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The Green Planet > Plant Genome Manipulation > Flashcards

Plant Genome Manipulation Flashcards

1
Q

What is Genetic Transformation?

A

Introduction of genes into genomes by means other than gamete fusion or somatic cells

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2
Q

How does transformation differ from sexual reproduction?

A

DNA can be introduced from unrelated sources, and can introduce single genes other than whole genomes

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3
Q

What is transient transformation?

A

DNA has not been integrated into the genome and so may be lost when cell divides
DNA integrated into genome will be passed down by meiosis and mitosis

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4
Q

What is crown gall disease?

A

Tumour like growth often caused by insects at the crown of the plant (where root meets stem)
Caused by soil bacterium Agrobacterium tumefaciens which detects and infects wounded cells (common at crown where bending occurs)

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5
Q

What causes tumour production?

A

Agrobacterium alters the host cells by introducing factors into them
Tumours become independent of bacteria presence after long enough (4 or so days?)
High levels of IAA (Auxin) and CK (cytokinin) in gall tissues suggest that the cells have acquired the capacity for autonomous growth by permanent activation of growth substance synthesising systems

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6
Q

What is the difference between culturing normal plant tissue and Gall tissue?

A

Normal plant tissue need exogenous factors to live indefinitely in medium
Gall tissue does not

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7
Q

What unusual metabolites are present in the gall tissue of Jerusalem artichoke

A

Octopine and Nopaline

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8
Q

What metabolites are found in all gall tissues?

A

Opines (Arginine derivatives)
Different opines depending on agrobacterium strain

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9
Q

What is the effect of opine production on the plant cell?

A

Opines can’t be metabolised by the plant only the bacterium
Locks away nutrition from the plant cells for bacterial nutrition

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10
Q

What causes the Agrobacterium to be virulent?

A

The tumour inducing (Ti) plasmid
This plasmid is absent from avirulent strains (cannot form tumours)
Transferring the plasmid to Avr strain transfers virulence
Ti plasmid is present in DNA from tumour cells

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11
Q

What is the structure of the Ti plasmid?

A

T-DNA (transfer DNA)
T-DNA is flanked by 2 25bp repeat border sequences
Virulence genes which are required for T-DNA movement into plant cell

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12
Q

What induces Vir gene expression?

A

Small plant derived molecules such as Acetosyringone
Acetosyringone likely perceived by VirA protein encoded on Ti plasmid
VirA and VirG induce other vir genes in response to plant signals

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13
Q

How is the T-DNA transferred?

A

By a multi-subunit type IV secretion system (secretion through a protein complex that traverses the cell envelope) made up of different vir gene products

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14
Q

What does opine production induce in the Agrobacterium?

A

Expression of genes required for opine uptake and catabolism (permeate and hydrolase enzymes)

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15
Q

Why is tumour formation beneficial for the bacterium?

A

More cells producing opines?

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16
Q

How does the T-DNA region produce tumours?

A

Contains genes for auxin and cytokinin production
Helps to induce tissue growth and tumours

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17
Q

What industrial use does Agrobacterium have?

A

As a vector for plant transformation
Modify Ti plasmid to make it non-oncogenic and non opine producing
Regenerate transgenic plant from transformed cells to produce full plant

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18
Q

How can the altered plasmid be put into the Agrobacterium?

A

Electroporation
Conjugation

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19
Q

How can the Ti plasmid be made easier to work with?

A

Usually >200kb per plasmid so is susceptible to mechanical damage when extracted
Also low number of copies per cell

Can be split into two plasmids:
One helper plasmid which contains the vir genes (stays in agrobacterium)
And the T-DNA plasmid which is manipulated in vitro

20
Q

Requirements for transformation?

A

-Means of getting the DNA into plant cells and stably incorporated into host chromosome
-Means of selecting transformed cell, often using gene for antibiotic resistance
-Need to regenerate transgenic plant from transformed cells, need to make them divide, normally done via tissue culture (except arabidopsis where egg cells can be transformed)

21
Q

How many genes does arabidopsis have?

A

26,000
Can predict protein sequence and function by DNA sequence
Large percentage of the genome has unknown function

22
Q

What is a side effect of T-DNA insertions?

A

they don’t require homologous recombination, are not targeted to specific locations on the genome
They can insert into existing genes and disrupt them
This creates a mutant plant
Can determine gene which has been inserted into by looking at the mutant (genome/phenotype)

23
Q

What is a method for easily transforming arabidopsis seeds?

A

Floral Dipping
Dip the flowers from a flowering arabidopsis into Agribacterium culture
Produces mutant seeds
Can grow the mutant seeds to maturity
High efficiency T-DNA transformation

24
Q

Why is floral dipping possible in arabidopsis?

