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

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

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

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

Single guided RNA (sgRNA)

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

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

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

~100 bp

~400 bp

M P sgP PP Psg sgsgsg

M = 1 Kb DNA Ladder

P = Promoter

Sg = sgRNA

~100 bp

~400 bp

M P sg

M = 1 Kb DNA Ladder

P = Promoter

Sg = sgRNA

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

~500 bp

M

1000bp2000bp

Gate way cloning

Licor for measuring photosynthesis and also for

measuring CO2 in Setaria viridis

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

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

Thank you