水分生理

Materials and methods
Plant materials and treatments
seed sterilization germination cultured in growth chambers
PEG6000 treatment treatment with signalling molecules(ABA,MeJA,SA,auxin,H2O2) Tungstate fluridone For inhibitor or scavenger treatment (DMTU)， imidazole
Analysis of RWC,IL,MDA,ROS accumulation and antioxidant enzyme activity Sequence analysis
Statistical analysis
results
Cloning and sequence analysis of the full-length TaASR1 CDNA TaASR1 cDNA comprised of 544 bp with a 414 bp open reading frame ， the deduced TaASR1 protein contained 137 amino acid residues with a predicted molecular mass of 15.3 kDa. BLASTX analysis showed that TaASR1 shared a high degree of sequence identity with ASRs from other plant species: 86% sequence identity with BdASR3 from B.distachyon , 80%with ZmASR2 from Zea mays ,72%with MaASR from Musa ABB Group and 70%with OsASR from Oryza sativa Japonica Group.
Introduction
Plants are frequently challenged by various harsh environmental factors , among which drought stress is adverse because it limits plant growth, development and crop productivity. The major response of plants to drought stress include perception of stress signals and their transduction that activate various stress-related genes and synthesis of proteins with diverse functions resulting in physiological and metabolic responses .
Interestingly ,a recent study showed that rice ASR protein OsASR1,could function as an effective reactive oxygen species(ROS)scavenger and its expression in yeast cells enhanced acquired tolerance of ROS-induce doxidative stress through induction of various cell rescue proteins . However , whether ASRs confer drought stress tolerance by regulating ROS homeostasis through the antioxidant system is yet to be elucidated in plants.
Generation of transgenic tobacco overexpressing TaASR1
Transgenic tobacco plants overexpressing TaASR1 under the control of CaMV 35S promoter were generated. A total of 14 transgenic lines(T1)were identified by hygromycin-resistance analysis and PCR using primers specific to TaASR1 and GFP. Among the T1 lines , three(OE2,OE5 and OE12)segregated at a ratio of 3:1 based on hygromycin resistance. seedlings from all three transgenic T2 lines grew well on MS medium containing 40 mg L-1 of hygromycin. Among the lines OE5 and OE12 had higher TaASR1 expression levels .
The results suggested that TaSAG3 exhibited a resultspattern suggested thatTaASR1 the up- after similar These expression with To various exploreregulation whether theof up-regulation of TaASR1 under TaASR1 by PEG with treatments and that treatment PEG6000 treatment involves ABA H2 O signalling, treatment possibly involves H O tungstate and fluridone had no and effect on the up22 2 signalling regulation of TaSnRK2.7 under PEG treatment. These results implied that up-regulation of TaASR1 by PEG possibly involves ABA signalling
transplanted in containers for two weeks
water withholding for 30 d
Collection of leaves to measure RWC, IL, MDA, H2O2 ,SOD .CAT , POD.
One hundred and fifty surface-sterilized seeds from each transgenic line or control were sown on MS with or without 150 or 300 mM mannitol for 8 d to detect germination rate
cultured on MS for 1 week
transplanted to MS with or without 150 or 300 mM mannitol for 1 week
measure root length , ROS accumulation , and SOD and CAT activities.
Stress tolerance of the WT , vector control(VC) and transgenic lines
For drought stress tolerance assay
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For osmotic stress tolerance
cultured on MS for 1 week
TaASR1,a transcription factor gene in wheat confers drought stress tolerance in transgenic tobacco
introduction materials and methods results discussion
Cloning and bioinformatics analysis of TaASR1 RT-PCR，qRT-PCR Subcellular localization of TaASR1 protein Plant transformation and generation of transgenic plants
These results suggested that TaASR1 transcript waspatterns strongly of TaASR1 to determine the expression induced by PEG6000,ABA and in different wheat tissue,qRT-PCR was carried 2O2. out with H mRNA from different tissues
ASR protein have low molecular weight and are heat-stable and highly hydrophilic observed specifically in plants. Although the precise physiological function of ASRs remains unknown , they are presumed to have functional duality in plants. Firstly ,ASRs are classified to group 7in the late embryogenesis abundant(LEA)protein family with high hydrophilicity , which correlates to the high glycinecontent for direct plant protection .Secondly , ASRs have been suggested to be transcriptional regulators because of their ability to bind DNA in a sequence-specific and Zn2+dependent manner during the transition from a disordered to an ordered state.
conserved regions were observed in TaASR1:a small N-terminal sequence with 18–20 amino acids and a longer C-terminal region with at least 80 amino acids. The N-terminal consensus sequence has a stretch of six His residues in a 10 amino acid sequence that is typical for Zn-binding. In the C-terminal region , there are two Ala-rich regions , one site for N-myristoylation, a putative nuclear targeting signal an ABA/WDS domain that is described in ABA-,water , stressand ripening-induced proteins.