本文主要研究内容
作者王丹阳（2019）在《酵母转录因子Cbfl蛋白K271位点泛素化修饰及其功能研究》一文中研究指出：目的:作为酵母细胞中的一种关键转录因子,Cbf1（Centromere-binding protein1）参与并调控了包括呼吸作用、脂质合成以及甲硫基酸合成等多种通路相关基因的转录激活。课题组前期研究发现,相对于免疫沉淀（IP）策略富集细胞内所有的Cbf1,仅采用DNA Pulldown方法以富集Cbf1转录复合体,可以鉴定到Cbf1蛋白的K271位点被高度泛素化,暗示仅少部分Cbf1可发生泛素化修饰,且K271位点泛素化修饰可能参与转录激活调控。因此,本课题以酿酒酵母为研究对象,旨在研究Cbf1蛋白K271位点泛素化修饰特异性参与某些生物学过程的调控机制,并验证其生物学功能。方法:1.JMP024-cbf1△、JMP024-3HA-Cbf1和JMP024-3HA-Cbf1K271菌株的构建1.1 JMP024-cbf1△菌株的构建在SGD数据库中获取Cbf1蛋白的基因序列,合成含有Cbf1基因上游和下游同源序列和卡那霉素ORF两端序列的引物,然后以卡那霉素抗性基因为模板,采用PCR扩增技术扩增敲除元件。将敲除元件转化到JMP024感受态细胞中,涂布于YPD-G418平板上,筛选转化子,并通过菌落PCR筛选阳性克隆。1.2 JMP024-3HA-Cbf1菌株的构建首先合成用于扩增3xHA片段和Cbf1片段的上、下游引物,分别以pRS316质粒和酵母基因组DNA为模板扩增3xHA片段和Cbf1片段,然后通过融合PCR扩增得到3xHA-Cbf1片段。将融合扩增得到的3xHA-Cbf1片段连接到pMD18-T载体上。转化细胞,提取质粒并测序。以测序正确的质粒为模板扩增3xHA-Cbf1片段,转化到JMP024-cbf1△细胞中,涂布于SD-Met培养基中,筛选转化子,并通过菌落PCR筛选阳性克隆。1.3 JMP024-3HA-Cbf1K271R菌株的构建设计合成Cbf1K271R点突变引物,以3HA-Cbf1-T质粒为模板进行PCR扩增。反应结束后,加入Dpn I限制性内切酶消除模板质粒DNA,转染细胞,提取质粒并测序。以测序正确的质粒为模板扩增得到3HA-Cbf1K271R片段,转化到JMP024-cbf1△感受态细胞中,涂布于SD-Met平板上筛选转化子,通过菌落PCR筛选阳性克隆。2.Cbf1点突变和野生型菌株中Cbf1表达量和菌株生长状况比较2.1 Cbf1表达量检测培养Cbf1点突变和野生型菌株,在变性条件下提取细胞总蛋白并测定蛋白浓度。通过Western Blot和SILAC定量蛋白质组学检测Cbf1点突变菌株和野生型菌株中Cbf1含量是否有差异。2.2生长状况表征YPD培养Cbf1点突变和野生型菌株,以相同起始OD600转接到SC和SD+Met液体培养基中,每隔2 h测菌液OD600,每株菌设置两个平行生物学重复并计算标准差。取指数生长期的菌体(OD600=1.0)于离心管中,倍比稀释为四种浓度,点在SC和SD+Met平板上,每12小时拍照,观察表型。3.Cbf1 K271位点泛素链在甲硫氨酸合成通路中作用3.1 SILAC定量蛋白质组学利用SILAC正反标策略培养Cbf1点突变和野生型菌株,并设置生物学重复。取等量轻、重标酵母细胞混合后变性条件下提取全细胞蛋白,经胰蛋白酶消化后通过StageTip快速分离方法制备质谱样品进行鉴定,使用MaxQuant软件对数据进行分析。3.2实时定量PCR在SD+Met培养基中培养Cbf1点突变和野生型菌株,置换到新的SD-Met培养基中培养0min、15min、30min、45min收集菌体置于液氮中速冻。研磨法提取RNA,利用反转录试剂盒合成cDNA。设计目的基因引物序列,进行RT-PCR扩增。4.Cbf1蛋白K271位点特异性调控底物蛋白筛选通过SILAC定量蛋白质组学方法得到蛋白定量信息,设置卡值标准,筛选差异蛋白。分析差异蛋白功能,并挑选差异蛋白通过RT-PCR进行转录水平验证;通过点板表型实验和生长曲线测绘检测生长状况。5.Cbf1 K271位点泛素化的生物学功能合成生物素修饰的上、下游引物,以Met4-2367-T质粒为模板进行PCR扩增并回收产物,然后将回收的DNA片段与Dynabeads?M-280 Streptavidin beads充分结合后,beads与Cbf1点突变和野生型菌株全细胞蛋白混合,以富集与该DNA序列存在相互作用的转录复合物相关蛋白。将富集的蛋白经胶内胰蛋白酶消化后进行质谱鉴定和数据分析。结果:1.JMP024-cbf1△、JMP024-3HA-Cbf1和JMP024-3HA-Cbf1K271R菌株的构建通过菌落PCR和琼脂糖凝胶电泳验证各菌株条带大小符合预期,菌株构建成功。2.点突变和野生型菌株Cbf1表达量和生长状况检测Western Blot结果显示,Cbf1点突变和野生型菌株中Cbf1蛋白含量在全细胞蛋白水平并无明显差异。SILAC实验定量结果显示Cbf1蛋白在点突变和野生型菌株中表达量无明显差异。生长曲线测绘和点板表型实验结果显示,点突变和野生型菌株的生长状况较为一致。3.Cbf1 K271位点泛素链在甲硫氨酸合成通路中作用探究SILAC定量结果显示,除Met22蛋白外,甲硫氨酸合成通路相关蛋白在Cbf1点突变和野生型菌株中表达量基本无差异。RT-PCR结果显示,Met22在点突变和野生型菌株中的转录水平几乎无差异。通路下游位置的蛋白受到缺甲硫氨酸（Methionine）刺激时,在点突变和野生型菌株中转录激活水平无明显差异。