MolecularcloningofTupaiabe(精)
水稻细胞分裂氧化酶的原核表达
水稻细胞分裂氧化酶的原核表达吴云华;方玉姣【摘要】The total RNA from var nipponbare was extracted and the cytokinin oxidase gene( CKO)was amplified by PCR using the reverse product cDNA as template,then CKO was ligated to vector pET-28a after restriction and a recombinant plasmid pET 28a-CKO was constructed. Finally the rice cytokinin oxidase was expressed in E. coli BL21. The results showed that the recombinant plasmid pET-28a-CKO was successfully constructed and the prokaryotic expression of rice cytokinin oxidase was achieved.%从水稻日本晴幼叶中提取 RNA，以反转产物 cDNA 为模板 PCR 扩增得到了细胞分裂素氧化酶基因（CKO），酶切连接到 pET-28a载体上，构建了重组载体 pET-28a-CKO，在大肠杆菌 BL21中表达了细胞分裂素氧化酶。
【期刊名称】《中南民族大学学报（自然科学版）》【年(卷),期】2015(000)004【总页数】4页(P37-40)【关键词】细胞分裂素氧化酶;原核表达;载体pET-28a;日本晴【作者】吴云华;方玉姣【作者单位】中南民族大学生命科学学院，武陵山区特色资源植物种质保护和利用湖北省重点实验室，武汉430074;中南民族大学生命科学学院，武陵山区特色资源植物种质保护和利用湖北省重点实验室，武汉430074【正文语种】中文【中图分类】Q51细胞分裂素作为一种植物激素，是目前人们已经知道的5大类植物激素(即生长素、赤霉素、细胞分裂素、脱落酸和乙烯)之一，它具有腺嘌呤环结构.这类物质的共同特点是在腺嘌呤环的第六位置氨基上有特定的取代物，对细胞的分裂、芽的分化、侧芽的发育、果实的发育等起重要作用.植物中细胞分裂素(CTK)[1]的分解，很大程度上依赖于一种特殊酶的作用实现.这种酶以分子氧为氧化剂，催化CTK的N6上不饱和侧链裂解而使其彻底丧失活性，此反应不可逆，这种酶是细胞分裂素氧化酶(cytokinin oxidase，CKO)，它能作用于体内CTK代谢和调节CTK与生长素之间的平衡等.玉米[2]、拟南芥[3]、石斛兰[4]、大麦[5]、小麦[6]等的细胞分裂素氧化酶基因目前已经被克隆.转基因实验表明：通过改变植物细胞分裂素氧化酶基因的表达可以调控内源激素CTK的含量，进而影响植物的发育.Ashikari M等[7]发现在水稻花序分生组织中的细胞分裂素氧化酶基因OsCKX2的表达受抑制时，会导致细胞分裂素水平增加、发育旺盛、穗粒数明显增多，从而提高水稻产量.但目前关于水稻细胞分裂素氧化酶CKO的原核表达并未见报道，本文利用pET-28a为载体，探索水稻细胞分裂素氧化酶CKO在大肠杆菌BL21中的表达，为细胞分裂素氧化酶CKO生物传感器的制备和应用研究奠定基础.1.1 材料和仪器水稻日本晴种子、载体pET-28a、大肠杆菌E.coli.DH5α和E.coli.BL21(DE3) 由中南民族大学武陵山区特色资源植物种质保护和利用湖北省重点实验室提供. Trizol Reagent(Ambion)，RevertAid First Strand cDNA SynthesisKit(Thermo scientific)，限制性内切酶Nde I、EcoR I、Xho I, T4 DNA连接酶、DNA Maker、蛋白Maker(TaKaRa)，KOD-plus-试剂盒(TOYOBO司)，DNA凝胶回收试剂盒、DNA清洁回收试剂盒(AXYGEN)，卡那霉素(上海生工公司)，二硫苏糖醇(DTT)和异丙基硫代-β-D-半乳糖苷(IPTG, Alfa Aesar)，苯甲基硫酰氟(PMSF,丁香园)，引物合成和基因测序(擎科生物有限公司)，其他试剂均为国药集团化学和天津市广成化学试剂有限公司等的分析级AR试剂.PBS缓冲液(pH 7.0, 1 L)：0.27 g KH2PO4，1.42 g Na2HPO4，8 g NaCl，0.2 g KCl，加ddH2O至900 mL混匀，调节pH至7.0，再加ddH2O定容至1L，高压灭菌.Buffer A(1 L)：171.1 g蔗糖，0.186 g EDTA·2Na，12.1 g Tris溶于900 mL ddH2O混匀，用冰乙酸调pH至7.6，再定容至1 L，过滤除菌，存于4℃备用. Buffer B(1 L)：加少量ddH2O溶解1.286 g四水乙酸镁，加入200 mL甘油，13.4 mL 1mol/L KH2PO4, 86.8 mL 1mol/L K2HPO4，加ddH2O至900 mL混匀，调节pH至7.6，再加ddH2O定容至1L，存于4℃备用[8].精密电子天平(ME204E /02, 上海梅特勒-托利多仪器有限公司)，PCR仪(PTC-200, 美国Bio-Rad公司)，琼脂糖胶电泳仪(DYY-5, 北京六一仪器厂)，恒温摇床(HQ45Z, 武汉中科仪技术有限公司)，凝胶成像系统(TEL-40Gelvue UV Transilluminator, 英国)，核酸蛋白测定仪(HD-4, 上海泸西分析仪器厂有限公司)，PH计(pHS-3C, 上海仪电科学仪器股份有限公司).1.2 原核表达载体pET-28a-CKO的构建用水泡适量日本晴种子，待其发芽后移栽到土中生长，待长出5～6片幼叶后，取日本晴新鲜幼叶，用Trizol法提取叶片的RNA，用反转试剂盒将RNA反转，用水稻Actin引物对cDNA进行检测，以该cDNA为模板，用设计的特异性引物F(5′-ATCCAT ATGGAGGTTGCCATGGTCTGC-3′)和R(5′-CCGCTCGAGGCTATAGCTTGCAAATGCGCC-3′)，设计的酶切位点是Xho I和Nde I，用KOD-plus-试剂盒进行PCR扩增，将扩增所得产物与质粒pET-28a一起先用Nde I在37 ℃酶切过夜，酶切后片段用清洁回收.再分别用Xho I进行37 ℃酶切6 h，酶切的目的片段用清洁回收，质粒pET-28a酶切产物切胶回收后，用T4连接酶将载体和目的片段于16 ℃连接12 h，构建出重组体pET-28a-CKO[9]. 将重组体pET-28a-CKO和空载pET-28a分别通过热激转化法转化到相应的感受态细胞E.coli.BL21(DE3)中，其中空载作为对照，分别取200 mL菌液涂布到含抗性的LB平板(含50 μg/mL卡那霉素)上，37 ℃培养箱中过夜培养(12～18 h)，挑取单克隆菌落进行菌落PCR检测，菌落PCR有目的带后对应的单克隆再提取质粒，进行质粒PCR和双酶切鉴定，检测正确的质粒测序.鉴定正确的重组体挑取单克隆于3 mL LB液体培养基(含50 μg/mL卡那霉素)中，在摇床中以37℃、180r/min培养过夜，将培养好的3 mL菌液接种于500 mL TB(含卡那霉素)液体培养基中，培养2～3 h检测其OD值，使其OD600达到约0.6，加入1 mol/L IPTG 使其终浓度为1 mmol/L，15℃、180 r/min诱导表达8 h.1.3 重组蛋白的提取及SDS-PAGE、Western-blot检测分析于15 ℃8 h，诱导50 mL的菌液，菌液于4℃、5000 g离心15 min，弃上清，收集菌体.冰浴放置30 min后，在4℃、5000 g离心20 min，弃上清，收集菌体，加入Buffer A重悬 (70 mg湿细胞/ mL)，再加入与Buffer A等体积的无菌水，同时加入溶菌酶至其终浓度为0.1 mg/mL，用移液器吹打混匀，冰浴在摇床上轻摇80 r/min 30 min，于4℃、5000 g离心20 min后，弃上清，沉淀中加入Buffer B重悬(0.5 g湿细胞/mL)，并现加DTT至终浓度为0.01 mmol/L，备用的蛋白冻存于-80 ℃.待破碎的，加PMSF至终浓度为1 mmol/L，在冰浴条件下超声破碎，功率为16 W，每次破碎4 s，间歇6 s，一共破碎处理约8 min，使其成为均一的液体.破碎产物于4℃、 14000 r/min离心10 min，吸取出上清备用，沉淀加入PBS缓冲液(pH 7.0)，在冰上放置1 h，对处理好的的上清、混合、沉淀进行SDS-PAGE分析检测和Western-blot分析检测[10].2.1 目的片段CKO的扩增用Trizol法从日本晴叶片提取RNA，经反转录试剂盒反转出产物cDNA，用水稻actin引物进行检测，检测结果(见图1)正确，可作为扩增模板用于目的片段的扩增.以检测好的cDNA为模板，用KOD-plus-酶扩增CKO片段，由于第1次扩增的片段很弱，以第1次PCR产物为模板进行第2次PCR条件摸索后，第2次PCR产物的浓度可用于后续的实验(见图2).2.2 构建原核表达载体pET-28a-CKO提取载体质粒pET-28a，用EcoR I单酶切检测.用Nde I分别酶切目的片段和载体质粒pET-28a，37℃过夜，回收第一次酶切产物；再用Xho I酶切6h，将第二次酶切的产物回收后，用T4连接酶16℃连接过夜，连接产物和空载pET-28a分别热激转化大肠杆菌DH5a感受态细胞.对长出的单菌落进行构建载体重组体的检测，结果见如图3.将构建好的载体经菌落和质粒PCR检测，目的片段大小约为1500 bp，再将重组载体用Nde I和Xho I双酶切检测，结果显示有约5500 bp和1500 bp的条带(见图4)，将鉴定正确的重组载体测序，测序结果100%正确，可用于后续实验. 2.3 CKO蛋白的SDS-PAGE和Western-blot分析15 ℃下用1 mmol/L IPTG诱导原核表达菌体表达8 h，以空载pET-28a为对照，诱导蛋白通过SDS-PAGE检测，与对照相比，可见蛋白大小在55～70 kDa，进一步进行Western-blot分析检测，分析结果在55～70 kDa处有带His标签的融合蛋白，该蛋白就是目的蛋白CKO，说明CKO重组蛋白成功得到了表达(见图5). 本研究利用水稻日本晴为材料，成功构建了pET-28a-CKO重组载体, 采用大肠杆菌菌株BL21(DE3)作为表达菌株，实现了水稻细胞分裂素氧化酶(CKO)的原核表达.表达产物经SDS-PAGE检测和Western-blot分析，目的蛋白获得了成功表达.本研究为后续结合纳米材料放大效应，构建相应的生物传感器和蛋白生理功能研究奠定了基础.[1] 邓江明,潘瑞炽.细胞分裂素氧化酶[J].植物生理学通讯,1997，33(5):370-375.[2] Houba-Hérin N, Pethe C, Alayer J, et al. Cytokinin oxidase from Zeamays: purification, cDNA cloning and expression inmoss protoplasts[J].Plant J, 1999, 17(6): 615-626.[3] Bilyeu K D, Cole J L, Laskey J G, et al. Molecular and biochemical characterization of a cytokinin oxidase from maize[J].Plant Physiol, 2001, 125(1):378-386.[4] Yang S H, Yu H, Goh C J. Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid[J].Plant Mol Biol, 2003,51: 237-248.[5] Petr G, Jitka F, Tomas W, et al. Cytokinin oxidase/dehydrogenase genes in barley and wheat: Cloning and heterologous expression[J]. FEBS J, 2004, 271(20): 3990-4002.[6] 马信, 林爱玲, 封德顺，等. 小麦细胞分裂素氧化酶基因TaCKX1原核表达载体的构建及表达[J]. 生物技术, 2009, 19(1):10-13.[7] Ashikari M, Sakakibara H, Lin S, et al. Cytokinin oxidase regulates rice grain production[J].Science, 2005, 309(5735): 741-745.[8] Li Z, Liu X, Wang C, et al. Expression, purification and direct eletrochemistry of cytochrome P450 6A1 from the house fly, Musca domestica[J]. Protein Expres Purif, 2010,71(1):74-78.[9] 陈思礼,梅菊.鱼腥藻PCC 7120染色体上relBE同源基因的克隆及表达[J].中南民族大学学报: 自然科学版, 2012, 31(1):16-19.[10] 吴云华, 邓欢. 尖镰胞菌细胞色素 P450 55a1 基因超表达载体的构建和水稻日本晴遗传转化[J]. 中南民族大学学报: 自然科学版, 2014, 33(2): 32-35.。
极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用
Re search Paper研究报告微生物学报Acta Microbiologica Sinica 49(8):1040-1047;4August 2009ISS N 0001-6209;C N 11-1995ΠQ http :ΠΠ Πactamicrocn基金项目:国家自然科学基金(30671141);国家“863计划”(2006AA09Z 401)3通信作者。
T el ΠFax :+86210264807472;E 2mail :xiangh @作者简介:苗荻(1983-),女,北京人,硕士研究生,主要从事微生物分子遗传学研究。
