Download ppt - Department of Plant Sciences

Lecture 9 Recombinant DNA,
Vector Design and Construction
Neal Stewart and Dave Mann
Discussion questions
1. What basic elements should be included in the design
and construction of an efficient ubiquitous and
constitutive plant gene expression vector?
2. Discuss the advantages and disadvantages of
recombination cloning technologies versus traditional
restriction digestion and ligation technology.
3. Describe a novel strategy to generate a T-DNA vector
that allows the expression of several genes from a single
position in the genome.
4. Discuss the advantages and disadvantages of using
plastid vectors for plant transformation and gene
expression.
5. Describe ways in which transgene technology could be
made more acceptable to the public.
Nucleotide base pairing
A’s pair with T’s
G’s pair with C’s
Nucleotide base pairing occurs
through “hydrogen bonding”
Strands have directionality from
5’ to 3’ and when paired strands
are in “antiparallel” orientation
Figure 8.1
Transgenic plantsAgrobacterium
Any gene, any organism
The new plant will pass the transgene
to its progeny through seed.
Biolistics
Steps to make transgenic plants—
lotta transformation
• Make transformation cloning plasmid
vector
• Transform bacteria (usually Escherichia
coli) to maintain clone
• Characterize plasmid (restriction digest
and sequencing)
• Transform Agrobacterium (if using
Agrobacterium) and characterize
• Transform plant
Recombinant DNA history
1966 The genetic code is deciphered when biochemical analysis reveals
which codons determine which amino acids.
1970 Hamilton Smith, at Johns Hopkins Medical School, isolates the first
restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide
sequence. Over the next few years, several more restriction enzymes will be
isolated.
1972 Stanley Cohen and Herbert Boyer combine their efforts to create
recombinant DNA. This technology will be the beginning of the
biotechnology industry.
1976 Herbert Boyer cofounds Genentech, the first firm founded in the United
States to apply recombinant DNA technology
1978 Somatostatin, which regulates human growth hormones, is the first
human protein made using recombinant technology.
1983 Kary Mullis does PCR. 1985 Kary Mullis publishes method. Patents
follow.
2000 Gateway cloning
http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/sidebarmilestone.html
Key enzymes
• Restriction endonuclease
• DNA ligase
• Taq DNA polymerase
Cloning platforms
• Restriction enzymes/ligase
• PCR-based methods
• Gateway and other site specific
recombination methods
• Golden Gate and other “parts-based”
modular construction methods
Restriction enzyme-ligation
Figure 8.3
Table 8.1
Enzyme
Restriction Endonucleases
Source
EcoRI
Escherichia coli RY13
BamHI
Bacillus amyloliquefaciens
HindIII
Haemophilus inflenzae
KpnI
Klebsiella pneumoniae
NotI
Nocardia otitidis
PstI
Providencia stuartii
SmaI
Serratia marcescens
SacI
Streptomyces achromogenes
SstI
Streptomyces stanford
TaqI
Thermophilus aquaticus
XbaI
Xanthomonas campestris
Recognition
sequence
GAATTC
CTTAAC
GGATCC
CCTAGG
AAGCTT
TTCGAA
GGTACC
CCATGG
GCGGCCGC
CGCCGGCG
CTGCAG
GACGTC
CCCGGG
GGGCCC
GAGCTC
CTCGAG
GAGCTC
CTCGAG
TCGA
AGCT
TCTAGA
Cut
G
CTTAA
G
CCTAG
A
TTCGA
GGTAC
C
GC
GGCCGC
CTGCA
G
CCC
GGG
GAGCT
C
GAGCT
C
T
AGC
T
AGATCT
AGATC
Ends
AATTC
G
GATCC
G
AGCTT
A
C
CATGG
CGCCGG
CG
G
ACGTC
GGG
CCC
C
TCGAG
C
TCGAG
CGA
T
CTAGA
T
5overhangs
5overhangs
5overhangs
3overhangs
5overhangs
3overhangs
Blunt ends
3overhangs
3overhangs
5overhangs
5overhangs
Figure 8.2
Figure 8.4
Figure 8.5
Transformation vector requirements
•
•
•
•
Origin of replication
Bacterial selectable marker
Gene constructs of interest
T-DNA borders and other Agrobacterium
genes if using Agrobacterium
• Compatible with helper plasmid if using
Agrobacterium
Figure 8.6
Figure 8.6 The Ti plasmid of
Agrobacterium tumefaciens showing the
origin of replication, the region encoding
the virulence (vir) genes, and the
transfer-DNA (T-DNA). The T-DNA is
flanked by 25-bp direct repeats, known
as the left and right border sequences
(LB and RB, respectively). The vir genes
are required for T-DNA processing and
transfer to the plant cell. The T-DNA is
stably integrated into the nuclear
genome of the plant cell, and genes
encoded within it, necessary for the
biosynthesis of the plant growth
hormones, cytokinin and auxin, result in
the formation of the characteristic
tumorous growth associated with crown
gall disease. The T-DNA also encodes
opines (nopaline and octapine) that
provide the Agrobacterium with an
exclusive nitrogen source. This provides
Agrobacterium carrying the Ti plasmid
with a competitive advantage over
Agrobacterium that do not.
