february 20, 2005 sharp – what and why davis – mapping genes clark - maize rootworm bohnert -...
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February 20, 2005
Sharp – what and why
Davis – mapping genes
Clark - maize rootworm
Bohnert - get genes/transcripts
Tao – dynamics of genes
Springer – making sense of all
Plant Genome &
Gene Complexity,
Gene Regulation,
Place & Timing,
and how to sort the
PlayersNSF DBI #0211842
The Plant Genome DiploidPolyploid
Chrysanthemum species illustrate the phenomenon.
Monoploid number (the basic set) = 9 chromosomes
In Chrysanthemum species, the number of chromosomes fall into 5 categories: 18 chromosomes = diploid (2 copies of the monoploid) 36 chromosomes = tetrapoid (4 copies of the monoploid) 54 chromosomes = hexapoid (6 copies of the monoploid) 72 chromosomes = octaploid (8 copies of the monploid) 90 chromosomes = decaploid (10 copies of the monoploid)
50% of all flowering plants are polyploid.
• Ploidy changes - a recurring process• Many ‘diploid’ species have gone through ploidy changes• Fusions of related species new species
See: Arabidopsis
PIP2;2
Ch-1
PIP1;3 TIP3;2 NIP3;1 TIP2;xpseudo TIP3;1 NIP6;1
Ch-5
SIP1;2 NIP4;1 NIP4;2 TIP2;3 PIP2;4
5 10 20 30Mb
Ch-4
PIP1;4 TIP1;3 NIP5;1 TIP2;2NIP1;1NIP1;2 PIP1;5
Ch-3
TIP1;2TIP2;1NIP7;1SIP1;1 TIP5;1
PIP1;1
PIP2;1
PIP2;5
SIP2;1
Ch-2
PIP1;2TIP4;1NIP2;1pseudoNIP3;1pseudo NIP2;1
TIP1;1
PIP2;6
PIP2;3
PIP2;8
PIP2;7
(15)
(4)
(14)
(3)
(12)
- duplicated regions that include AQPs.
rDNA
AQP are distributed over all Chromosomes - a few clusters, many duplications
Figure 3
control
O3
CO2
Columbia grown in Soy-FACE
Field ona dish!
Arabidopsis – model plant
small, fast, prolific,mutants, lines, ecotypes,genome sequence
Arabidopsisgrowing
in the fieldin high
CO2 and/or ozone
downthere
concept
plant performance inthe future earth’
atmosphere (~2040)-
also: soy, corn, weeds
FACE-rings
Plants in silico? Sure! And then: Plant Design from Scratch
Ecosystem – population – species – ecotype (breeding line)
Organism – organ – tissue – cell – compartment
Nucleus – envelope & pore – nucleoplasm, nucleolus & chromosomes
Euchromatin & heterochromatin – gene islands – gene
Promoters – 5’-regulatory –
introns & exons - coding region –
3’-regulatory regions
The Plant Genome
The Plant Genome ControlsControls for Gene Expression – many Switchboards
• Chromatin condensation state
• Local chromatin environment• Transcription initiation• Transcript elongation• mRNA splicing • mRNA export• mRNA place in the cell• RNA half-life• Killer microRNAs• Ribosome loading• Protein transport/targeting• Protein modifications• Protein turnover
Levels of regulation that
affect what we call
“gene expression”
The Plant Transcriptome
Killer RNAs(there are micro-genes)
no protein-
gene isessentially“silenced”
5 years ago, we did not know that
such a control system existed!
microRNAs
The Plant Transcriptome
How to sample the transcriptome?
Morphological dissection remember Bob Sharp’s talk! (root, leaf, flower - epidermis, guard cell, etc.)
Cell sorting make single cells, send through cell sorter (size, color, reporter gene)
Laser ablation micromanipulation of laser to cut individual cells
Biochemical dissection chloroplasts, mitochondria, ribosomes, other membranes
Painting cellswith a
reporter gene-
here this isGFP
GreenFluorescence
Protein
The Endodermis of the root tip
is highlighted in transgenic
plants using pSCR::mGFP5.
Painting tissuesthen isolating desired cells
Enzymatic staining
The Plant Transcriptome
Cell-Specific Cell-Specific GFP ExpressionGFP Expression
• Catalog of available transgenic Arabidopsis lines.
• Lines are available from the stock centers.
• However, the molecular basis for the observed phenotype is usually uncharacterized.
The Plant Transcriptome
> cDNA libraries
• “neat”
• normalized
• subtracted
> SAGE libraries
cDNA – complementary DNA
converts messenger RNA into
double-stranded DNA
“Normalization” removes mRNAs
for which there are many copies
in a cell – thus enriching for
“rare mRNAs” (not so much sequencing to do)
Subtraction removes cDNAs which you already know
(less sequencing)
Total RNA
Poly(A)+ RNA
1st strand cDNA
ds-cDNA
Size-selected double stranded cDNA (>500 bp)
Ligate to EcoRI adapters/digest NotI
Clone (EcoRI/NotI) digested pBSII/SK+ & adaptored cDNA
Primary cDNA Library
Primary (neat) library may be used for “normalization”
Library Normalization
primary cDNA library
ss-DNA
DNA “tracer”
PCR inserts by T7 and T3
standard primers
DNA “driver”tracer/driverhybridization
column chromatogr.(double-strands stick)
Non-hybridized DNA from flow-through = normalized clones
make ss-DNA out of primary
library
cDNA Libraries
Cloning ofroot RNAs
from segmentsS1 – S4root tip
(Sharp lab)
sequenced~18,000 clones
found~8,000 unique
and~130 novel genes
How many genesmake a root?
The Plant Transcriptome
Isolate total RNA from cells or tissue
Isolate small regions (SAGE tags) of each
mRNA transcript in a cell
Digest tags and ligate into concatamers for sequencing
Reference sequencing results against public databases
SAGE – Serial Analysis of Gene Expression – an Overview
The Plant Transcriptome
Quantitative PCRin 384-well plates
(96 primer pairs,3 repeats each)
Taking SAGE & cDNA
sequences together-
corn roots
“express”
20-23,000 genes(i.e., mRNA is made)
-
The entire corn genomeis expected to include
~50,000 genes
The Plant Transcriptome
Why are we doing this?• Genes expressed in well-watered conditions,
how many, where and which?
• Changes during drought episodes?
• Variation in different lines or land races?
• Breeders to cross and select for tolerance!
• Proteins and substances (metabolites) made?(how to make a cell wall, how to defend against rootworms)
• Make corn with thicker (modified) cell walls!