E 2mail :miaous @1631com 收稿日期:2009203216;修回日期:2009204203极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用苗荻,孙超岷,向华3(中国科学院微生物研究所,微生物资源前期开发国家重点实验室,北京 100101)摘要:【目的】利用有自主知识产权的嗜盐古菌θ型复制质粒和启动子,构建在极端嗜盐古菌模式菌株西班牙盐盒菌(Haloarcula hispanica )中方便使用、功能完善的基因表达载体。
【方法】以pSC M201的最小复制子为基础,通过引入莫维诺林抗性基因,大肠杆菌质粒复制子以及氨苄抗性基因,构建了一个新的嗜盐古菌2大肠杆菌穿梭载体。
利用定点突变和末端补平法依次将其中多余的酶切位点去除后,再添加hsp 5启动子核心序列、人工合成的多克隆位点以及蛋白纯化标签His ・T ag 等重要元件成功构建了嗜盐古菌表达载体pSC M307。
将报告基因bgaH 插入到该载体的多克隆位点中并转化H .hispanica AS2049,通过X 2gal 平板筛选和β2半乳糖苷酶酶活实验检测pSC M307的表达能力。
【结果】pSC M307具有独立的自主复制能力,其多克隆位点方便实用,报告基因bgaH 在hsp 5启动子控制下实现了高效表达。
高一英语生物名词单选题40题
高一英语生物名词单选题40题1.Which animal has a long tail and can climb trees?A.LionB.TigerC.MonkeyD.Elephant答案：C。
猴子有长尾巴且能爬树。
狮子和老虎尾巴相对较短且主要在地面活动。
大象尾巴短且体型庞大不擅长爬树。
2.Which animal is known for its black and white stripes?A.ZebraB.HippoC.GiraffeD.Lion答案：A。
斑马以黑白条纹著称。
河马是灰色的。
长颈鹿有花纹但不是黑白条纹。
狮子是黄色的。
3.Which animal has a long neck?A.PandaB.KangarooC.GiraffeD.Monkey答案：C。
长颈鹿有长脖子。
熊猫脖子短。
袋鼠脖子也不长。
猴子脖子不长。
4.Which animal can swim and fly?A.DuckB.CatC.DogD.Pig答案：A。
鸭子可以游泳和飞行。
猫、狗、猪都不能飞行。
5.Which animal is a carnivore?A.RabbitB.DeerC.LionD.Sheep答案：C。
狮子是肉食动物。
兔子、鹿、羊是食草动物。
6.Which animal hibernates in winter?A.BearB.TigerC.LionD.Elephant答案：A。
熊在冬天冬眠。
老虎、狮子、大象都不冬眠。
7.Which animal has a pouch?A.KangarooB.PandaC.GiraffeD.Monkey答案：A。
袋鼠有育儿袋。
熊猫、长颈鹿、猴子都没有育儿袋。
8.Which animal is known for its speed?A.TurtleB.SnailC.CheetahD.Rabbit答案：C。
猎豹以速度著称。
乌龟和蜗牛速度很慢。
兔子速度较快但不如猎豹。
植物学专有名词
前沿·热点Research Hot/Frontiers种系发生Phylogeny生命起源Origins of Life群落多样性Community diversity生物多样性Biodiversity外来物种Alien species物种共存Species coexistence生态系统健康Ecosystem HealthDNA条形码DNA barcoding信号转导Signal transduction细胞周期调控Cell cycle regulation细胞增殖cell proliferation细胞凋亡Cell Apoptosis生物钟Biological clock生物适应性Biochemical adaptation网络生物学Network biology生物控制论Biological Cybernetics染色体重排Chromosomal rearrangements 进化论The theory of evolution自然选择natural selection人工选择artificial selection细胞分裂cell division选择育种selective breeding遗传漂变Genetic drift种系发生phylogeny脱氧核糖核酸DNA生命起源Origin of life古生物学paleontology种系遗传学phylogenetics表型学phenetics共同祖先common descent物种形成Speciation古生菌archaea灵长类primates板块构造论plate tectonics无树大草原savannah寄生parasitism共生symbiosis生命之树Tree of life遗传分类学cladistics开放系统open system动态平衡dynamic equilibrium查尔斯·达尔文Charles Darwin动物Animals植物plants真菌fungi原生生物protists细菌bacteria原核生物prokaryote昆虫类insects病毒viruses多细胞生物multicellular organism热血动物warm-blood生态系统ecosystems系统发生树Phylogenetic tree新陈代谢metabolism遗传学Genetics遗传heredity变异variation核苷酸nucleotides氨基酸amino acids遗传密码genetic code泛生论pangenesis染色体chromosomes遗传连锁genetic linkage螺旋结构helical structure信使RNAmessenger RNA氨基酸序列amino acid sequence遗传的特征 Features of inheritance孟德尔Gregor Mendel等位基因alleles纯合子homozygote杂合子heterozygote基因型genotype表现型phenotype显性dominance隐性recessiveness不完全显性incomplete dominance共显性codominance有性繁殖sexual reproduction谱系图pedigree charts异位显性epistasis遗传力heritability遗传的分子基础 Molecular basis for inheritance 腺嘌呤adenine胞嘧啶cytosine鸟嘌呤guanine胸腺嘧啶thymine双螺旋double helix碱基对base pairs染色质chromatin核小体nucleosomes组蛋白histone单倍体haploid二倍体diploid性连锁遗传病sex-linked disorder无性生殖asexual reproduction有丝分裂mitosis配子gametes生殖细胞germ cells连锁图linkage map分子生物学中心法则central dogma of molecular biology 镰状细胞性贫血sickle-cell anemia核糖体RNArRNA转移核糖核酸tRNA微RNAmicroRNA苯（丙）酮尿症phenylketonuria转录因子Transcription factors大肠杆菌Escherichia coli色氨酸tryptophan负反馈negative feedback阻遏物repressor胞间信号intercellular signals副突变paramutation遗传改变Genetic change诱变mutagenesis紫外辐射UV radiationDNA修复DNA repair减数分裂meiosis适合度fitness果蝇Drosophila群体遗传学Population genetics适应adaptation分子钟molecular clock进化树evolutionary trees模式生物model organisms医学遗传学Medical genetics孟德尔随机化Mendelian randomization同源基因orthologues药物遗传学pharmacogenetics连接酶ligase分子克隆molecular cloning重组DNArecombinant DNA遗传多样性genetic diversity生物量biomass分子生物学Molecular biology分子筛molecular sieve核糖核酸RNA蛋白质的生物合成protein biosynthesis 生物大分子biomolecules复制replication翻译translation转录transcription克隆clone聚合酶链式反应PCR质粒plasmid载体vector启动子元件promoter elements抗生素耐性antibiotic resistance结合conjugation转导transduction转染transfection电穿孔electroporation显微注射microinjection三级结构tertiary structure定量多聚酶链反应QPCR电泳electrophoresis毛细管作用capillary action胚胎干细胞株embryonic stem cell lines SDS-PAGEnorthern blot放射自显影autoradiography增强子Enhancer抗体antibody蔗糖梯度sucrose gradient粘度测定法viscometry保护生物学Conservation biology生物多样性biodiversity种群动态population dynamics稀有种rare species自然保护运动conservation movement 灭绝extinction脊椎动物vertebrates无脊椎动物invertebrates人口过剩overpopulation砍伐森林deforestation放牧过度overgrazing刀耕火种法slash and burn应用生态学Applied ecology濒危物种Endangered species环境保护论Environmentalism迁地保护Ex-situ conservation基因污染Genetic pollution就地保护In situ conservation发育生物学Developmental Biology细胞生长cell growth形态发生morphogenesis分化differentiation染色体畸变chromosomal aberrations个体发生ontogeny细胞凋亡apoptosis先天性疾病congenital disorders内稳态homeostasis神经胚形成neurulation胚胎发生embryogenesis性别决定sex determination原肠胚形成gastrulation适应能力adaptive capacity衰老senescence细胞信号传导Cell signaling信号转导Signal transduction器官发生organogenesis胚胎发生embryogenesis形态发生morphogenesis生态学Ecology次级生产力secondary productivity捕食predationecozones食物链food chains大气圈atmosphere生物群落biocoenosis分解者Decomposers互利共生mutualism光合作用photosynthesis初级生产者primary producers初级消费者primary consumers三级消费者tertiary consumers食肉动物carnivores杂食动物omnivores生境biotope生物地球化学循环biogeochemical cycle氮循环nitrogen cycle陆地生态系统terrestrial ecosystems森林生态系统forest ecosystems水圈hydrosphere生物圈biosphere岩石圈lithosphere生态位ecological niche海洋生态系统marine ecosystems生态危机Ecological crisis血亲consanguinity植物学Botany/Plant Biology(Plant Science)真菌类fungi被子植物angiosperms藻类algae双子叶植物dicotyledon单子叶植物monocot裸子植物gymnosperm分类学taxonomy形态学morphology藓类Mosses苔类liverworts园艺学Horticulture花粉Pollen植物病理学Phytopathology植物生理学Plant physiology草本植物Herbs温室气体greenhouse gas地衣类lichens水循环water cycle古植物学家Paleobotanists果菜类fruit vegetable食性层次trophic level食品安全food security作物育种plant breeding人类植物学Ethnobotany遗传学定律genetic laws四氢大麻酚tetrahydrocannabinol跳跃基因jumping genes细胞生物学Cell biology显微镜microscope核糖体ribosomes细胞质cytoplasm膜蛋白membrane proteins内质网endoplasmic reticulum高尔基体Golgi apparatus细胞骨架cytoskeletal线粒体mitochondria细胞核nucleus叶绿体chloroplasts溶酶体lysosomes主动运输Active transportmRNA剪接mRNA splicing自吞噬Autophagy纤毛Cilia微管microtubule鞭毛Flagella脂质双层Lipid bilayerImmunostaining原位杂交In situ hybridization免疫沉淀反应Immunoprecipitation免疫组化immunohistochemistry生命周期life cycles神经生物学Neurobiology计算神经科学computational neuroscience 认知神经科学cognitive neuroscience行为神经学behavioral neuroscience生物精神病学biological psychiatry神经病学neurology神经心理学neuropsychology神经心理学neuropsychology树状突dendrites光受体photoreceptors动作电位action potentialsquid giant axon突触synapses神经系统nervous system多巴胺Dopamine去甲肾上腺素Norepinephrine肾上腺素Epinephrine黑色素Melanin神经递质neurotransmitters褪黑激素MelatoninP物质Substance P兴奋性突触后电位EPSP抑制性突触后电位IPSP神经元neuron听力系统auditory system嗅觉系统olfactory system视觉系统visual system胶质细胞glial cell神经板neural plate系统生物学Systems BiologyDNA微阵列DNA microarrays蛋白质组学Proteomics质谱测定法mass spectrometry高性能液体色谱HPLC phosphoproteomics糖蛋白质组学glycoproteomics代谢物组学Metabolomics Biomicsprocess calculi信息提取information extraction转录组学transcriptomics代谢组学metabolomics酶动力学enzyme kinetics糖组学Glycomics生物学的其他学科Other fields太空生物学Astrobiology微生物学Microbiology古生物学Paleontology寄生虫学Parasitology生理学Physiology动物学Zoology。
