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!
Bergman et al. 1992
Trends in Plant Science, 10:
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
Inhibit Nostoc growth and hormogonia formation
Test the hypothesis Trends in Plant Science, 10:
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 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
Test the Primers PCR
Procedures PCR Extracted DNA from Agarose Gel Ligation of DNA to pGEM-Easy Plasmid Transformation Sequence Clones
Ligation of DNA to pGEM-Easy Plasmid PLASMID DNA LIGATION
After Sequencing How many genes are there for CHS enzyme? GENE1 GENE2GENE3GENE4
RNAi 1.Find Conserved Domains 2.Generate Primers for those regions. 3.Knock down expression of all sequence possessing homology to CHS.
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 0.1mM0.005mM0.01mM 0.001mM *Naringenin inhibited differentiation at conc. ≥.01mM
Flavonoids polyphenolic compounds with 15 Carbon atoms, 2 benzene rings connected by a 3 carbon chain. Flower pigments in angiosperms