TABLE 8.2 Commonly Used Bacterial Selectable Marker Genes
Antibiotic
Antibiotic Resistance Gene
Gene
Source
Organism
E. coli
Streptomycin/
Spectinomycin
Aminoglycoside adenyl
transferase gene
aadA
Kanamycin
nptII (neo) E. coli Tn5
Ampicillin
Neomycin phosphotransferase
gene
Chloramphenicol
acetyltransferase gene
-Lactamase
Tetracycline
Tetracycline/H+antiporter
Chloramphenicol
cat
E. coli Tn5
bla
E. coli Tn3
tet
E. coli Tn10
Typical components of transformation vector:
making a construct
• Selectable marker cassette (with promoter
and terminator)
• Gene of interest cassette (with promoter
and terminator)
• Scorable marker cassette (with promoter
and terminator)
What happens if the promoter is missing?
Is there ever a time when a promoterless
construct is desirable?
Figure 8.7
Figure 8.8
Bacterial
selection
marker
(Kanr)
pVS1 ori
Plant
transformation
vector
ColE1 ori
T-DNA
Figure 8.8 A generic plant binary
vector with two origins of replication,
the pVS1 ori for propagation in
Agrobacterium and the ColE1 ori for
propagation in Escherichia coli. The
backbone of the vector contains an
antibiotic resistance gene for
bacterial selection (kanamycin
resistance), and the T-DNA contains
a plant selectable marker and the
gene of interest (GOI).
Figure 8.9
PCR videos!
How PCR works:
http://youtube.com/watch?v=_YgXcJ4n-kQ
PCR song!
http://youtube.com/watch?v=x5yPkxCLads&feature=related
What is cloning?
http://upload.wikimedia.org/wikipedia/commons/thumb/6/66/Scissors.sv
g/540px-Scissors.svg.png
So, you’ve cut out a gene… Now what?
Gene for blue flowers
How did you amplify
this gene?
What are
essential
components
of vector
DNAs?
Digest
vector DNA
with
restriction
enzyme
Ligate
gene
into
vector
Plasmid
vector
Extract plasmid DNA
Transform plant
Solving problems of insert orientation
Problems with conventional cloning
• Inconvenient restriction sites
• Vector construction is laborious
• Time-consuming reactions
Blunt-end Ligation
EcoRI
G
CTTAA
Klenow
Fragment
PstI
ACGTC
G
EcoRI
G
C
Ligase
PstI
C
G
GC
CG
Site-specific Recombination:
Gateway™ Cloning
Bacteriophage λ
Bacteriophage λ
Figure 8.11
Figure 8.12
Gateway™ cloning
Figure 8.13
Transform into
DH5α E. coli
cells
Transform into
DH5α E. coli
cells
http://media.invitrogen.com.edgesuite.net/presentations/gateway/index.html?icid=fr-gwcloning-7
Why do we need so many types of vectors?
What are some different applications in plants?
• Functional analysis of open reading frame (ORFs)
• Overexpression and knockdown (RNAi) of specific
genes.
• Multigenic traits for crop improvement
• Analysis of the expression level/specificity/
inducibility of promoters
RNA interference pathway
Figure
8.14
Figure 8.14 Plant gene expression vectors
for conventional cloning using restriction
digestion and ligation (a) and Gateway®
recombination cloning (b). The first vectors
shown in (a) and (b) are designed to allow a
gene to be ectopically expressed in a plant
cell. The second vectors shown for each
category contain the GFP (green fluorescent
protein) gene. These vectors are designed
to effect protein fusions with GFP to help
identify the subcellular target of a protein
under investigation. Ideally, three vectors for
each type are frequently made, one for each
reading frame, to ensure that a perfect
fusion between the GOI and the marker
gene is made. The insert DNA must be in an
“open” ORF configuration (described in the
text) so that no stop codon is present
between the GOI and the marker gene.