KLF6 Induces Apoptosis in Prostate Cancer Cells through Up-regulation of ATF3
KLF6Induces Apoptosis in Prostate Cancer Cells through Up-regulation of ATF3*□SReceived for publication,April 1,2008,and in revised form,August 5,2008Published,JBC Papers in Press,August 28,2008,DOI 10.1074/jbc.M802515200Xiangwei Huang 1,Xia Li 1,and Bin Guo 2From the Department of Pharmaceutical Sciences,College of Pharmacy,North Dakota State University,Fargo,North Dakota 58105KLF6(Kruppel-like factor 6)is a zinc finger transcription fac-tor and a tumor suppressor that is frequently mutated in pros-tate cancer.KLF6suppresses tumor growth and induces apopto-sis in cancer cells through mechanisms still not defined.Here we show that KLF6induces apoptosis in prostate cancer cells by ATF3(activating transcription factor 3)expression.KLF6binds directly to and activates the ATF3promoter.ATF3induced apo-ptosis when ectopically expressed in cells,whereas knockdown of ATF3by small interference RNA blocked KLF6-induced apo-ptosis.KLF6mutants derived from clinical prostate cancers failed to activate the ATF3promoter and were unable to induce apoptosis.Furthermore,stress conditions (exposure to stauros-porine and hypoxia induced by sodium azide)caused significant increase in ATF3expression and induced apoptosis,whereas knockdown of KLF6by small interference RNA blocked the increase of ATF3as well as the induction of apoptosis in these conditions.Thus,ATF3is a key mediator of KLF6-induced apo-ptosis in prostate cancer cells.KLF6(Kruppel-like factor 6)is a tumor suppressor protein that is down-regulated or mutated in several types of cancers,including prostate cancer (1–3).KLF6is a zinc finger transcrip-tion factor that binds to a GC box and regulates the expression of target genes.It has been shown that KLF6suppresses tumor growth through activating p21WAF1/Cip1,an inhibitor of the cyclin-dependent kinases,in both cultured cells and a trans-genic mouse model (2,4).KLF6also directly interacts with cyclin D1to suppress cyclin-dependent kinase 4and causes cell cycle arrest (5).Besides growth inhibition,KLF6has also been shown to induce apoptosis in non-small cell lung cancer cells (1).However,the mechanism of KLF6-induced apoptosis is still not known.ATF3(activating transcription factor 3)is a member of the ATF/cAMP-response element-binding protein family of tran-scription factors (6).ATF3is rapidly up-regulated under vari-ous stress conditions,including hepatotoxicity,UV/ionizing radiation,and exposure to DNA-damaging agents (7).ATF3can also be induced by ischemia and hypoxia (8–10).ATF3is a pro-apoptotic protein.It induces apoptosis in ovarian cancer cells (11)and enhances etoposide-or camptothecin-induced apoptosis in HeLa cells (12).Although transgenic mice express-ing ATF3in beta cells develop abnormal islets and defects sec-ondary to beta cell apoptosis,primary islets derived from ATF3knock-out mice were partially protected from cytokine-and nitric oxide-induced apoptosis (13).Additionally,fibroblasts from ATF3knock-out mice were partially protected from UV-induced apoptosis (10).On the other hand,it was shown that ATF3overexpression promoted invasiveness of prostate tumor cells in vitro and significantly enhanced spontaneous lung metastasis without affecting primary tumorigenicity in a severe combined immunodeficient mouse model (14).Interestingly,it was also shown that ATF3has a dichotomous role in cancer development,while enhancing apoptosis in the untransformed MCF10A cells,ATF3inhibited apoptosis in the more aggres-sive MCF10CA1a cells and enhanced cell mobility (15).We found that KLF6induces apoptosis when ectopically expressed in prostate cancer cells.To understand the mecha-nism of KLF6-induced apoptosis,we employed microarray gene expression analysis and identified ATF3as one of the tar-get genes regulated by KLF6.We further demonstrated that ATF3is a key mediator of KLF6-induced apoptosis in prostate cancer cells.KLF6and ATF3are required for cancer cell apo-ptosis in stress conditions.EXPERIMENTAL PROCEDURESCells and Transfection —Human prostate cancer cell lines PC-3and LNCaP were purchased from American Type Culture Collection.The cells were cultured in RPMI 1640media con-taining 10%fetal bovine serum.For transient transfection,plas-mids were transfected into cells using Lipofectamine TM Plus reagent (Invitrogen)following the manufacturer’s protocol.Drugs and Chemicals —Staurosporine and sodium azide were purchased from Sigma.Staurosporine was dissolved in dimeth-yl sulfoxide (DMSO)at a concentration of 1m M .Sodium azide was dissolved in PBS 3at concentration of 4M .In all studies,an equivalent amount of diluent (DMSO or PBS)was added to the culture media as a negative control.Plasmid Construction —Human cDNAs encoding full-length KLF6gene and SV2variant were obtained by PCR amplification*This work was supported,in whole or in part,by National Institutes of HealthGrants 5R21CA111765(from NCI)and 2P20RR015566(from the NCRR).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertise-ment ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.□SThe on-line version of this article (available at )contains supplemental Figs.1and 2.1Both authors contributed equally to this work.2To whom correspondence should be addressed.Tel.:701-231-5164;Fax:701-231-8333;E-mail:Bin.Guo@.3The abbreviations used are:PBS,phosphate-buffered saline;siRNA,small interference RNA;ChIP assay,chromatin immunoprecipitation assay;EMSA,electrophoretic mobility shift assay;KB,KLF6binding region;PARP,poly(ADP-ribose)polymerase.