Hunting Genes. 7 “characters” equal seven genes What is a Gene? A locatable region of genomic...

Hunting Genes

7 “characters” equal seven genes

What is a Gene?

A locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed Regions and/or other functional sequence regions. Sequence ontology consortium, Berkeley 2006

Dystrophin (human) 2.5 mio bp 11.000 coding bpycf1 (chloroplast) 6800 bp 6800 coding bpMRP1 (plant) 13400 bp 4870 coding bp

Plants 25,000Humans 25,000Fly 12,000Yeast 6,000E.coli 5,000DNA virus 10-300

Nomenclature:Gene unit of inheritance

Mutation changes in gene DNA sequence that may lead to changes in phenotype

Phenotype observable (measurable) characteristics of a plant

Genotype all genes or alleles that determine a plant’s phenotype

recessive phenotype not visible in heterozygous stage

dominant phenotype visible in heterozygous stage

Wildtype phenotype of the majority in a population

RNA

RNA

RNA

protein

AAAAAAAm7G cap

Gene regulation and signal transduction

What is a gene?

Gene of interest

T-DNALB RB

Protein

mRNA

T-DNALB RB

Gene o f interest

T-DNALB RBGene o f interest

T-DNALB RBGene o f interest

mutant wildtype

Transposable genes in corn

Transposable elementsall over the place

Plant transposable elements

Transposon tagging and gene cloning

Expression analysis through microarrays

Hunting a maize dwarf gene

N. R. Fuller, NSF

Plant architecture: one of the greatest improvements

of crop yield in the history of agriculture

Important regulators influencing yield in maizePlant growth

–gibberellic acid (GA):–auxin:–brassinosteroids (BR):

Sex determination:–jasmonic acid (JA):

17

teosinte

corn

Phenotype of brts1 mutants

brts1 WT brts1 WT 18

Mesocotyl elongation in brts1 mutantsmesocotyl elongation in darkness is reduced in an1 (GA) and brts1

exogenous GA treatment does not reverse inhibition in brts1

Ligh

t/D

ark

Dar

k -/

+ G

A

19

GA inhibitor reduces brts1 mesocytl elongation in the dark

20

WT brts1 d5

scale bar 10 cm; uniconazole (UCZ)

UCZ [µM] 0 35 0 35 0 35

brts1 on Chr. 3L

21

brts1 encodes DWF6/DET2 homolog

• Mu8 insertion in ORF of maize AtDWARF6/DET2 homolog

• single gene; 50% aa identity with AtDET2

• 5α-steroid reductase: conversion of 4-en-3-one to 3-one BR biosynthesis

dwarf6 / det2 in Arabidopsis

WT det2

WT det2

brts1 gene model

Alleles of brts1/na1

5’UTR ATG TAG

Exon1 Exon2

3’UTRB73 reference

5’UTR ATG TAG

Exon1 Exon2

3’UTR

unknown insertion in na1-1

Mu8 insertion in brts1

Mu4 insertion in na1-3

Mu8 insertion in na1-4

EMS induced G to A exchange in na1-2

+489 STOP

3-oxo-5--steroid 4-dehydrogenase

?

?

ubiquitin

DET2

WT brts1-1 na1-1 na1-2 H2O

RT-PCR

brts1 na1-1

H2O 5 µM PCZ

WT

brts1 d5

H2O 5 µM PCZ

propiconazole (PCZ) [µM]

heig

ht (%

)

propiconazole treatment mimic brts1

propiconazole induces „tasselseed“

24

WT

PCZ - -

brts1 WT brts1

+ +

TS - + + ++

propiconazole (PCZ), tasselseed phenotype (TS)

Gene regulation and signal transduction

What is a reporter gene reporting?

Here we are!!!!

Gene regulation and signal transduction

What is a reporter gene reporting?

non transformed wildtypeGTL-GUS transformant