G. Manicata (Anthophyta) CB colonize and penetrate glands produced below each leaf Immature gland; 8- day old seeding Mucus covered gland Inoculation of gland with CB How are Gunnera’s gland cells different from normal plant cells?
BROAD IMPACT Save the world and revolutionize agriculture!! Create productive symbiosis between cyanobacteria and crop plants!
N-starvation Myb-transcription factor Flavonoidsynthesis glandprimodiainhibit 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 Flavonoidsynthesis High flavonoids at the glandprimodiainhibit 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
Test the hypothesis Trends in Plant Science, 10: 236-242
Methods Create 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? ANSWER Conserved regions across various species.
Methods Make primers using conserved regions residing in a wide range of dicots. > NCBI
Methods Align all of those various sequences and locate the conserved regions. > BIOBIKE
Conserved Regions 0 1190 352-376 1129-1154 421-444 913-941 F1 R1 F2 R2 Chalcone Synthase Sequence
Degenerate Primer F1-Outside Primer GCN 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
The Degenerate Primer GCA ATA AAA GAA TGG GGC CAA CCA AA C C G G G G C G T T T T