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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