LOGO 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 Introduction 1. Materials and methods 2. discussion 4. results 3.

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. ThlaspicaerulescensThlaspicaerulescens Gel blot analysis GenomicDNAGenomicDNA semi-quantitativeRT-PCRanalysis RNAisolationRNAisolation Performance line Ⅱ

Hybridized with wild-type T.caerulescens leaves probe Hybridized with Cd-treated T.caerulescensle aves probe Cd-treated T.caerulescens cDNA library Differential screening of a cDNA library negative positive

result

Construction of vector and transformation  The PCR product was digested with Xba Ⅰ and BamH Ⅰ and ligated to construct the expression vector 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 T3 seeds 4 days 5% sorbitol 4-week-old 20% PEG6000

result

Espression patterns of some osmotic stress related genes in transgenic plants ERF5EREBP-1LEA5 Transcription factors Function gene 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 Thlaspicaerulescens 4℃4℃4℃4℃ NaCl PEG h 2h 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.

LOGO