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.283,NO.44,pp.29795–29801,October 31,2008©2008by The American Society for Biochemistry and Molecular Biology,Inc.Printed in the U.S.A.at Wayne State University Libraries on December 25, 2008 Downloaded from/cgi/content/full/M802515200/DC1Supplemental Material can be found at:using an EST clone (I.M.A.G.E.clone ID 3623401)as template.KLF6and KLF6-SV2cDNAs were subcloned into pCMV-Tag2(Stratagene)vector to express a FLAG-tagged protein.For doxycycline-inducible expression using the Tet-On advanced system,KLF6was subcloned into pTRE-Tight vector (Clon-tech).The pCG and pCG-ATF3expression vectors were kindly provided by Dr.Tsonwin Hai at Ohio State University.The ATF3promoter reporter plasmids Luc-1850,Luc-632,Luc-111,and Luc-84were kindly provided by Dr.Shigetaka Kitajima of Tokyo Medical and Dental University.Luc-258was generated by PCR.Luc-632and Luc-258carrying KLF6-bind-ing site mutants (CC to AA)and KLF6mutants were created by PCR using the QuickChange II site-directed mutagenesis kit (Stratagene),following the supplied protocol.Cell Viability and Apoptosis Assays —Trypan blue dye exclu-sion assay was used to measure cell viability.Briefly,cells were suspended 1:1with 0.4%trypan blue solution.Dead cells (stained blue)were counted under a microscope using a hemo-cytometer.Apoptosis was measured using the Cell Death Detection ELISA PLUS kit (Roche Applied Science)following the manufacturer’s protocol.This assay determines apoptosis by measuring mono-and oligonucleosomes in the lysates of apo-ptotic cells.The cell lysates were placed into a streptavidin-coated microplate and incubated with a mixture of anti-histone/biotin and anti-DNA/peroxidase.The amount of peroxidase retained in the immunocomplex was photometri-cally determined with 2,2Ј-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid as the substrate.Absorbance was measured at 405nm.Western Blot Analysis —Cells were lysed in RIPA buffer (1%Nonidet P-40,0.5%sodium deoxycholate,0.1%SDS in PBS).Complete protease inhibitor mixture (Roche Applied Science)was added to lysis buffer before use.Protein concentration was determined by the DC protein assay (Bio-Rad).Protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane.The membrane was blocked in 5%nonfat milk in PBS overnight and incubated with primary antibody and sub-sequently with appropriate horseradish peroxidase-conjugated secondary antibody.Signals were developed with ECL reagents (Amersham Biosciences)and exposure to x-ray films.Anti-ATF3polyclonal antibody was purchased from Santa Cruz Bio-technology (Santa Cruz,CA).Anti--tubulin monoclonal anti-body was purchased from Sigma.Anti-KLF6monoclonal antibody was purchased from Invitrogen.Anti-KLF6poly-clonal antibody was purchased from Santa Cruz Biotechnology.Anti-cleaved caspase-9and anti-cleaved PARP polyclonal anti-bodies were purchased from Cell Signaling Technologies (Dan-vers,MA).siRNAs and Transfection —Silencer TM pre-designed siRNAs and negative control siRNA (catalog number 4611)were pur-chased from Ambion (Austin,TX).The sequence for KLF6siRNA is:sense (5Ј-GCCCGAGCUUUUGUUACAAtt-3Ј)and antisense (5Ј-UUGUAACAAAAGCUCGGGCtg-3Ј).The sequence for ATF3siRNA is sense (5Ј-UCACAAAAGCCGA-GGUAGCtt-3Ј)and antisense (5Ј-GCUACCUCGGCUUUUG-UGAtg-3Ј).The sequence for KLF6-SV1siRNA is sense (5Ј-GCUUUUCUCCUUCCCUGGCtt-3Ј)and antisense (5Ј-GCC-AGGGAAGGAGAAAAGCct-3Ј).The siRNAs were transfe-cted into PC-3cells using X-tremeGENE siRNA transfection reagent (Roche Applied Science)following the manufacturer’s protocol.Cells were cultured and transfected in 6-well plates (1ϫ105cells per well),and the final siRNA concentrations were 100n M .Protein samples were collected 48h after transfe-ction for Western blot analysis.Real-time PCR —The mRNA level of ATF3was measured by real-time PCR using TaqMan gene expression assay (catalog number Hs00231069)from Applied Biosystems (Foster City,CA).Total RNA was isolated from PC-3cells using RNeasy kit (Qiagen).5g of total RNA was used in reverse transcription reaction.The cDNAs were used as templates to perform PCR on an Applied Biosystems 7500real-time PCR system following the manufacturer’s protocol.Luciferase Assay —PC-3or LNCaP cells in 6-well plates were cotransfected with reporter plasmids and pCMV-Tag2-KLF6or empty expression vectors.To reduce serum-induced activa-tion of the ATF3promoter (16),cells were incubated in media containing 0.1%serum after transfection.Caspase inhibitor benzyloxycarbonyl-VAD was added to the culture media (20M )to prevent cell death caused by KLF6expression.Superna-tants of cell extracts were assayed for luciferase activity using a luciferase assay system (Promega).Chromatin Immunoprecipitation Assay —ChIP assay was performed using the ChIP assay kit from Millipore (Billerica,MA),following the supplied protocol.Immunoprecipitations were performed using anti-KLF6or control IgG antibodies.PCR was performed with the following primers for two regions on the ATF3promoter:an upstream randomly selected region (RND)from Ϫ1418to Ϫ1219as the negative control,5Ј-CTG-CGGCCGCGCAGGTCTCC-3Јand 5Ј-GATTCGAGCTGA-GACCTCAG-3Ј;and KLF6binding region (KB)from Ϫ370to Ϫ120,5Ј-GCCGGTAACCGTGTGGATTC-3Јand 5Ј-GAC-TAGGTGAGGCTGGGAAG-3Ј.Electrophoretic Mobility Shift Assay (EMSA)—Recombinant KLF6protein (Abnova,Taiwan)was incubated with the follow-ing biotin-labeled DNA probes for 30min:Ϫ167TGCCCCCT-CTCTCCACCCCTTCGGC Ϫ191;Ϫ202CTGGGCTGGCTCC-TCCCCGAACTT Ϫ225.EMSA was performed using LightShift Chemiluminescent EMSA Kit (Pierce)following the manufac-turer’s protocol.Statistical Analysis —Differences between the mean values were analyzed for significance using the unpaired two-tailed Student’s test for independent samples;p Յ0.05was consid-ered to be statistically significant.RESULTSKLF6Induces Apoptosis in PC-3and LNCaP Cells —When ectopically expressed,KLF6induced apoptosis in non-small cell lung cancer cells (1).Because KLF6is frequently mutated in prostate cancer (2,17),we determined whether KLF6could also induce apoptosis in prostate cancer cells.KLF6was overex-pressed in PC-3prostate cancer cells by transient transfection