gst-exo84质粒的构建和exo84-2酿酒酵母突变菌株的构建

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1、山西医科大学硕士学位论文GST-Exo84质粒的构建和Exo84-2酿酒酵母突变菌株的构建中文摘要目的1.试图构建Exo84p质粒表达载体，为体外乙酰化组蛋白是否可以与Exo84p结合做准备。2.制成组蛋白敲除/exo84-2变异菌株以观察DYN2基因内含子的剪接状况。方法：1.从酵母基因中，经PCR扩增出所需的Exo84p，经纯化后用XmaⅠ酶切，琼脂糖凝胶回收后与pGEX-2T载体连接，导入感受态细胞DH5α中，挑选出正确的克隆。2.用PCR方法扩增出带LEU的基因片段，将扩增产物转入目的酵母X中，得到Exo84-2变异菌株。用RT

2、-PCR技术检测Exo84-2突变体对DYN2基因第二外显子的保留/切除情况。结果我们构建出Exo84质粒与Exo84-2变异菌株，并在Exo84-2变异菌株的背景下观察DYN2基因第二外显子保留/剪接的情况。结论1实验成功构建出Exo84-2变异菌株。2在Exo84-2变异菌株中，DYN2基因第二外显子被保留的效率比野生型低。关键词Exo84基因；DYN2基因；mRNA剪接II山西医科大学硕士学位论文英文摘要Objective1.WemanagedtoconstructtheExo84pexpressedplasmid,inorder

3、toperformtheexperimenttoconformthatifacetylatedhistoneH3interactwithexo84pinvitro.2.Wealsomadethetemperaturesensitiveexo84-2mutantforstrainofSaccharomycescerevisiae,andtestedtheratioofinclusionofsecondexoninDYN2gene(YDR424C)andSCR1.TheminigeneforDYN2genewereconstructedby

4、uspreviously.Methods1.WechosethepGEX-2TasavectoranddesignedapairofprimersfortheEXO84genewhichcouldbeamplifiedfrombuddingyeastgenomicDNA.TheamplifiedEXO84geneDNAweresubclonedintopGEX-2TviaxmaIsite.2.WedesignedapairofprimersforamplifyingtheLeugene,andtransformedthePCRprodu

5、ctintobuddingyeaststrainwhichcontainingtheDNY2minigene.ThetransformantswerescreenedbySc-Trp-Leu.Afterwegotthemutant,Theyeaststrainwereculturedintopermisivetemperature(30°C)andthenputintononpermisivetemperature(37°C)for1.5hand3h.Afterharvestingtheyeastcellsweextractedthem

6、RNAandperformtheRT-PCRtodetectetheratioofinclusionofsecondexonofDYN2genezndScr1mRNA.ResultsWegottheplasmidscontainingtheexo84gene.Wealsomaketheexo84-2mutantstrainandobservedtheinclusion/exclusionofsecondexonofDYN2geneintheexo84-2mutationbackground.ConclusionsTheefficienc

7、yofmRNAsplicingforDYN2genebecomeslowinexo84-2background.Keywords：Exo84gene;DYN2gene;mRNAsplicingIII山西医科大学硕士学位论文英汉缩略语名词对照英文缩写英文全称中文全称AMPAmpicillin氨苄青霉素bpBasepair碱基对ddH2ODoubledistillationwater双蒸水DNADeoxyribonucleicacid脱氧核糖核酸dNTPDeoxynucleosidestriphosphate脱氧核糖核苷三磷酸EBEthid

8、iumbromide溴化乙锭EDTAethylenedinitrilotetraaceticacid乙二胺四乙酸LBLuria-bertanimediumLB培养基KbKilobase千碱基对ODOptic