Siddra Ijaz, PhD Assistant 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 Blumwald Professor of Cell Biology and Will W. Lester Endowed Chair Dept of Plant Sciences, University of California Davis, USA
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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
Members of Blumwald’s Lab
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
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
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
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)
Transplantation of plants in vermiculite
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
*
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
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
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
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
qPCR (Real Time PCR)
CRISPR-Cas9 vector Cloning
CRISPR: Clustered Regularly Interspaced short Palindromic Repeats
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
overlapped
WX
YZ
overlappedoverlapped
Target sequence (20bp)
Promoter (400bp)
Adapters (15-20bp)Adapters (15-20bp)
~ 500 bp
sgRNA (80 bp)
500 bp
M
M = 1 Kb DNA Ladder
overlapped
WX
YZ
overlappedoverlapped
~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
~500bp
~4000bp
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