using pCMV-Tag2-KLF6expression vector (Fig.1A ).KLF6expression significantly reduced the viability of PC-3cells (Fig.1B ).PC-3cells died through apoptosis as evidenced by the enzyme-linked immunosorbent assay measuring mono-and oligonucleosomes in the lysates of apoptotic cells (Fig.1C )andKLF6Induces Apoptosis through ATF3at Wayne State University Libraries on December 25, 2008Downloaded fromWestern blotting of cleaved PARP and cleaved (activated)caspase-9(Fig.1D ).Overexpression of KLF6also induced apo-ptosis in another prostate cancer cell line LNCaP cells (Fig.1,E and F ),suggesting a common pro-apoptotic activity of KLF6in prostate cancer cells.KLF6Up-regulates the Expression of ATF3—To investigate the molecular mechanism of KLF6-induced apoptosis in pros-tate cancer cells,we compared the gene expression profiles between empty pCMV-Tag2vector and pCMV-Tag2-KLF6-transfected PC-3cells using microarray analysis.Among the genes that were up-regulated by KLF6overexpression,we selected ATF3for further study because of its known involve-ment in apoptosis (10,13).We verified the up-regulation of ATF3expression by KLF6using real-time PCR.As shown in Fig.2A ,overexpression of KLF6by transient transfection caused about 94%increase of ATF3mRNA expression,com-pared with cells transfected with empty vector.Furthermore,KLF6transfection also caused significant increases in ATF3protein expression in a dose-dependent manner,as determined by Western blotting (Fig.2B ).Overexpression of KLF6also induced ATF3expression in LNCaP cells (Fig.2C ).Using the Tet-On inducible gene expression system (Clontech),we were able to control the expression of KLF6in PC-3cells with doxy-cycline.As shown in Fig.2D ,doxycycline-induced KLF6expression (started at 8h after adding doxycycline)preceded the induction of ATF3protein expression (which occurred at 24h after doxycycline addition).Consistently,measurable apoptosis was only detected at the same time when the ATF3protein level was increased at 24h after adding doxycycline but not when KLF6was initially induced at 8-or 16-h time points (Fig.2E ).KLF6Binds to and Activates the ATF3Promoter —To deter-mine whether KLF6directly regulates the expression of the ATF3gene,we performed an ATF3gene reporter assay.Fig.3A illustrates the promoter elements of the ATF3gene promoter and the structures of the ATF3promoter reporters containing various deletions.As shown in Fig.3B ,when KLF6was expressed in LNCaP cells by co-transfection with the reporter plasmids,the ATF3promoter was activated.The deletion mutant down to Ϫ632was still activated by KLF6.In contrast,further deletions down to Ϫ111and Ϫ84completely abolished the effects of KLF6.The region between Ϫ632and Ϫ111con-tains the putative AP-1and SRF motifs.Similarly in PC-3cells,KLF6was able to activate the ATF3promoter,and deletions down to Ϫ111and Ϫ84abolished the effects of KLF6(supple-mental Fig.1A ).By carefully examine the DNA sequence in the region of Ϫ632to Ϫ111,we identified a potential KLF6-binding site (Ϫ202to Ϫ225)that contains the sequence of CCTCCCC (18).Electrophoretic mobility shift assay confirmed that KLF6can directly bind to this sequence,whereas a nearby sequence (Ϫ167to Ϫ191)failed to interact with KLF6(Fig.3C ).To fur-ther determine whether KLF6binds directly to this promoter region in vivo ,we performed a chromatin immunoprecipitation (ChIP)assay,using PCR primers designed to target the endog-enous ATF3promoter region.In LNCaP cells overexpressing KLF6(by transient transfection),anti-KLF6antibody was able to immunoprecipitate the Ϫ370to Ϫ120region of the ATF3promoter (KB),but not the randomly selected upstream region (RND)between Ϫ1418and Ϫ1219(Fig.3D ).A similar result was obtained in PC-3cells (supplemental Fig.1B ).The control IgG did not immunoprecipitate with any fragments of the DNA.We mutated the KLF6-binding motif CCTCCCC to CCTCCAA and tested the effects of this mutation onKLF6-FIGURE 1.KLF6induces apoptosis in prostate cancer cells.A,overexpress-ing KLF6by transient transfection.PC-3cells were transfected with 1.0g of empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids in 6-well plates.Western blotting was done using anti-FLAG antibody with cell lysates collected at 24h after transfection.Vec ,vector.B,KLF6expression reduced the viability of PC-3cells.Cells were transfected with 1.0g of empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids in 6-well plates.Cell viability was determined by trypan blue assay 24h after transfection.C,KLF6induced apoptosis in PC-3cells.Cells were transfected with 1.0g of empty pCMV-Tag2or pCMV-Tag2-KLF6plas-mids in 6-well plates.Apoptosis was analyzed using the Cell Death Detection ELISA PLUS kit at 24h after transfection.The average results from three inde-pendent experiments are shown.D,PC-3cells were transfected with 1.0g of empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids.Cell lysates were analyzed by Western blotting with anti-cleaved caspase-9and anti-cleaved PARP anti-bodies.E,LNCaP cells were transfected with 1.0g of empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids in 6-well plates.Western blotting was done using monoclonal anti-KLF6antibody with cell lysates collected at 24h after trans-fection.F,LNCaP cells were transfected with 1.0g of empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids in 6-well plates.Apoptosis was analyzed as described in C.FIGURE 2.KLF6up-regulates ATF3expression.A,KLF6-activated transcrip-tion of ATF3mRNA.PC-3cells were transfected with empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids.Total RNA was isolated from cells at 24h after transfection and analyzed by real-time PCR as described under “Experimental Procedures.”The average results from three independent experiments are shown.B,KLF6up-regulated ATF3protein expression.PC-3cells were transfected with empty pCMV-Tag2or various amounts of pCMV-Tag2-KLF6vectors (Vec ).ATF3and -tubulin protein levels were detected by Western blotting.C,LNCaP cells were transfected with empty pCMV-Tag2or pCMV-Tag2-KLF6plasmids.ATF3and -tubulin protein levels were detected by Western blotting.D,PC-3cells were transfected with pTet-On Advanced and pTRE-Tight-KLF6for doxycycline-inducible expression of KLF-6.At