Investigation of flavonoids in the development of plant...
Transcript of Investigation of flavonoids in the development of plant...
Meeks & Elhai 2003
Hornworts(Anthrocerophyta)
Bryophytes
Azolla (Pterophyta)
Water ferns
Cycads (Cycadophyta)
Gymnosperms
G. Manicata(Anthophyta)
CB colonize and CB colonize and penetrate glands penetrate glands produced below produced below each leafeach leaf
Immature gland; 8-day old seeding
Mucus covered gland
Inoculation of gland with CBWhat’s so special about Gunnera’s What’s so special about Gunnera’s
relationship with CB?!!relationship with CB?!!
It’s the only intracellular It’s the only intracellular endosymbiotic relationship endosymbiotic relationship known b/n angiosperm and known b/n angiosperm and
cyanobacteria!!!cyanobacteria!!!
How are Gunnera’s gland cells different from normal plant cells?
BROAD IMPACT
• Save the world and revolutionize agriculture!!
Create productive Create productive symbiosis between symbiosis between cyanobacteria and crop cyanobacteria and crop plants!plants!
N-starvation
Myb -transcription factor
Flavonoid synthesis
gland primodia inhibit
auxin efflux
Localized auxin accumulation
Activation of cell division and gland development
Remobilization of nutrients
from leaves to the stem
N-starvation
Myb -transcription factor
Flavonoid synthesis
High flavonoids at the
gland primodia inhibit
auxin efflux
Localized auxin accumulation
Activation of cell division and gland development
Remobilization of nutrients
from leaves to the stem
10 mM nitrate
0.1 mM nitrate
MethodsCreate flavonoid deficient mutant in Gunnera
!!!There’s a Problem!!!
Gunnera’s genome has never been fully sequenced.
How can I clone an enzyme when I don’t know it’s sequence?
ANSWERConserved regions across various species.
Degenerate PrimerF1-Outside PrimerGCN ATH AAR GAR TGG GGB CAR CCH AA
F2-INSIDE PRIMER
GAC ATG CCY GGY …
R1-OUTSIDE PRIMER
ACV GTV ARN CCN …
R2-INSIDE PRIMER
GCN GGV CCD CCH …
B= C or G or T
D= A or G or T
H= A or C or T
K= G or T
M= A or C
N= A or C or G or T
R= A or G
S= C or G
V= A or C or G
W= A or T
Y= C or T
ProceduresPCR
Extracted DNA from Agarose Gel
Ligation of DNA to
pGEM-Easy Plasmid
Transformation
Sequence Clones
Transformation
www.biochem.arizona.edu/.../Ligation-2.gif
RNAi
1. Find Conserved Domains2. Generate Primers for those regions.3. Knock down expression of all sequence
possessing homology to CHS.
http://www.hhmi.org/biointeractive/rna/rnai/
Near Future
• Create full cDNA library enriched for genes expressed during and/or in early colonization of symbiont.
Effect of the Flavoniod Naringenin (naringin) on Differentiation
Control
0.1mM 0.005mM0.01mM
0.001mM
*Naringenin inhibiteddifferentiation at conc. ≥ .01mM