Stress Tolerance Analysis And Subcellular Localization Of Cpcor413pm1 Gene

Chimonanthus praecox(L.)Link(Wintersweet)is a plant which belongs to calycanthaceae, chimonanthus and it has a strong resistance for abotic stress(cold,drought).Cpcor413pm1 gene from chimonanthus praecox,expresses not only induced by cold stress,but also by drought,salality and ABA.therefor,Cpcor413pm1 may be associated with stress tolerance.So we performed functional analysis,and the results are as follow:1.Cloning of RD29A promoter from ArabidospsisAccording to the sequence published in Genebank(number is D 13044),we designed specific PCR primers to clone RD29A promoter using genomic DNA from Arabidospsis as template.Sequence of the clone is completely the same as that of D13044 and it contains four ABRE elements,two DRE elements,a TATA-box,which suggested that the clone is a intact RD29A promoter.2.Construction of Cpcor413pm1 plant expression vector2.1 Construction of pBL616-RD29A- Cpcor413pm1 plant expression vectorBecause Cpcor413pm1 can not transfered directly from pTriplEx2 plasmid into pBL616 plant expression plasmid,so digested pTriplEx2-Cpcor413pm1 plasmid and modified pMD18-T plasmid for sfil endonuclease,then connect them with T4 ligase forming pMD18-Cpcor413pm1.Digested pMD18-Cpcor413pm1 plasmid and pBL616 plasmid for Xbal,Smal endonuclease,then connect them with T4 ligase forming pBL616-Cpcor413pm1.Digested pBL616-Cpcor413pm1 plasmid and pMD18-RD29A plasmid for HindⅢ,Xbal endonuclease,then connect them with T4 ligase forming pBL616-RD29A-Cpcor413pm1 plasmid.2.2 Construction of pC-Cpcor413pm1:GFP1 plant expression vectorAmplify Cpcor413pm1 without terminor by PCR using pfu polymerase,then Cpcor413pm1 was cloned into pMD18-T vector forming pMD 18-Cpcor413pm1.Digested pMD 18-Cpcor413pm1 vector and pCAMBIA1302 vector for Ncol,Spel endonuclease,then connect them with T4 ligase forming pC-Cpcor413pm1:GFP which do not changes the open reading frame of GFP.3.Obtain transgenic tobaccoRD29A-Cpcor413pm1 and Cpcor413pm1:GFP were introduced into tobacco by Agrobacterium-mediated Transformation,and then selected the single plant lines which growed in the medium containing antibiotics.4.Tolerance analysis of transgenic tobacco4.1 Analysis of cold toleranceAfter 4℃treatment for 24 hours,increase of electrical conductivity in transgenic tobacco was more than that in the control,suggesting that the membrane stability of transgenic tobacco is better than that of the control,which means that Cpcor413pm1 gene could enhances the stability of membrane;chlorophyll content desreased respectively 60%,38%in transgenic tobacco and the control,indicating that Cpcor413pm1 gene could protect chlorophyll from degradation;MDA content in the control is as more than twice as that in transgenic tobacco,which means that Cpcor413pm1 gene could reduce membrane lipid peroxidation;SOD activity of transgenic tobacco is more one times higher than that of the control,suggesting that Cpcor413pm1 gene could protect anti-oxygen protective enzyme system..4.2 Analysis of drought toleranceIt is founded that both transgenic tobacco and the control are all wilting that the degree are the same,after no watering for a month.Then give them watering,and the degree of recovery are also the same.So we deduced Cpcor413pm1 gene could not work in the process of drought stress.4.3 Analysis of salt toleranceThe injury degree and death rate of transgenic tobacco are the same as of non-transgenic tobacco when treated in 250mm NaCl solution for the same time,suggesting that Cpcor413pm1 gene do not play a vital role in salt-stress.5.Subcellular localization of Cpcor413pm1Cpcor413pm1 protein is predicted to locate in plasma membrane by bioinformatics,and we could see the green fluorescence in plasma membrane of root cell of transgenic tobacco,but not in the control.Therefore,we made a conclusion that Cpcor413pm1 protein locates in plasma membrane playing a important role when low temperature comes.