vari-ous time points after adding 1g/ml doxycycline,cell lysates were collected for Western blot analysis.E,PC-3cells were transfected with plasmids as described in D,and KLF6expression was induced by adding 1g/ml doxycy-cline.At various time points after doxycycline addition,apoptosis was meas-ured using the Cell Death Detection ELISA PLUS kit.The average results from three independent experiments are shown.KLF6Induces Apoptosis through ATF3at Wayne State University Libraries on December 25, 2008Downloaded frominduced ATF3promoter activity.To determine the role of AP-1site,we also created a reporter plasmid Luc-258,in which the AP-1site is deleted.As shown in Fig.3E ,in the absence of AP-1site,KLF6was able to activate the Luc-258reporter,but Luc-258KLF6-binding motif mutant reporter was not activated by KLF6.In the presence of AP-1site,mutation of the KLF6-binding motif failed to block KLF6activa-tion of the reporter,indicating other transcription factors could also acti-vate this promoter (potentially through binding to the AP-1site).ATF3Induces Apoptosis in PC-3Cells —To determine whether ATF3plays a role in apoptosis in PC-3cells,we transiently overexpressed ATF3in PC-3cells.Transient trans-fection of pCG-ATF3plasmids into PC-3cells resulted in a significant increase in the ATF3protein level (Fig.4A ),which was accompanied by a significant induction of apopto-sis in these cells (Fig.4B ).Thus,ATF3has strong pro-apoptoticactivity in PC-3cells.ATF3Mediates KLF6-inducedApoptosis —To determine whether ATF3is a critical factor that mediates KLF6-induced apoptosis in PC-3cells,we employed an ATF3-targeting siRNA to specifically knock down the expression of ATF3in PC-3cells.As shown in Fig.4C ,ATF3siRNA was able to significantly down-regulate the level of ATF3induction by KLF6(in which cells were transfected with pCMV-Tag2-KLF6vectors and control siRNA or ATF3siRNA,compare 2nd and 4th lanes ).Although KLF6induced apoptosis in cells transfected with the negative control siRNA,it failed to induce apoptosis in cells where ATF3was knocked down by -siRNA (Fig.4D ).KLF6Mutants Are Unable to Up-regulate ATF3or Induce Apoptosis —KLF6mutations have been identified in as high as 71%of human prostate cancer samples (2).Functional studies have shown that these KLF6mutants have lost the ability to inhibit cell proliferation,a key function of the wild-type KLF6asa tumor suppressor (2).To determine the effects of tumor-derived mutations on KLF6function in ATF3regulation and apoptosis,we generated cDNAs encoding three KLF6mutants that have been detected in clinical prostate cancer samples,A123D,S137X ,and L169P (2).When transiently transfected into PC-3cells,all three KLF6mutant constructs expressed their respective proteins at levels higher than that of the wild-type KLF6protein (Fig.5A ).Although the wild-type KLF6was able to activate the ATF3promoter,none of the three mutants was active in the promoter reporter assay (Fig.5B ).In contrast to the wild-type KLF6,none of these mutant proteins was ableto up-regulate ATF3in PC-3cells (Fig.5C ).Consistently,all FIGURE 3.KLF6binds to and activates ATF3promoter.A,structures of ATF3promoter reporter plasmid and thosecontaining various deletions.The positions of regions RND and KB in the ChIP assay are shown on the map.B,KLF6activatedthe ATF3promoter.LNCaPcellswerecotransfectedwithreporterplasmidsandpCMV-Tag2-KLF6orempty pCMV-Tag2.Cells were incubated in media containing 0.1%serum after transfection.Cell extracts were assayed forluciferase activity.The experiment has been repeated three times.C,recombinant KLF6protein was incubated with biotin-labeled DNA probes (Ϫ167to Ϫ191and Ϫ202to Ϫ225on the ATF3promoter)for gel shift assay.In the 3rd and 6th lanes ,200-fold molar excess unlabeled probes were used to compete with biotin-labeled probes.D,KLF6binds directly to the ATF3promoter.ChIP assay was performed with KLF-6-transfected LNCaP cells using anti-KLF6antibody.Promoter regions RND (Ϫ1418to Ϫ1219)and KB (Ϫ370to Ϫ120)were amplified by PCR.E,LNCaP cells were cotransfected with wild-type or mutant reporter plasmids and pCMV-Tag2-KLF6or empty pCMV-Tag2.Cells were incubated in media containing 0.1%serum after transfection.Cell extracts were assayed for luciferase activity.The experiment has been repeated three times.Mutation of CCTCCCC to CCTCCAA was created in the KLF6-binding sites of the Luc-632and Luc-258reporterplasmids.FIGURE 4.ATF3mediates KLF6-induced apoptosis.A,overexpression of ATF3in PC-3cells.PC-3cells were transfected with empty vector (Vec )or pCG-ATF3vectors.ATF3and -tubulin protein levels were detected by Western blotting.B,ATF3induces apoptosis in PC-3cells.PC-3cells were transfected with empty vector or pCG-ATF3vectors.Apoptosis was analyzed using the Cell Death Detec-tion ELISA PLUS kit 24h after transfection.The average result from three independ-entexperimentsisshown.C,knockingdownofATF3bysiRNAblockedKLF6-inducedATF3expression.ATF3targeting siRNA or negative control siRNA were transfected into PC-3cells as described under “Experimental Procedures.”At 24h post-siRNA transfection,empty pCMV-Tag2or pCMV-Tag2-KLF6vectors were transfected into cells.Down-regulationofATF3wasconfirmed by Western blotting of protein sam-plescollectedat48haftersiRNAtransfection.D,knockingdownofATF3bysiRNA blocked KLF6-induced apoptosis.PC-3cells were transfected with ATF3siRNA or negative control siRNA,followed by empty pCMV-Tag2or pCMV-Tag2-KLF6vec-tors as described in C ,and apoptosis was analyzed 48h after siRNA transfection using the Cell Death Detection ELISA PLUS kit.KLF6Induces Apoptosis through ATF3at Wayne State University Libraries on December 25, 2008Downloaded fromthree KLF6mutants failed to induce apoptosis when transiently trans-fected into PC-3cells (Fig.5D ).KLF6Mediates Stress-induced Apoptosis through Up-regulation of ATF3—ATF3plays an important role in physiological stress condi-tions (6,7).We hypothesized that by controlling ATF3expression,KLF6may have a critical function in stress-induced apoptosis in prostate cancer cells.To test this hypothesis,we examined the effects of KLF6knockdown by siRNA on PC-3cell apoptosis induced by two agents as follows:staurosporine,a relatively nonselective protein kinase