Transcript Vector Construction II - Department of Plant Sciences

Lecture 11 Chapter 7
Vector Construction II
Gateway cloning
David Mann
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 7.11
Figure 7.12
Gateway™ cloning
Figure 7.13
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
Site-specific Recombination:
Creator™ Cloning
Cre/loxP Recombination System:
The Jackson Laboratory (http://www.jax.org/index.html)
Figure 7.14
Creator™ cloning
Figure 7.15
Figure 7.16
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
Conventional cloning
Gateway cloning
Site-specific DNA
recombination
Creator cloning
Univector cloning
Figure 7.17
RNA interference pathway
Figure 7.18
Vectors for RNAi
Figure 7.21
Figure 7.22
Multisite Gateway allows several DNA fragments
to be cloned into a single construct
Figure 7.19
Katzen 2007. Expert Opin. Drug Discov. 2(4): 571-589
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
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)
Figure 7.23
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
37
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: