

• Encodes a transcription factor which represses cell cycle progression genes
• Expressed in buds
• Expressed at higher levels in maize vs. teosinte

Question 6

Describe the function of the RHT1 gene

Answer 6

• Reduced Height 1 gene (Orthologue of maize dwarf-8 gene and Arabidopsis GAI gene)
• Functions in giberllin sensing and controls shoot length and dormancy

Question 7

Give an example of a plant which has been modified by green revolution genes

Answer 7

Basmati Rice

(Peng et al 1999)

• Reduced height of plant means less resources available to seeds
• Conventional breeding can acheive shortening of the plant but rice quality is low
• Transformation of rice with Arabidopsis gai gene produces shorter plants but maintains rice quality

Question 8

Describe the mechanim of gibberellin signalling

Answer 8

In the absence of Gibberellin:

• Reduced Height (RHT) gene encodes a DELLA domain which acts as a transcriptional repressor in the absence of gibberellin

In the presence of Gibberellin:

• Binding of gibberellin to its receptor triggers the degredation of the repressor

(Deletion (gene KO) of the DELLA domain prevents gibberellin induced degredation and produced very tall plants)

Question 9

Describe a single nucleotide polymorphism (SNP) which leads to loss of seed shattering

Answer 9

SNP in the promoter of qSH1

qSH1 codes for a transcription factor that is needed in the abscission layer of the grain

The single base change eliminates its expression in the abscission layer

(Konishi et al 2006)

Question 10

Describe a way of engineering insect resistance in plants and describe ways to prevent insect resistance.

Answer 10

Inserting the Bacillus Thuringensis (Bt) toxin gene into plants

Avoiding resistance

Use multiple toxins - Toxin resistance develops more slowly when two different Bt toxins are expressed

Use of refuges - Allows resistance alleles to remain heterozygous

Suppression of resistance by releasing sterile insects

Question 11

Give ways of engineering herbicide resistance

Question 12

Give examples of biotic and abiotic stressors

Answer 12

Biotic: Pathogens, Pests, Wounding, Reactive Oxygen Species, Oxidative Stress

Abiotic: Light, Herbicides, Ozone, Temperature, Water-deficit (freezing, drought, salinity etc.), flooding, heavy metals

Question 13

Give exmaples of two plants that could be exploited to improve crop resistance to drought and salt

Answer 13

Ressurection plat (Graterostigma plantagineum) - extreme drought tolerance

Ice plant (Mesembryanthemum crsyallineum) - extreme salt tolerance - can pump out salt which then forms as crystals of sodium chloride on the outside oft he plant

Question 14

Give three examples of protective strategies against drought and salt used by plants

Answer 14

1) Reduced transpiration
2) Accumulation of non-toxic osmolytes (osmoprotectants)
3) Sequestration of salt outside the cytoplasm

Question 15

Give some examples of osmoprotectants and and example of their use

Answer 15

Trehalose, Proline, Mannitol, Sorbitol

Garg et al. 2002 - Trehalose accumulation in rice plants confers tolerance to salt and drought stress

Transgenic rice created by transfer of enzymes required to produce trehalose. Two different transgenic lines performed well under salt and drought conditions compared to non-transformed control. Trehalose accumulation (and therefore upregulation) was significantly higher in transgenic plants than controls

Question 16

Give studies which show the ability of plants to sequester salt outside the cytoplasm

Answer 16

Apse et al (1999) - salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in arabidopsis - allows tolerance of 200mM NaCl solutions

_{Zhang and Blumenwald (2001) transferred Na+/H+ antiport into tomatoes. Conferred salt tolerance up to 200 mM NaCl (Zhang et al 2001 did the same with oil seed rape)}

Gaxida et al (2001) - overexpression of Arabidopsis vacuolar pyrophosphatase 1 (AVP1) H+pump confers salt and drought tolerance in arabidopsis (Pumps sodium into vacuole)

Shi et al (2000) -the arabidopsis salt tolerance gene SOS1 (salt overly sensitive) encodes a putative Na+/H+ antiporter. Removal of SOS1 reduces ability of plant to grow even in low salt concentrations

Shi et al (2003) Overexpression of the SOS1 Na+/H+ antiporter improves salt tolerance in Arabidopsis. Mutant plants show reduced Na+ accumulation. ^{NB: Experimentally, the transformation was done using a constitutive promotor, however, when mutant plants were exposed to no salt, SOS1 mRNA was not present instead of a constitutive expression as would be expected. SOS1 mRNA is degraded when NaCl is not present, and the system is therefore inducible.}

Question 16

Give an example of a way gene silencing can regulate stress tolerance

Answer 16

Borsani (2005) - Natural siRNAs regulate stress tolerance

In the absence of salt stress, P5CDH gene which encodes a dehydrogenase (which is involved in the degredation of proline) is constitutively expressed.

