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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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