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Isolation And Transient Expressional Analysis Of The Promoter Of CaSC1in Tobacco

Posted on:2013-11-12Degree:MasterType:Thesis
Country:ChinaCandidate:C Y LiFull Text:PDF
GTID:2230330374462730Subject:Cell biology
Abstract/Summary:PDF Full Text Request
CaSC1:(Sesquiterpene cyclase of Capsicum annuum), a key enzyme whichinvolved in Metabolic process of Capsidiol, FPP catalytic synthesis of5-epi-Aristolochene which play important roles in the regulation of plant growth,development and stresses responses. Our previous study found that CaSC1is a keycyclase which involved in Metabolic process of Capsidiol,FPP catalytic synthesis of5-epi-Aristolochene.The expression of CaSC1can be induced by UV light treatment.However the molecular mechanism of the transcription expression of CaSC1against UV light lightning remains unclear. In this study, we isolated the promoter ofCaSC1from the genomic DNA of pepper leaves. Deletion analysis of the CaSC1promoter in tobacco leaves against different stresses was carried out via anAgrobacterium-mediated transient expression assay. The results are as followings:(1) The612bp upstream regular sequence of the CaSC1was isolated from thegenomic DNA of pepper using genome walking. Bioinformatics analysis of thepromoter sequences showed that the TATA-box (TATATAA) of the promoter regionsfrom-140bp to-136bp, and many cis-acting elements including W-box, TCA(SA-responsive element), and ABRE responsive to different stresses were found in thepromoter.(2)Transient expression in tobacco leaves of the promoter-GUS fusionexpression vector addicted that the-681bp region upstream of CaSC1was a fullypromoter region. The GUS gene of CaSC1promoter can be induced by UV-B.JA.SAtreatment, in according to the conclusion that the transcript of CaSC1gene can beinduced under the UV-B.JA.SA treatment. Further deletion analysis of the CaSC1promoter fuse to the GUS reporter gene in tobacco leaves showed that the UV-Brelative cis-acting element of the CaSC1promoter was located between-612bp and-543bp, the SA relative cis-acting element of the CaSC1promoter was locatedbetween-181bp and-127bp and the JA relative cis-acting element of the CaSC1promoter was located between-543bp and-336bp. (3) Using particle bombardment in onion epidermis cell through, the peppertranscription factors CaWRKY2, CaWRKY3and CaWRKY5can be binding to thepromoter of CaSC1and driving the expression of downstream GUS gene. The threetranscription factors above play positive and negative role in plant defense reactionagainst pathogen implies that the expression of CaSC1may be regulated by the threegenes above.
Keywords/Search Tags:Genome-walking, promoter, Gateway, Agroinfiltration, chemicalorganization analysis
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