In the presence of salt stress, ROS are upregulated, casuing the transcription of SRO5, whose transciptional unit overlaps with that of P5CDH. This creates double stranded RNA which is then marked for degredation, forming siRNA which causes degredation of P5CDH, therefore prevention the destruction of proline.

Question 17

Describe a study which shows an increase in salt tolerance by reducing shoot Na+ accumulation

Answer 17

Moller et al (2009) - used an enhancer trap mecahnism to specifally express a sodium transporter in root stele. Works because the toxicity of Na+ to many crop plants is correlated with overaccumulation of Na+ in the shoot and overexpression of the sodium transporter in the root leads to reduced root-shoot Na+ transport

Question 18

Describe pathways of cold acclimation

Answer 18

1) Low temperature causes an increase in abscisic acid (ABA), this causes the formation of cADP ribose, which causes the upregulation of cytolsic free Ca2+ which acts as a signalling ion to begin downstream gene expression of cold-regulated gene (COR)
2) CBF (c repeater binding factor) binds to the regions of the promotor in the regulator genes of COR genes
3) Proline accumulation mediated by Eskimo 1 gene

Question 19

Describe the phenotype of Eskimo1

Answer 19

• Constitutively freezing tolerant
• Does not depend on the expression of cold-regulated genes (both wt and esk1 genotypes only express COR genes after slow acclimatisation)
• eskimno1 grows smaller and slower than the wt

Question 20

Give an example of how inducible vs. constitutive overexpression of a trait can incur growth defects

Answer 20

Overexpression of CBF1 in brassicae causes drought and freezing tolerance. Constitutive overexpression of CBF3 causes reduced growth in arabidopsis, however induced overexpression confers drought, salt and freezing tolerance.

Question 21

Give ways to reduce Aluminium toxicity

Answer 21

Excretion of organic acids to form complexes with Al3+ ions (e.g. Hydrangea)

Increase in rhizoshpere pH (formation of Al hyroxides)

Question 22

Describe how T-DNA is transfered from Agrobacterium to a plant

Answer 22

Wounded plant cell sends out TF which can activate virulence genes in agrobacterium. T-DNA is transfered to plant via plant delivery system. T-DNA is integrated randomly into the genome in the nucleus, it is then stable and inherited in a mendelian manner - this method is not targeted

Question 23

Describe the binary vector system

Answer 23

Agrobacterium plasmid changed to binary vector plasmid - remove virlence genes. keep T-DNA, border regions and a selectable marker (i.e. antibiotic resistance genes)

Question 24

Give two ways of recovering successful transformations

Answer 24

1. Regeneration of plants from transformed cells (Plant tissue/protoplasts -> callus -> shoots and roots)
2. Floral Dip (High efficacy in Arabidopsis only) - Dip inflorescence in Agrobacterium culture -> let plants seed -> plate seeds on selective medium

Question 25

Give and explain two types of mechanism for the regulation of gene expression

Answer 25

Genetic mehanisms - Depend on nucelotide sequence, can be controlled precisely by construct design (e.g. promotor sequence, splice signals etc.)

Epigenetic mechanisms - do not depend on nucleotide sequence, can not be controlled by construct design, often depend on the site of transgene integration and locus organisation (e.g. chromatin structure, DNA methylation etc.)

Question 26

Give three ways in which transcription initiation can be regulated

Answer 26

Constitutive promtotors - active in plant cells most of the time, useful for maximal accumulation of recombinant (e.g. cauliflower mosaic virus (CaMV) 35S promotor, maize ubiquitin-1 promotor)

Regulated promotors (cell-specific/stage specific) - activity is restricted to particular tissues or developmental stages e.g. maize zein (seed specific), rice sucrose synthase (phloem specific), histone (meristem specific)

Regulated promotors (inducible) - active in response to external signals, examples of native inducible promotors: rbsC promotor (light induced), PR-1a promotor (chemically induced)

Question 27

Outline a method to create tissue specific expression of a gene of interest

Answer 27

1. Utilise an enhancer trap to identify tissue specific expression (enhancer in conjunction with a minimal promotor causes a trascription factor to transcribe a reporter gene. Look in the tissue for specific cell types highlighted by the reporter, pick the cell type that you wish to express the GOI in)
2. Cross breed with gene of interest (Gene of interest line needs to be under control of marker gene promotor - therefore when the enhancer and promotor is activated, ther transcription factor acts on both the marker and the GOI)