Figure
8.15
Figure 8.15 Gene silencing in
plants can be achieved using
inverted repeat transgene constructs
that encode a hairpin RNA (hpRNA).
Using Gateway® cloning technology,
the production of such invertedrepeat transgene constructs can be
achieved efficiently, since DNA
fragment orientation during the
excision and integration process is
maintained and the Gateway®
recombination cassettes are
arranged in opposite orientations
with respect to each other.
Figure 8.16
Figure 8.16 Multisite Gateway® allows
several DNA fragments to be cloned within
a single vector construct. More recent
advances in the design of new att
recombination sites have permitted the
assembly of up to five DNA molecules
within a single vector construct, but none
have been designed as yet for plant
transformation
Multiple promoters
from the MRS2/MGT
gene family fused to
the GUS gene and
expressed in
Arabidopsis thaliana
Gebert et al., The Plant Cell,
Vol. 21: 4018–4030, December
2009
Figure 8.17
Figure 8.17 Excision of selectable marker gene following T-DNA insertion into the
plant genome. XVE is a chimeric transcription factor. It contains three functional
domains, a LexA DNA binding domain (X), the VP16 activation domain (V), and the
estrogen receptor binding domain (E). The G10-90 promoter drives the constitutive
and ubiquitous expression of XVE in transformed plant cells. The XVE protein is then
bound as a monomer in the cytosol of the cell by a chaperone protein HSP90, and
the target gene is transcriptionally inactive. Application of -estradiol causes a
conformational change in E, which leads to the release of HSP90 and dimerization of
the receptor. On dimerization, the receptor is activated, allowing the protein to
translocate to the nucleus of the cell where it binds OLexA binding sites of the
promoter that is placed upstream of the Cre recombinase. The VP16 activation
Figure
8.18
Figure 8.18 Site-specific
integration is achieved by two
homologous recombination events,
one on either side of the DNA
fragment to be integrated. During
insertion, the targeted region of the
vector replaces the targeted region
of the plastid genome, and the
vector backbone is lost. The inserted
DNA fragment contains a selectable
marker (here, the aadA gene
encoding aminoglycoside 3adenylyltransferase, providing
spectinomycin resistance) and can
contain either a single gene flanked
by independent 5 and 3 regulatory
regions, including a promoter; a 5
UTR and a 3 UTR; or, as is the case
here, multiple genes with a single
promoter that regulates the
Vectors derived from plant
sequences
• Public acceptance of GMOs linked to
concerns of the origin of DNA employed
• Ironically, wild-type plant cells already
contain bacterially-derived genomes
• T-DNA could be replaced with P-DNA
• Replace viral promoters with plant
promoters
Figure 7.20
A
loxP-FRT
loxP-FRT
LB
RB
LAT52 FLP Recombinase NOS
Pollen-specific
promoter LAT52
activates
recombinase in
tobacco pollen
excision
35S p GUS:NPTII NOS
Pollen
genome
loxP
FRT
LAT52 FLP Recombinase NOS
35S p GUS:NPTII NOS
loxP-FRT
RB
LB
Pollen genome
B
Luo et al. 2007. Plant Biotechnology Journal
5(2): 263 - 274. (Courtesy of Moon’s poster)
Example of a plant expression
vector set
•
•
•
•
pANIC
Made for switchgrass transformation:
BioEnergy Science Center (BESC)
http://plantsciences.utk.edu/stewart.htm
The pANIC vector set
• Functional in switchgrass and rice
• Overexpression (OE) and suppression (RNAi) of genes
– ZmUbi1
– CaMV 35S
RNAi of lignin biosynthetic genes in switchgrass
• Protein tag for OE – AcV5
Shen & Dixon, Noble Foundation
Mann et al. Plant Biotechnology Journal 2012
53
pANIC - Reporter
cassette
GUS staining photos courtesy of Zach
King
• PvUbi1 promoter
• Histochemical
– GUSPlus
• Fluorescent
Brightfield – 5ms
– pporRFP – novel RFP
– Comparable to DsRed
– Ex/Em = 578 nm/595
nm
RFP – 2s
DsRed
pporRFP
GFP – 10s
pANIC Vectors:
Modular “parts”
based cloning
Golden Gate is one method
https://www.neb.com/applications/cloning-andsynthetic-biology/dna-assembly-andcloning/golden-gate-assembly