| The AP2/EREBP (ethylene responsive element binding protein) family of transcriptionfactors are known unique to plants, and they are mainly involved in cell cycle, growth,development, and the regulation of abiotic and biotic stress related gene’s expression inplants. According to the number of AP2/ERF domain, the AP2/EREBP superfamily couldbe divided into AP2, RAV and ERF three family. The ERF family proteins, which containone AP2/ERF domain, could be divided into CBF/DREB subfamily, ERF sufamily andother proteins. DREB proteins which belongs to the CBF/DREB subfamily,couldspecifically bind to the dehydration responsive element, response to drought, highsalt, low temperature, water and other abiotic stresses, and regulate the expression of genesinduced by stress conditions. The ERF subgroup proteins, which areinvolved in planthormone (ethylene) molecular response, also play a role in abiotic stress. The considerablestudies of AP2/EREBPfamily genes were madein the model plant Arabidopsis, while fewstudies have been carried on in wild plant resources which have the excellent resistancecharacters. Therefore, to speed up the mining and functional characterization of theexcellent resistance gene resources inwild plants has the important application value andtheoretical significance to the breeding of transgenic crops.EeAP2.2and EeAP2-1.1are two AP2/EREBP family high abundance expression genesagainst abiotic stress, which were cloned from resistance excellent Elongatum in ourlaboratory. In this study, bioinformatics technology, was used to predict the function ofEeAP2.2and EeAP2-1.1; semi-quantitative RT-PCR (sqRT-PCR) assay was used to detectthe changes of the two genes’ transcript abundance under salt and drought stresses; anda preliminary study of EeAP2.2function of resistance was conducted through thetransformation of tobacco; the non-invasive micro test (NMT) technique was used toexplore the mechanism of resistance of EeAP2.2over-expression in tobacco. At the sametime, pSB187-EeAP2.2: GFP fusion expression vector was constructed, and the optimumhygromycin concentration was tested for screening the transgenic tobacco plants. Theresults are shown as follows: 1. EeAP2.2and EeAP2-1.1contain two conserved YRG and RAYD domains, belong toEREBP family. EeAP2.2belongs to DREB subfamily of ERF family, and EeAP2-1.1belongs to the ERF subfamily of ERF family.2. EeAP2-1.1expression in the roots was significantly lower than EeAP2.2under normalnon-stres condition, drought and high salt stresses can induce the upregulation of bothgenes, the two genes’ expression levels were significantly increased in the roots andleaves of Elytrigia elongata after4hours of stress, which continue increase to12hoursof stress, but the mRNA level of EeAP2-1.1was basically maintained atthe level ofafter4hours stress in the PEG stress condition.3. EeAP2.2transgenic tobacco increased K~+influx after drought stress, and deterred themassive outflow of K~+and H~+under high salt stress condition, while the speed anddirection of H~+and K~+currents were unusual after cold stress, but when transferred theplants to normal temperature condition, all wild plants died, and the EeAP2.2transgenic plants survived better.4. PCR analysis of the transformed plants showed EeAP2.2was successfully integratedinto the tobacco genome. Under normal growth conditions, transgenic tobacco plantsshowed dwarf phenotype. Their average height was reduced by about44%, the totalnumber of leaves was reduced by41.4%, and the leaf area was decreased nearly38%,but the number of green leaves at flowering time was increased.5. Wild-type tobacco seeds germinate slowly and grow weaker, compared with transgenictobacco seeds when they are planted in the MS medium containing250mM NaCl.After30days of drought stress, wild-type tobacco plant grow smaller and weaker thantransgenic tobacco plant, with more yellow leaves, and its bottom leaves become wither.The optimum concentration of hygromycin for screening pSB187carrier transgenictobacco plants was approximately4mg/L or so. |
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