Cloning And Function Analysis Of The Drought-induced Promoter From Arabidopsis Thaliana

In the field of agricultural production,drought has been a natural disasters threatto the growth of crops, affecting food production. In the field of plant geneticengineering, Functional gene expression and regulation in higher plants has been hotand cutting-edge.Because the promoter has a unique structure and features, itregulates gene expression time and space of higher plant.It provides a new way ofthinking of functional genomics research and precise regulation of plant metabolismthrough genetic engineering technology. The promoter is divided into tissue specificpromoter, a constitutive promoter and an inducible promoter. Constitutive promoterdriving expression of the exogenous gene does not have the time and tissue specificity.It makes accumulation of excess exogenous gene expression product in plant,resulting in phenotypic, metabolic severely affected. Specific promoters and induciblepromoters is more valuable.In plant science, including the study of genetics and plantdevelopment Arabidopsis is important model plant.The Arabidopsis MYB gene family is a typical gene family which has aresilience function. In this study, Arabidopsis is the experimental material. We selectMYB35gene promoter, and named it Atmyb35We prediction its structure andfunction.We cloned the promoter sequence specific primers eukaryotic expressionvector, GUS staining analysis of Agrobacterium-mediated infection the tobacco seriesof research, clear the promoter, to provide the basis for future cultivation of newvarieties of drought-resistant crops.The results of this study are as follows:1. We obtained sequences of promoter Atmyb35from the the Arabidopsisthaliana online database (TAIR). The promoter Atmyb35sequence bioinformaticsanalysis results show that the sequence contains a variety of higher plants promoterUniversal promoter homeopathic role element TATA-BOX, CAAT-BOX has the basic characteristics of eukaryotic promoters. Also found that the originals involved in theABA response MYB binding sites, involved in drought-induced reaction involved invarious stress responses originals involved in the original response methyl jasmonate,function prediction results show that the change start sub having a drought-inducedproperties.2. Specific primers were designed for the cloning of promoter Atpmyb35sequence, using enzyme digestion, PCR experiments, broth sequencing resultsshowed that the results obtained in the promoter sequence. The plasmid constructed inour laboratory with the purpose of promoter fragment reorganization build the theeukaryotic plant expression carrier pCAM∷Atmyb3, and useAgrobacterium-mediated transformation of tobacco leaf and tobacco callus. We useGUS staining, and the transient expression of the GUS gene were analyzed bycomparing the wild-type, PEG6000treatment group, untreated group, the resultsshowed promoter activity, and activity was significantly enhanced by the droughtinduced.3. The deletion primer gradient promoter was designed. By5,end of the deletionanalysis, clear shows tissue expression promoter and regulatory region of the start.The eukaryotic expression vectors were built, and named them pCAM-Atmyb35I，PCAM-Atmyb35II，pCAM-Atmyb35III,pCAM-Atmyb35IV,pCAM-Atmyb35V. Thefluorescence quantitative test results showed that the core region of the promoter isAtmyb35V section. The strongest activity is Atmyb3I paragraph.4. In this study, Agrobacterium-mediated leaf disc transformation method is usedand we successfully obtained transgenic tobacco plants. By GUS histochemicalstaining method we analyze the expression of GUS gene, the results show that thepromoter can stable expression in tobacco. Transgenic tobacco plants afterdrought-induced promoter activity was significantly enhanced.