点板表型实验结果显示,Cbf1点突变菌株对多种甲硫氨酸浓度刺激不敏感。因此在甲硫氨酸合成通路激活前后K271位点泛素化修饰对该通路相关基因转录激活无明显影响。4.Cbf1蛋白K271位点特异性调控底物筛选及验证根据卡值标准筛选到SC培养基中K271R点突变菌株相对野生型菌株有32个差异蛋白,主要参与细胞应激、葡萄糖代谢和生物合成等生理过程。RT-PCR结果显示,GPP2、GRX1和TDH1在Cbf1K271R点突变菌株中转录水平明显下调。生长表型实验结果显示,在含有不同浓度AgNO3固体平板上,Cbf1野生型菌株生长明显受到阻滞。因此,在正常培养条件下,Cbf1K271位点的泛素化修饰可能参与上述蛋白的转录激活过程的调控;在AgNO3刺激条件下,Cbf1 K271位点泛素化修饰发挥阻碍细胞应答的作用,具体机制有待探究。5.Cbf1 K271位点泛素化修饰功能DNA Pulldown纯化结果显示,转录复合体上Cbf1蛋白在野生型菌株中含量超过Cbf1K271R点突变菌株。结论:1.Cbf1 K271位点上泛素化修饰不影响自身稳定性,Cbf1 K271位点泛素链并不介导蛋白质降解功能。2.Cbf1 K271位点泛素化修饰并不参与甲硫氨酸合成通路相关基因的转录激活调控。3.Cbf1 K271位点泛素化修饰可能通过影响Cbf1与某些蛋白编码基因的启动子区域的结合能力,进而影响基因的转录激活。体现了泛素化修饰在调控转录因子靶点特异性过程中的重要作用。
Abstract
mu de :zuo wei jiao mu xi bao zhong de yi chong guan jian zhuai lu yin zi ,Cbf1（Centromere-binding protein1）can yu bing diao kong le bao gua hu xi zuo yong 、zhi zhi ge cheng yi ji jia liu ji suan ge cheng deng duo chong tong lu xiang guan ji yin de zhuai lu ji huo 。ke ti zu qian ji yan jiu fa xian ,xiang dui yu mian yi chen dian （IP）ce lve fu ji xi bao nei suo you de Cbf1,jin cai yong DNA Pulldownfang fa yi fu ji Cbf1zhuai lu fu ge ti ,ke yi jian ding dao Cbf1dan bai de K271wei dian bei gao du fan su hua ,an shi jin shao bu fen Cbf1ke fa sheng fan su hua xiu shi ,ju K271wei dian fan su hua xiu shi ke neng can yu zhuai lu ji huo diao kong 。yin ci ,ben ke ti yi niang jiu jiao mu wei yan jiu dui xiang ,zhi zai yan jiu Cbf1dan bai K271wei dian fan su hua xiu shi te yi xing can yu mou xie sheng wu xue guo cheng de diao kong ji zhi ,bing yan zheng ji sheng wu xue gong neng 。fang fa :1.JMP024-cbf1△、JMP024-3HA-Cbf1he JMP024-3HA-Cbf1K271jun zhu de gou jian 1.1 JMP024-cbf1△jun zhu de gou jian zai SGDshu ju ku zhong huo qu Cbf1dan bai de ji yin xu lie ,ge cheng han you Cbf1ji yin shang you he xia you tong yuan xu lie he ka na mei su ORFliang duan xu lie de yin wu ,ran hou yi ka na mei su kang xing ji yin wei mo ban ,cai yong PCRkuo zeng ji shu kuo zeng qiao chu yuan jian 。jiang qiao chu yuan jian zhuai hua dao JMP024gan shou tai xi bao zhong ,tu bu yu YPD-G418ping ban shang ,shai shua zhuai hua zi ,bing tong guo jun la PCRshai shua yang xing ke long 。1.2 JMP024-3HA-Cbf1jun zhu de gou jian shou xian ge cheng yong yu kuo zeng 3xHApian duan he Cbf1pian duan de shang 、xia you yin wu ,fen bie yi pRS316zhi li he jiao mu ji yin zu DNAwei mo ban kuo zeng 3xHApian duan he Cbf1pian duan ,ran hou tong guo rong ge PCRkuo zeng de dao 3xHA-Cbf1pian duan 。