A

In early developing arabidopsis flowers the carpels are open
Agribacterium can enter and transform ovules

25
Method for detecting transgenic embryos resulting from floral dipping?
Antibiotic resistance markers
26
Primary transfortmants are hemizygous for each inserted gene. What does this mean?
It only has the one copy of the gene on one of its chromosomes, no other copy/allele on the other homologous chromosome. This means that if a gene is disrupted only one copy of it is.
27
Why does the primary transformant not usually have a mutant phenotype?
Only one copy of the gene is disrupted (due to hemizygosity)
28
How can you get a homozygous mutant phenotype from a primary transformant?
If the primary transformant is self pollinated then 1/4 of the progeny will be homozygous mutant 1/2 will be heterozygous and so still antibiotic resistant (3:1 segregation as trait is controlled by 1 gene) Need to screen phenotype of antibiotic resistant progeny to identify mutant phenotypes and select them
29
What is forward genetics?
Going from phenotype to finding the genotype If a trait shows heritability with 3:1 segregation this defines a single gene that controls the trait Identifying the DNA encoding the gene, can work out the sequence and therefore what protein it makes and how that controls the trait
30
What is reverse genetics?
Start from the genome sequence What phenotypes result from inactivating this gene Done by systematic isolation of flanking sequences of 100,000s T-DNA insertion (flanking sequences are the gene which was disrupted by the T-DNA insertion)
31
What is an example of a case where reverse genetics is better than forward genetics?
Identifying Sepallata genes SEP 1-3 exist, have redundancy if one is inactivated others can fill gap Mutant phenotype requires all 3 to be disrupted Can cross plants with disruptions in each to reveal mutant phenotype Can only be found in reverse screen as randomness of forward screening makes it unlikely to get an individual with all 3 mutations
32
What is the promoter and terminator sequence in DNA?
Comes directly before the transcribed region (which contains the coding region) and decides when and where the gene is expressed Terminator sequence comes after the transcribed region and ceases transcription (???)
33
What is a chimeric gene?
A gene where parts of different genes are mixed together Swapping promoters can change where a gene is expressed
34
What is ectopic expression
Expression of a gene in a place where it usually isn’t Can be caused by adding a promoter from a different gene
35
What is Over/Constitutive expression?
Usually driven by promoters from pathogens/viruses. Designed to express at high levels in all plant cells Most commonly used if 35S promoter from cauliflower mosaic virus
36
What is the result of ectopic expression of SEP3 in leaves?
If the promoter for SEP3 is changed to express in leaves, the leaves developed as petals
37
What are reporter genes?
Transcriptional: Reports where a promoter is active Translational: Reports where protein product is produced and where it’s localised in cell
38
Advantages and disadvantages of GFP?
Self sufficient fluorescence, doesn’t need substrate added to report Can see it live in plants Is small so can be fused with other proteins w/out disrupting function Many variant available in different colours Disadvantage is that it doesn’t quantify, only qualitataive Hangs around for a while in the cell
39
How does GFP fluoresce?
Fluorophore located on the central helix Deprotonated phenolate of Tyr66 causes fluorescence
40
When is overexpression or ectopic expression of a gene useful?
Plant genomes have many duplicated genes which act redundantly Loss of function mutations don’t help to identify gene as redundancy takes over Overexpressing the gene or expressing it ectopically allows you to see the effect of the gene where a loss of function mutation would not
41
What are the two different ways of expressing GFP?
As a transcriptional reporter by attaching the GFP gene to a specific promoter for the protein you’re looking at As a translational reporter by adding the GFP sequence into the end of the gene for the protein Protein is translated with GFP attached, can see where protein is localised in cell
42
How does luciferase enzyme report?
Firefly enzyme Breaks down luciferin substrate and produces light
43
Advantages and disadvantages of luciferase?
Can be viewed in live plants Luciferase is rapidly inactivated and so activity largeley reflects the newly synthesised protein. Good for monitoring genes with rapidly changing expression (circadian genes) Disadvantage is that luciferin is expensive and has to be constantly supplied, also cellular resolution is poor, not good for localisation
44
How does the E. Coli GUS gene work?
Encodes beta-glucoronidase Glycosidase enzyme that catalyses the production of coloured products if supplied with appropriate substrate
45
Advantages and disadvantages of E. Coli GUS?
It is very sensitive so is good for genes expressed at low levels Also gives quantitative results, good for measuring expression levels Disadvantage that plant usually has to be killed to assay GUS Enzyme and product are very stable so aren’t good for seeing dynamic changes in expression Also not good for seeing intracellular localisation

The Green Planet (8 decks)

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