Download - Siddra ijaz 300516
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Siddra Ijaz, PhDAssistant Professor
Centre of Agricultural Biochemistry and Biotechnology (CABB)
University of Agriculture Faisalabad, Pakistan
Visiting Research Scholar
Plant Reproductive Biology Lab, Department of Plant Sciences,
University of California Davis, USA
Supervisor: Prof. Dr. Eduardo BlumwaldProfessor of Cell Biology and
Will W. Lester Endowed Chair
Dept of Plant Sciences, University of California Davis, USA
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Members of Blumwald’s Lab
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Probing water stress tolerance in Setaria viridis L. based on characterizing
root hydraulics and expression profiling for contribution of Aquaporins
(AQPs) along with cloning of CRISPR-Cas9 vectors
Title:
Research Activities
Characterization of root hydraulics and
contribution of Aquaporins (AQPs) in green
millet (Setaria viridis L.) in water stress tolerance
Cloning of CRISPR-Cas vector
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Characterization of root hydraulics and the contribution of Aquaporins (AQPs)
in green millet (Setaria viridis L.) in water stress tolerance
Setaria viridis is a monocot model for C4 photosynthesis
Physiological responses to abiotic stress is not yet known
High root hydraulic system is correlated to more biomass production
This research has explored and characterized root hydraulic system in Setaria
Evaluated the contribution of aquaporins (water channel proteins) in water stress tolerance
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Parameters Accessions
Code Sv1 Sv2 Sv3 Sv4 Sv5 Sv6
Accession Ames 28193 PI 669942/
Ames 31045
PI 649320 PI 223677 PI 230135 PI 408811
Plant name 132 A10.1 98HT-80 Dekker 1851 Dekker 1850 UI 4833
Origin (country) Kazakhstan United States Mongolia Azerbaijan Iran China
Area Zhangiztobe Oklahoma Henti Aimag Astara Abali Shaanxi
Latitude 49.1286 NA 48.1339 38.4561 35.7624 34.2500
Longitude 81.1078 NA 110.2281 48.8786 51.9653 108.8667
Elevation (feet) 1,699 NA 3,369 900/72 6,000 1,329
Genotypes to be used
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Fig. 1. Physiological measurements of six Setaria viridis (Sv) accessions under WW, WS and HS conditions. (a) Leaf water potential (‘Ψleaf’) of accessions measured after 10 DPT using a pressure chamber instrument. Gas exchange measurements of (b) photosynthesis (‘A’) and (c) rates of transpiration (‘E’) and (d) stomatalconductance (‘gs’) after 15 DPT using a Li-6400 portable gas exchange system, respectively. Black, white and grey bars represent mean values (n = 9) ± standard error (SE) from well-water (WW), water stress (WS) and heat stress (HS) treated plants and letters on the bars indicate significant differences at P≤0.05 level as tested by Tukey-Kramer HSD. (Un published Data)
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Transplantation of plants in vermiculite
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Standardization of protocol and its parameters
Experiment done in triplicate
Plants were treated in three conditions
Normal fertilized water
Fertilized water containing 8mM H2O2
Fertilized water containing 1M NaCl
Water flux was observed
25 psi (0.17 MPa)
35 psi (0.24 MPa)
50 psi (O.34 MPa)
Hydrostatic hydraulic conductivity of roots
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*
P(MPa)
Jv(m
l h
-1 )
hy
dro
stati
c h
yd
rau
lic
con
du
ctiv
ity
of
roo
ts (
Lp
r-h
)
ml
g-1
h-1
Mp
a-1
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H2O2 is aquaporins inhibitor
It inhibits not all but most of the aquaporins
Aquaporins are water channel proteins and water moves
through it
Aquaporins have been shown to correlate with root
hydraulics
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hy
dro
sta
tic
hy
dra
uli
c co
nd
uct
ivit
y o
f ro
ots
(L
pr-
h)
ml
g-1
h-1
Mp
a-1
a
b
cc
AQP inhibitor
Expression profiling using qPCR was done to check the expression of aquaporins in roots
Aquaporins gene specific primers were used
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Small basic intrinsic proteins
Tonoplast intrinsic proteins
Noduli
n26
-lik
e in
trin
sic
mem
bra
ne
pro
tein
sP
lasma m
embran
e intrin
sic pro
teins
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qPCR (Real Time PCR)
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CRISPR-Cas9 vector Cloning