jiang rong ge kuo zeng de dao de 3xHA-Cbf1pian duan lian jie dao pMD18-Tzai ti shang 。zhuai hua xi bao ,di qu zhi li bing ce xu 。yi ce xu zheng que de zhi li wei mo ban kuo zeng 3xHA-Cbf1pian duan ,zhuai hua dao JMP024-cbf1△xi bao zhong ,tu bu yu SD-Metpei yang ji zhong ,shai shua zhuai hua zi ,bing tong guo jun la PCRshai shua yang xing ke long 。1.3 JMP024-3HA-Cbf1K271Rjun zhu de gou jian she ji ge cheng Cbf1K271Rdian tu bian yin wu ,yi 3HA-Cbf1-Tzhi li wei mo ban jin hang PCRkuo zeng 。fan ying jie shu hou ,jia ru Dpn Ixian zhi xing nei qie mei xiao chu mo ban zhi li DNA,zhuai ran xi bao ,di qu zhi li bing ce xu 。yi ce xu zheng que de zhi li wei mo ban kuo zeng de dao 3HA-Cbf1K271Rpian duan ,zhuai hua dao JMP024-cbf1△gan shou tai xi bao zhong ,tu bu yu SD-Metping ban shang shai shua zhuai hua zi ,tong guo jun la PCRshai shua yang xing ke long 。2.Cbf1dian tu bian he ye sheng xing jun zhu zhong Cbf1biao da liang he jun zhu sheng chang zhuang kuang bi jiao 2.1 Cbf1biao da liang jian ce pei yang Cbf1dian tu bian he ye sheng xing jun zhu ,zai bian xing tiao jian xia di qu xi bao zong dan bai bing ce ding dan bai nong du 。tong guo Western Blothe SILACding liang dan bai zhi zu xue jian ce Cbf1dian tu bian jun zhu he ye sheng xing jun zhu zhong Cbf1han liang shi fou you cha yi 。2.2sheng chang zhuang kuang biao zheng YPDpei yang Cbf1dian tu bian he ye sheng xing jun zhu ,yi xiang tong qi shi OD600zhuai jie dao SChe SD+Metye ti pei yang ji zhong ,mei ge 2 hce jun ye OD600,mei zhu jun she zhi liang ge ping hang sheng wu xue chong fu bing ji suan biao zhun cha 。qu zhi shu sheng chang ji de jun ti (OD600=1.0)yu li xin guan zhong ,bei bi xi shi wei si chong nong du ,dian zai SChe SD+Metping ban shang ,mei 12xiao shi pai zhao ,guan cha biao xing 。3.Cbf1 K271wei dian fan su lian zai jia liu an suan ge cheng tong lu zhong zuo yong 3.1 SILACding liang dan bai zhi zu xue li yong SILACzheng fan biao ce lve pei yang Cbf1dian tu bian he ye sheng xing jun zhu ,bing she zhi sheng wu xue chong fu 。qu deng liang qing 、chong biao jiao mu xi bao hun ge hou bian xing tiao jian xia di qu quan xi bao dan bai ,jing yi dan bai mei xiao hua hou tong guo StageTipkuai su fen li fang fa zhi bei zhi pu yang pin jin hang jian ding ,shi yong MaxQuantruan jian dui shu ju jin hang fen xi 。