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A novel WRKY transcriptional factor from Thlaspi caerulescens negatively
regulates the osmotic stress tolerance of transgenic tobacco
A novel WRKY transcriptional factor from Thlaspi caerulescens negatively
regulates the osmotic stress tolerance of transgenic tobacco
Plant Cell Rep (2008) Wei wei
报告人:季琳
contents
Introduction1.
Materials and methods2.
discussion4.
results3.
Introduction
The WRKY gene family represents one of the major groups of plant-specific transcriptional regulators.
WRKY proteins bind specifically into the W-box. W-boxes have been identified in the
promoters of many genes involved in defense and in response to environmental stress.
W-boxes also present within WRKY gene promoters themselves.
Introduction
The most prominent feature of all WRKY
proteins is the WRKY domain, a 60 amino
acid region containing a highly conserved
WRKYGQK motif at the N-terminus, and a
zinc-binding motif at the C-terminus.
Introduction
WRKY genes are active during pathogen defense and abiotic stress in a number of different plant species.
To adapt Adverse environmental conditions, plants have developed elaborate mechanisms for surviving these challenges. The transcription factors adopt pivotal roles in controlling the expression of specific stress-related genes.
Introduction
Thlaspi caerulescens is known as a heavy-metal hyperaccumulator that grows in soils contaminated with zinc, lead, and cadmium.
Materials and methods
T.caerulescens
Differential screening of a cDNA library
Nicotiana tabacum
Construction of a TcWRKY53 expression vector and transformation into the plant
Nicotiana tabacum
Osmotic stress
tolerance in transgenic
plants and espression
patterns of some
osmotic stress related
genes in transgenic
plants
Performance line Ⅰ
Plants were treated with PEG6000 or NaCl or were kept at 4 ℃
and SA 0.5h.
Plants were treated with PEG6000 or NaCl or were kept at 4 ℃
and SA 0.5h.
ThlaspiThlaspicaerulescenscaerulescens
ThlaspiThlaspicaerulescenscaerulescens
Gel blot analysis
GenomicGenomic DNADNA
GenomicGenomic DNADNA
semi-quantitativesemi-quantitative RT-PCR RT-PCR analysisanalysis
RNA RNA isolationisolation
RNA RNA isolationisolation
Performance line Ⅱ
Hybridized with wild-type T.caerulescensleaves probe
Hybridized with Cd-treated T.caerulescensleaves probe
Cd-treated T.caerulescenscDNA library
Differential screening of a cDNA library
negative positive
result
result
Construction of vector and transformation
The PCR product was digested with Xba and ⅠBamH and ligated to construct the expression Ⅰvector pBI121-TcWRKY53.
The recombinant plasmid was introduced into the Agrobacterium tumefaciens EHA105 strain.
This strain was then used to transform Nicotiana tabacum NC89 by the Agrobacterium-mediated transformation method.
Osmotic tress tolerance in transgenic plants
Osmotic tress tolerance
T3seeds
4 days
5% sorbitol
4-week-old
20%PEG6000
result
Espression patterns of some osmotic stress related
genes in transgenic plants
ERF5 EREBP-1 LEA5
Transcription
factors
Function
gene
Environmental stresses
Environmental stresses
Dehydration, Osmotic,Low temperature
results
Leaves of T.caerulescens were harvested
Leaf
Shoot
root
一
SA
0.5h
二
Young
senescence
三
Leaves of T.caerulescens were harvested
ThlaspiThlaspicaerulescenscaerulescens
44℃℃
NaClNaCl
PEG6000PEG60000
1h2h 4h 8h 12h
0h
四
result
Genomic DNA gel blot analysis
Isolate genomic DNA of T.caerulescens leaves via a modined CTAB extraction method.
Genomic DNA hybridization was performed with a full-length TcWRKY53 cDNA probe labeled with digoxigenin-Dutp.
result
Discussion
Comparison of the genomic sequence with the cDNA sequence showed the TcWRKY53 gene contains two introns.
TcWRKY53 is present as tow copies and up-regulated by abiotic stresses in T.caerulescens .
Expression of TcWRKY53 in tobacco negatively modulates the osmotic stress tolerance and the expression of ERF protein.
Discussion
the individual WRKY factor can respond to different stress signals, while, different members in the same family of WRKY factors display distinct roles in plant defense and development processes.
it is reasonable to hypothesize that TcWRKY53 might also function in plant pathogen infection and heavy metal response.
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