CRISPR: Clustered Regularly Interspaced short Palindromic Repeats
Cas9: CRISPR associated protein 9
Sequence specific nucleases (SSN)
Generate double stranded break
Single guide RNA (sgRNA)
o CRISPR RNA (crRNA sequence)
oTrans-activating crRNA (tracrRNA)…….. Cas9 nuclease-recruiting sequence
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Single guided RNA (sgRNA)
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Select genomic target:
Target sequence is ~20 bp sequence followed by the PAM sequence (NGG)
PAM: Protospacer adjacent motif
Essential targeting component
Follow the DNA sequence targeted by the Cas9 nuclease
Cas9 will not successfully bind to or cleave the target DNA sequence if it is
not followed by the PAM sequence
Design Single guided RNA (sgRNA)
Guide sequence should match the target sequence
First nucleotide may be “G” or “A”
G….U6p
A…..U3p
Assembled Cas9/sgRNA construct
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overlapped
WX
YZ
overlappedoverlapped
Target sequence (20bp)
Promoter (400bp)
Adapters (15-20bp)Adapters (15-20bp)
~ 500 bp
sgRNA (80 bp)
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500 bp
M
M = 1 Kb DNA Ladder
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overlapped
WX
YZ
overlappedoverlapped
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~500bp
W X Y Z
~4000bp
VM
M = 1 Kb DNA Ladder
V = Linearized Vector
W = Fragment 1
X = Fragment 2
Y = Fragment 3Z = Fragment 4
Gibson cloning and InFusion cloning for assembling fragments
• primers designed to amplify fragments (and/or vector) with appropriate overlaps•PCR amplify fragments using CloneAmp™HiFi PCR premix•Prepare linearized vector by PCR amplification using a CloneAmp™HiFi PCR premix
PCR Amplification
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~500bp
~4000bp
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Linearized Vector
Fragment W
Fragment X
Fragment Y
Fragment Z
Gibson Assembly master mix
H2O
Total volume 10µL
Linearized Vector
Fragment W
Fragment X
Fragment Y
Fragment Z
In fusion HD enzyme primer mix
H2O
Total volume 5µL
Gibson cloning reaction InFusion cloning reaction
Incubation time: 50°C for 60 minutes Incubation time: 50°C for 15 minutes
These product were transferred into E coli cells using heat shock methods
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Amplification of promoters and sgRNAs
Target sequence (-)containing chimeric primersFirst PCR
Target sequence (+)containing chimeric primers
U6 PromotersgRNA
gR-R
U-F
Amplified promoter Amplified target-sgRNA
PpsPgs
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~100 bp
~400 bp
M P sgP PP Psg sgsgsg
M = 1 Kb DNA Ladder
P = Promoter
Sg = sgRNA
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~100 bp
~400 bp
M P sg
M = 1 Kb DNA Ladder
P = Promoter
Sg = sgRNA
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Fusion using PCR
First PCR
Target sequence (+)containing chimeric primers
~ 500 bp
Overlapped region
(Target sequence)
U6 PromotersgRNA
gR-R
U-F
Target sequence (-)containing chimeric primers
Amplified promoter Amplified target-sgRNA
Promoter (400bp)
Target sequence (20bp)
sgRNA (80 bp)
PpsPgs
Second PCR
PpsBsa1
Pgs Bsa1
Bsa1Bsa1
Bsa1 Bsa1
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~500 bp
M
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1000bp2000bp
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Gate way cloning
Licor for measuring photosynthesis and also for
measuring CO2 in Setaria viridis
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Course Audited:
BIT161A Genetics & Biotechnology Lab
Professor: Diane M. Beckles
Online Courses:
UC Laboratory Safety Fundamentals
Nursery/Green house General safety training January 15, 2016
Initial Health and safety training December 7, 2015
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Food, Ag & Health solution summit December 2-3, 2015
Forum presentation with speaker Dr. Etienne Rabe of the wonderful Company December 10, 2015
Special Event Roundtable January 14, 2016
Featuring Pacific Biosciences of California, Afingen and Trace Genomics February 11, 2016
Grain legume breeding in California and East Africa: contrasting endeavors speaker Dr. Paul Gepts, February 11, 2016
Hybrid Rice: A global perspective, speaker Michael Gumina, April 14, 2016
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Thank you