Question 28

Outline the process of desiging a synthetic promotor

Answer 28

1. Analyse all genes expessed under certain conditions (of choice) by microarray analysis
2. Find common elements and isolate them
3. Produce new combinations of elements

Question 29

Give three factors that affect the efficiency of initiation of protein synthesis

Answer 29

1. “Kozak” Rules - Purine in position -3 and a G in +4 (pyrimindines in these positions result in at least a 10 fold decrease in efficiency)
2. Non-AUG start codons in good context (-3 A/G +4 G) may be as effective as AUG in a poor context (no -3 A/G or +4 G)
3. Upstream open reading frames woth good context AUGs may prevent GOI from being translated

Question 30

Describe the process of 454 pyrosequencing

Answer 30

Pool of DNA fragments generated from genomic DNA

single strand is attached to a bead which is the amplified in a microarray (10 million copies)

A single bead is placed in each well (400,000 wells) and nucleotides are sequentially run over the wells.

As a nucleotide binds, pyrophosphate is released and this is linked to a luciferase based light emisioon which is recorded by the CCD camera

signal strength is proportional to the number of nucleotides incorportaed

Question 31

Describe the method of marker assisted breeding

Answer 31

Identify molecular markers (SNPs, SSRs, AFLPs)

Construct genetic linkage map

Idebntify qunatative trait loci (QTL) based on co-segregation

Use markers that associate with positive QTLs for tracking traits and selecting individuals with good genetic background

Question 32

Describe forward and reverse genetics and give some pros and cons for each

Answer 32

Forward: Screen for mutants with phenotype of choice, characterise the mutabt, backcross to recurring parent

Pro: No prior knowledge of genes required

Con: Screens laborious/impractical, small gene families etc. mean that single gene mutations may have no phenotype

Reverse: Start with gene sequence, identify a mutation in that geen and see what effect this has on plant

Pro: for HDM/TILLING muttions can be detected in heterozygous state and crooses and selfing used to produce homozygous single or double mutants

Con: Extensive knowledge of candidate genes required

Question 33

Describe the process of TILLING

Answer 33

1. Mutagenise seeds with ethylmethanesulphanate (EMS) which introduces single base changes
2. Self fertilise M1
3. isolate genomic DNA
4. Pool 8-10 samples and amplify target gene using PCR (mutated gene forms heteroduplex bubble, Cel 1 enzyme cuts at bubble, cleaved fragment allows ID of plat carrying mutation
5. Self M2 seed with tartget mutation
6. Cross M2 and 3 and profile metabolites

Question 34

Describe the action of EMS

Answer 34

Attacks guanine bases and causes G to bind to T. After translation, causes a GC - AT change

Question 35

State how to calculate mutation frequency and its relevance to mutation load

Answer 35

population size * 1 or 2 (haploid/diploid) * gene size (in kb) / no. of independant mutations

Mutation load can be high therefore backcrossing is needed to reduce load

Question 36

Describe how opium can be genetically engineered

Answer 36

Challange - Increase noscapine production (anti -tumour agent)

Method - Transcription analysis: revealed 10 genes which are exclusively expressed in a high noscapine producing cultivar

Develop PCR based dominant molecular markers to follow segregation of each of the genes - found that the 10 genes are tighly linked and form a gene cluster for noscapine producation. Noscapine is produced at alow level when the gene cluster is in the heterozygous state

Question 37

Give pros and cons of bacteria as a recombinant protein host

Answer 37

Pro: Rapid and easy to transform

High levels of production

Con: Poor with GC rich genes

Lacks post translational mods

Question 38

Give pros and cons of Yeast as a recombinant protein host

Answer 38

Pro: Eukaryotic

Used in food

Con: Lower yields than bacteria

Doesn’t process all mammalian or plant proteins correctly

Question 39

Give pros and cons of Filamentous fungi as a recombinant protein host

Answer 39

Pro: Active and powerful degrader

Con: Low transformation efficiency

secretes lots of proteases

Question 40

Give pros and cons of insect cells as a recombinant protein host

Answer 40

Pro: Processes plant and animal cells correctly

Con: Low yield

Not stable when transformed

Expensive

Question 41

Give pros and cons of mammalian cells as a recombinant protein host

Answer 41

Pro: Authentic, high value protein

Con: Expesnive and difficult to maintain

Low yield

Question 42

Give 5 things to consider when choosing an expression strategy for a recombinant protein

Answer 42

Stability of protein

Yield

Recovery of protein

Correct processing

Containment

Question 43

Give 4 different types of promotor

Answer 43

Consitutive promotor

RbcL -> Chloroplast

USP -> Seed specific

RbcS -> leaf specific