3.2shi shi ding liang PCRzai SD+Metpei yang ji zhong pei yang Cbf1dian tu bian he ye sheng xing jun zhu ,zhi huan dao xin de SD-Metpei yang ji zhong pei yang 0min、15min、30min、45minshou ji jun ti zhi yu ye dan zhong su dong 。yan mo fa di qu RNA,li yong fan zhuai lu shi ji he ge cheng cDNA。she ji mu de ji yin yin wu xu lie ,jin hang RT-PCRkuo zeng 。4.Cbf1dan bai K271wei dian te yi xing diao kong de wu dan bai shai shua tong guo SILACding liang dan bai zhi zu xue fang fa de dao dan bai ding liang xin xi ,she zhi ka zhi biao zhun ,shai shua cha yi dan bai 。fen xi cha yi dan bai gong neng ,bing tiao shua cha yi dan bai tong guo RT-PCRjin hang zhuai lu shui ping yan zheng ;tong guo dian ban biao xing shi yan he sheng chang qu xian ce hui jian ce sheng chang zhuang kuang 。5.Cbf1 K271wei dian fan su hua de sheng wu xue gong neng ge cheng sheng wu su xiu shi de shang 、xia you yin wu ,yi Met4-2367-Tzhi li wei mo ban jin hang PCRkuo zeng bing hui shou chan wu ,ran hou jiang hui shou de DNApian duan yu Dynabeads?M-280 Streptavidin beadschong fen jie ge hou ,beadsyu Cbf1dian tu bian he ye sheng xing jun zhu quan xi bao dan bai hun ge ,yi fu ji yu gai DNAxu lie cun zai xiang hu zuo yong de zhuai lu fu ge wu xiang guan dan bai 。jiang fu ji de dan bai jing jiao nei yi dan bai mei xiao hua hou jin hang zhi pu jian ding he shu ju fen xi 。jie guo :1.JMP024-cbf1△、JMP024-3HA-Cbf1he JMP024-3HA-Cbf1K271Rjun zhu de gou jian tong guo jun la PCRhe qiong zhi tang ning jiao dian yong yan zheng ge jun zhu tiao dai da xiao fu ge yu ji ,jun zhu gou jian cheng gong 。2.dian tu bian he ye sheng xing jun zhu Cbf1biao da liang he sheng chang zhuang kuang jian ce Western Blotjie guo xian shi ,Cbf1dian tu bian he ye sheng xing jun zhu zhong Cbf1dan bai han liang zai quan xi bao dan bai shui ping bing mo ming xian cha yi 。SILACshi yan ding liang jie guo xian shi Cbf1dan bai zai dian tu bian he ye sheng xing jun zhu zhong biao da liang mo ming xian cha yi 。sheng chang qu xian ce hui he dian ban biao xing shi yan jie guo xian shi ,dian tu bian he ye sheng xing jun zhu de sheng chang zhuang kuang jiao wei yi zhi 。3.Cbf1 K271wei dian fan su lian zai jia liu an suan ge cheng tong lu zhong zuo yong tan jiu SILACding liang jie guo xian shi ,chu Met22dan bai wai ,jia liu an suan ge cheng tong lu xiang guan dan bai zai Cbf1dian tu bian he ye sheng xing jun zhu zhong biao da liang ji ben mo cha yi 。