inhibi-tor;and sodium azide,an inducer of hypoxia condition.Exposure tostaurosporine or sodium azide induced significant increases in the level of ATF3protein in PC-3cells (Fig.6A ),which were accompanied by the induction of apoptosis (Fig.6,C and D ).To determine the role of KLF6in PC-3cell apoptosis in these conditions,we selected a KLF6siRNA that was able to down-regu-late KLF6protein almost completely (Fig.6B ).The KLF6siRNA was able to block ATF3induction caused by exposure to stau-rosporine or sodium azide,as well as to abolish apoptosis caused by these agents (Fig.6,C and D ).Thus,KLF6and its downstream target ATF3play important roles in PC-3cell apo-ptosis in stress conditions.KLF6Splicing Variants Also Up-regulate ATF3—KLF6vari-ant proteins,KLF6-SV1and KLF6-SV2,are expressed in pros-tate cancer cells through alternative splicing (19).The KLF6-targeting siRNA that we employed in the above studies will knock down the expression of both wild-type KLF6and the variant proteins.To determine whether the KLF6splicing vari-ants also regulate ATF3expression,we transiently transfected PC-3cells with pCMV-Tag2-KLF6-SV2.As shown in supple-mental Fig.2A ,overexpression of KLF6-SV2also induced ATF3protein expression.KLF6-SV2was also able to induce apoptosis in PC-3cells,although to a lower degree comparedwith wild-type KLF6(supplemental Fig.2B ).Furthermore,using a previously designed siRNA targeting KLF6-SV1(20),we were able to down-regulate KLF6-SV1in PC-3cells (supple-mental Fig.2C ).Down-regulation of KLF6-SV1by siRNA also blocked staurosporine-induced ATF3expression,as well as apoptosis in PC-3cells (supplemental Fig.2D ).DISCUSSIONThe KLF6gene is located on human chromosome 10p,a region deleted in about 55%of sporadic prostate adenocarci-noma (21,22).Mutations of KLF6gene have been identified in clinical prostate cancer samples (2,17),suggesting a tumor sup-pressor role for KLF6in prostate cancer.Indeed,decreasedFIGURE 5.KLF6mutants fail to up-regulate ATF3and are unable to induce apoptosis.A,expression of KLF6mutant proteins.FLAG-tagged KLF6mutant expression plasmids (in pCMV-Tag2)were transfected into PC-3cells,KLF6and -tubulin protein levels were detected by Western blotting.B,KLF6mutants failed to activate the ATF3promoter.LNCaP cells were cotransfected with reporter plasmid Luc-1850and pCMV-Tag2-KLF6or KLF6mutant constructs (in pCMV-Tag2).Luciferase assay was performed as described in Fig.3.C,KLF6mutants failed to up-reg-ulate ATF3protein expression.PC-3cells were transfected with empty pCMV-Tag2,pCMV-Tag2-KLF6,or KLF6mutant constructs in pCMV-Tag2vectors.ATF3and -tubulin protein levels were detected by Western blotting.D,KLF6mutantsfailedtoinduceapoptosisinPC-3cells.CellsweretransfectedwithemptypCMV-Tag2,pCMV-Tag2-KLF6,or KLF6mutant constructs in pCMV-Tag2vectors.Apoptosis was analyzed using the Cell Death Detection ELISA PLUS kit.The average result from three independent experiments isshown.FIGURE 6.KLF6mediates stress-induced apoptosis through ATF3.A,stau-rosporine (STS )and NaN 3up-regulated ATF3in PC-3cells.Cells were treatedwith 1M staurosporine for 8h or 50m M NaN 3for 24h.ATF3protein levels were determined by Western blotting.Ctr,control.B,knocking down of KLF6by siRNA.KLF6targeting siRNA and negative control siRNA were transfectedinto PC-3cells.Protein samples were collected at 48h after siRNA transfec-tion.Western blot was performed with anti-KLF6antibody.C,KLF6siRNA blocked STS-induced ATF3expression and apoptosis.PC-3cells were trans-fected with KLF6targeting siRNA or negative control siRNA.At 24h after siRNA transfection,cells were treated with 1M STS for an additional 8h.ATF3protein level was determined by Western blotting.Apoptosis was analyzed using the Cell Death Detection ELISA PLUS kit.D,KLF6siRNA blocked NaN 3-induced ATF3expression and apoptosis.PC-3cells were transfected with KLF6targeting siRNA or negative control siRNA.At 24h after siRNA transfec-tion,cells were treated with 50m M NaN 3for additional 24h.ATF3protein level was determined by Western blotting.Apoptosis was determined as described above.The average result from three independent experiments is shown.KLF6Induces Apoptosis through ATF3at Wayne State University Libraries on December 25, 2008Downloaded from。
上新世云南铁杉木化石的化学成分
viously ( Zhao et al . 2005 a, b , 2006 ; Giannasi and Niklas, 1977; Niklas and Giannasi, 1977 , 1978; ) . Abundant plant and animal fossils occured in complicated strata in west Yunnan Province, China ( Ge et al . 1999) , part of which were buried in coal-bearing basins . Tsuga dumosa is an economically important conifer indigenous to the Yunnan Province of China (Southwest College of Forestry , Yunnan Forestry Administration , 1988) . Sesquilignans and lignans from this genus have been reported, previously (Zhao et al . 2004 , 2005c ) . A piece of Pliocene-fossil wood of T. dumosa , which was preserved morphologically , was discovered in an
分子伴侣帮助蛋白质形成正确折叠状态的一类分子ppt(实用资料)
GroE染色体上的两个基因 conformation 构形
The lower region, (circled, bottom) is concerned with ATP binding.
GroEL和GroES编码翻译的 approximately 近似地
TRiC 型（ TCP-1 环状复合物）存在于古细菌和真核细胞质中，由双层 8 或 9 元环组成，亚基分子量约为 55K ，与小鼠中 TCP-1
1962年,意大利生物学家在研究果蝇 (Drosophila)的发育时,发现:当培养果蝇的 温度从正常的25℃上升到32℃时，幼虫 (larvae )细胞中的一些巨型染色体上的新 的位点变得异常活跃。研究表明，温度的
升高促使一些新基因的表达。这就是热激 反应（heat-shock response)的发现。
bacteriophage 噬菌体 phage 同上 evolve 进化 symmetrically 对称地 catalyze 促使…..起反应 polypeptide 多肽 intobic 疏水的 residue 残余物
droplet 小滴 soluble 可溶的 denature 变性 aggregate 集合物 crowbar 断裂 monomeric 单体 mitochondrial crystallography 结晶 chamber 小室 ethane 乙烷 cap 帽子
分子伴侣的种类
分类 核质蛋白家族 Nucleoplasmin Protein XLNO-38 NuleoplasminS Hsp70 Hsp70(Hsp40、Hsp24)
DnaK (DnaJ、GrpE) Ssa1-4 SsB1 SsC1 SsD1 Bip Hsp60家族 GroEL(GroES) Cpn60(cpn10) RBP
翻译沃森和克里克于1953年发表在《科学杂志》关于DNA双螺旋模型的论文
分子生物学作业：翻译沃森和克里克于1953年发表在《科学杂志》上的关于DNA双螺旋模型的论文Nature科学杂志Equipment,and to Dr. G. E. R. Deacon and the captain and officers of R.R.S.Discovery II for their part,in making the observations.Molecular structure of nucleic acids核酸分子结构A structure for Deoxyribose nucleic acid脱氧核糖核酸的结构We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A). This structure has novel features which are of considerable biological interest.我们希望去提出一种结构是刺激性的脱氧核糖核酸即DNA。
这个结构有一些新的特征对于生物学有很多重要的意义。
