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Study On Stress Tolerance Mechanism Mediated By AtbHLH112from Arabidopsis Thaliana

Posted on:2014-11-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y J LiuFull Text:PDF
GTID:1260330401479606Subject:Tree genetics and breeding
Abstract/Summary:PDF Full Text Request
The basic helix-loop-helix (bHLH) genes are a big group of transcription factors in plants. which is made up by60-100amino acids, containing a highly conserved DNA binding domain is followed by basic region and HLH.11domain. In plants, the vast majority of bHLH genes involved in regulating response by identifying the cis-acting element of G-box, such as growing development, nutrient absorption, signal transduction and adversity stress. In the present study, the T3transgenic Arabidopsis overexpressing AtbHLH112showed considerable stress tolerance under abiotic stress of ABA, NaCl and Manntiol, by comparing with T3transgenic Arabidopsis of AtbHLH112suppress expression, T3deletion mutant Arabidopsis of AtbHLH112, and wild type of Col-0. The recombinant AtbHLH112-GFP fused gene was transformed into onion epidermal cells by particle gun for subcellular location analysis. The results showed that AtbHLH112is located in nuclear of cell. The AtbHLH112promoter was fused with GUS. and introduced into wild-type Col-0by floral infiltration for promoter activity analysis. And GUS staining analysis indicated that the promoter of AtbHLH112has high activity in different plant tissues. PLACE and PlantCARE database prediction analysis suggested that there are many cis-elements which associated with signaling pathway and strss resistance of plants in the promoter of AtbHLH112, such as ARR1TA. GTGANTG10, MYCCONSENSUSAT. MYBCORE. MYBST1and WRKY71OS. We obtained that a AtPP2C gene (AtPP2C24) and a AtWRKY gene (AtWRKY66), using yeast one-hybrid analysis, can regulate the expression of AtbHLH112through recognizing E-box and W-box in the promoter of AtbHLH112, respectively. Furthermore, real-time quantification RT-PCR showed that AtPP2C24and AtWRKY66shared a similar expression patterns with AtbHLH112in response to adversity stresses, indicating that they may belong to the same expression regulation network. Yeast one-hybrid study showed that AtbHLH112can interact with G-box, and the DNA binding domain located in C-terminal of AtbHLH112. Agilent Arabidopsis oligo microarray analysis was employed to identify differences in gene expression between transgenic Arabidopsis and deletion mutant of AtbHLH112under normal and salt stress conditions. The results revealed that there are4159genes and2862genes significantly (P<0.05; FC>2) differentially expressed, respectively, under normal and salt stress conditions. The Agilent Arabidopsis oligo microarray results showed that AtbHLH112can regulate series related target genes, especially the related genes of ribosomal protein, heat shock protein, transduction grain protein, cell cycle protein, disease-resistance protein as well as a variety of transcription factors, such as NAC. WRKY. DOF, MYB, ZFP, ERF, TCP. and so on. Yest two-hybrid analysis revealed that the single form of AtbHLH112has transactivation activity, and the domain located in N-terminal of AtbHLH112; the fragment of AtbHLH112which do not contain transcriptional activation domain hybridized with the cDNA library of Arabidopsis thaliana to identified7interacting proteins, including2chlorophyll binding proteins,2ubiquitin-conjugating enzymes, a glutamine synthetase. a lipoxygenase and an unknown protein.
Keywords/Search Tags:Arabidopsis thaliana, AtbHLH112transeription factor, Abiotic stress, stressresistance, regulation of gene expression
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