RT-PCRjie guo xian shi ,Met22zai dian tu bian he ye sheng xing jun zhu zhong de zhuai lu shui ping ji hu mo cha yi 。tong lu xia you wei zhi de dan bai shou dao que jia liu an suan （Methionine）ci ji shi ,zai dian tu bian he ye sheng xing jun zhu zhong zhuai lu ji huo shui ping mo ming xian cha yi 。dian ban biao xing shi yan jie guo xian shi ,Cbf1dian tu bian jun zhu dui duo chong jia liu an suan nong du ci ji bu min gan 。yin ci zai jia liu an suan ge cheng tong lu ji huo qian hou K271wei dian fan su hua xiu shi dui gai tong lu xiang guan ji yin zhuai lu ji huo mo ming xian ying xiang 。4.Cbf1dan bai K271wei dian te yi xing diao kong de wu shai shua ji yan zheng gen ju ka zhi biao zhun shai shua dao SCpei yang ji zhong K271Rdian tu bian jun zhu xiang dui ye sheng xing jun zhu you 32ge cha yi dan bai ,zhu yao can yu xi bao ying ji 、pu tao tang dai xie he sheng wu ge cheng deng sheng li guo cheng 。RT-PCRjie guo xian shi ,GPP2、GRX1he TDH1zai Cbf1K271Rdian tu bian jun zhu zhong zhuai lu shui ping ming xian xia diao 。sheng chang biao xing shi yan jie guo xian shi ,zai han you bu tong nong du AgNO3gu ti ping ban shang ,Cbf1ye sheng xing jun zhu sheng chang ming xian shou dao zu zhi 。yin ci ,zai zheng chang pei yang tiao jian xia ,Cbf1K271wei dian de fan su hua xiu shi ke neng can yu shang shu dan bai de zhuai lu ji huo guo cheng de diao kong ;zai AgNO3ci ji tiao jian xia ,Cbf1 K271wei dian fan su hua xiu shi fa hui zu ai xi bao ying da de zuo yong ,ju ti ji zhi you dai tan jiu 。5.Cbf1 K271wei dian fan su hua xiu shi gong neng DNA Pulldownchun hua jie guo xian shi ,zhuai lu fu ge ti shang Cbf1dan bai zai ye sheng xing jun zhu zhong han liang chao guo Cbf1K271Rdian tu bian jun zhu 。jie lun :1.Cbf1 K271wei dian shang fan su hua xiu shi bu ying xiang zi shen wen ding xing ,Cbf1 K271wei dian fan su lian bing bu jie dao dan bai zhi jiang jie gong neng 。2.Cbf1 K271wei dian fan su hua xiu shi bing bu can yu jia liu an suan ge cheng tong lu xiang guan ji yin de zhuai lu ji huo diao kong 。3.Cbf1 K271wei dian fan su hua xiu shi ke neng tong guo ying xiang Cbf1yu mou xie dan bai bian ma ji yin de qi dong zi ou yu de jie ge neng li ,jin er ying xiang ji yin de zhuai lu ji huo 。ti xian le fan su hua xiu shi zai diao kong zhuai lu yin zi ba dian te yi xing guo cheng zhong de chong yao zuo yong 。
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