A structure for nucleic acid has already been proposed by Pauling and Corey2.鲍林和科瑞提出了核酸的结构。
they kindly made their manuscript available to us in advance of publication.在他们出版前，他们爽快的将对他们有用的手稿给我们。
Their model consists of three intertwined chains,with the phosphates near the fibre axis,and the bases on the outside.他们提出的模型由三个缠绕的链组成，以磷酸盐靠近纤维轴线并且盘绕在外部。
橡胶树炭疽菌(Colletotrichum siamense)疏水蛋白Hydr基因的克隆与原核表达
疏水蛋白是由丝状真菌特异产生的一类分泌型 g /o e <M />ori〇i<fes species c o m p l e x )和尖孢炭疽菌复合群
小分子蛋白质，大 小 通 常 为 100〜2 0 0 个氨基酸，相对 (C . a c u lo lu m species c o m p l e x ) , 其中 C -.s / o m c m e 又是 分子质量约8〜30 k D ,其特征在于其氨基酸序列中存 胶孢炭疽菌复合群中的主要类群(Lin et al.,2018)。本
C lo n in g and P ro k a ry o tic E x p re ssio n A n aly sis o f H y d ro p h o b in G en e Hydr fro m Colletotrichum siamense
W a n g Jiyuan 1,2 F a n g Siqi u L i a o X i a o m i a o 12 H e Q i g u a n g 12 Li X i a o 12 Liu W e n b o u Z h a n g Y u u Lin C h u n h u a 12* M i a o W e i g u o l>2*
A b s t r a c t In o r d e r to study the function o f H y d r in Colletotrichum siam en se, the g e n e o f h y d r o p h o b i n Hydr w a s c l o n e d b y h o m o l o g o u s m e t h o d ,a n d it w a s expr e s s e d b y prokaryotic expression s y s t e m .T h e results s h o w e d that the Hydr g e n e D N A s e q u e n c e w a s 520 b p ,a n d its c D N A s e q u e n c e w a s 417 b p .T h e g e n e cont a i n e d t w o introns a n d predicted to e n c o d e 139 a m i n o acids polypeptide,w h i c h cont a i n e d a signal peptide consisting o f 19 a m i n o acids at the N - t e r m i n u s o f the protein; T h e n b o t h fusion expression p l a s m i d H y d r -P E T ~32a w i t h s e q u e n c e e n c o d i n g a signal peptide a n d H y d r (-18aa)-P E T _32a w i t h o u t s e q u e n c e e n c o d i n g a signal peptide w e r e constructed,respectively. S D S - P A G E a n d W e s t e r n blot w e r e u s e d to detect the expression o f the fusion protein.T h e results s h o w e d that the protein with o u t signal peptide c o u l d b e successfully e xpressed in E sch rich ia coli u n d e r the induction o f I P T G at 0.4 m m o l /L ,0.6 m m o l /L ,0.8 m m o l /L , 1.0 m m o l / L a n d temperature o f 16 °C .H o w e v e r ,n o fusion protein w a s detected
内分泌干扰物与哺乳动物核受体的相互作用
生态毒理学报Asian Journal of Ecotoxicology第18卷第4期2023年8月V ol.18,No.4Aug.2023㊀㊀基金项目:国家自然科学基金资助项目(41876121)㊀㊀第一作者:史一姣(1997 ),女,硕士研究生,研究方向为生态毒理学,E -mail:********************㊀㊀*通信作者(Corresponding author ),E -mail:***************.cnDOI:10.7524/AJE.1673-5897.20221214001史一姣,田华,何杰,等.内分泌干扰物与哺乳动物核受体的相互作用[J].生态毒理学报,2023,18(4):162-173Shi Y J,Tian H,He J,et al.Interaction of endocrine disrupting chemicals with mammalian nuclear receptors [J].Asian Journal of Ecotoxicology,2023,18(4):162-173(in Chinese)内分泌干扰物与哺乳动物核受体的相互作用史一姣1,田华1,*,何杰2,王雪1,汝少国11.中国海洋大学海洋生命学院,青岛2660032.青岛国海浩瀚海洋工程咨询有限公司,青岛266100收稿日期:2022-12-14㊀㊀录用日期:2023-03-21摘要:内分泌干扰物(EDCs)作用于哺乳动物生殖轴或甲状腺轴,影响生长㊁发育㊁繁殖㊁免疫等生理过程并诱发疾病㊂核受体介导途径是EDCs 发挥内分泌干扰作用的最重要方式㊂在介绍EDCs 对哺乳动物的毒性效应与机制的基础上,详细归纳了EDCs 与核受体的相互作用,并总结了这一研究领域适用的研究方法㊂发现采用理论计算模拟㊁表面等离子共振㊁荧光偏振㊁细胞增殖㊁报告基因等技术方法,目前已经明确了邻苯二甲酸酯类㊁双酚类㊁有机氯农药等EDCs 能够竞争结合雌激素受体㊁雄激素受体和/或甲状腺激素受体,以此为作用靶点通过受体介导途径发挥内分泌干扰效应㊂基于目前的研究现状,我们认为未来的研究应更加注重EDCs 与孕激素受体及维甲酸受体的相互作用㊁膜受体介导途径以及体内实验与体外实验的有机结合㊂关键词:内分泌干扰物;雌激素受体;雄激素受体;甲状腺激素受体;膜受体文章编号:1673-5897(2023)4-162-12㊀㊀中图分类号:X171.5㊀㊀文献标识码:AInteraction of Endocrine Disrupting Chemicals with Mammalian Nuclear ReceptorsShi Yijiao 1,Tian Hua 1,*,He Jie 2,Wang Xue 1,Ru Shaoguo 11.College of Marine Life Sciences,Ocean University of China,Qingdao 266003,China2.Qingdao Guohai Haohan Ocean Engineering Consulting Co.,Qingdao 266100,ChinaReceived 14December 2022㊀㊀accepted 21March 2023Abstract :Endocrine disrupting chemicals (EDCs)act on the mammalian reproductive axis or thyroid axis to affect physiological processes such as growth,development,reproduction,immunity and induce disease.The nuclear re -ceptor -mediated pathways are the most important ways in which EDCs exert endocrine disrupting actions.In this paper,based on a brief introduction of the toxic effects and mechanisms of EDCs on mammals,the interactions be -tween EDCs and nuclear receptors are summarized in detail,and then the research methods applicable to this field of study are ing theoretical computational simulations,surface plasmon resonance,fluorescence polari -zation,cell proliferation,reporter genes and other technical approaches,it is now clear that EDCs such as phthalates,bisphenols and organochlorine pesticides can compete for binding to estrogen receptor,androgen recep -tor and/or thyroid hormone receptor as targets to exert endocrine disrupting effects through receptor -mediated path -第4期史一姣等:内分泌干扰物与哺乳动物核受体的相互作用163㊀ways.Based on the current state of research,we believe that future studies should focus more on the interaction be-tween EDCs and other nuclear receptors such as progesterone receptor and retinoic acid receptor,membrane recep-tor-mediated pathways,and the combination of in vivo and in vitro experiments.Keywords:endocrine disrupting chemicals;estrogen receptor;androgen receptor;thyroid hormone receptor;mem-brane receptor㊀㊀内分泌干扰物(endocrine disrupting chemicals, EDCs)是一类存在于环境中的天然或人工合成的外源性化学物质,通过干扰激素的合成㊁运输㊁转化㊁结合和代谢等过程来影响生物体的内分泌系统活动㊂EDCs种类众多㊁使用广泛[1-5]㊂一些EDCs属于持久性有机污染物,性质稳定,脂溶性强,进入动物体内容易蓄积在脂肪组织中,很难被分解和排出,其浓度会随着营养级的升高不断增大,导致即便进入环境中的浓度极为微量,也会使生物尤其是处于高营养级生物的内分泌功能受到损伤,特别是在发育易感期[3-4,6]㊂尽管有些EDCs已被禁止或限制使用,但它们仍然存在于环境中,其环境持久性与生物蓄积性使其暂时或长久地干扰内分泌系统的激素相关信号通路[7],从而影响生长㊁发育㊁繁殖㊁免疫等生理过程,并导致内分泌相关疾病的发生[8-9]㊂以哺乳动物细胞㊁大/小鼠动物模型为试验对象,针对EDCs的毒性效应与机制已开展大量系统研究,但有关EDCs的作用分子靶点的认知仍有待进一步明确㊂分子结构是决定化合物作用靶点的内因㊂多数EDCs与天然激素具有结构类似性,可作为配体与激素受体相互作用㊂本文以雌激素受体(estrogen receptor,ER)㊁雄激素受体(androgen recep-tor,AR)和甲状腺激素受体(thyroid hormone receptor, TR)为主,在介绍EDCs对哺乳动物的毒性效应与机制的基础上,聚焦 EDCs干扰激素与受体的结合 这一过程,对近年来EDCs与哺乳动物核受体的相互作用研究进行综述,以期为EDCs暴露的健康风险防范和科学干预提供理论依据㊂1㊀EDCs对哺乳动物的毒性效应与机制(Toxic effects and mechanisms of EDCs on mammals) EDCs对哺乳动物发挥内分泌干扰作用的主要对象是内分泌系统的下丘脑-垂体-性腺轴(hypothal-amus-pituitary-gonad,HPG)(简称生殖轴)和下丘脑-垂体-甲状腺轴(hypothalamus-pituitary-thyroid axis, HPT轴)(简称甲状腺轴)[6-7,10]㊂在生殖轴中,下丘脑通过分泌促性腺激素释放激素(gonadotropin-relea-sing hormone,GnRH),调节垂体促黄体生成素(lute-inizing hormone,LH)和促卵泡激素(follicle stimula-ting hormone,FSH)的合成与分泌,它们能够促进性腺合成并释放性激素如雌激素17β-雌二醇(17β-es-tradiol,E2)㊁雄激素睾酮(testosterone,T)和双氢睾酮(dihydrotestosterone,DHT)㊂哺乳动物的生殖主要受到生殖轴的调控[11]㊂EDCs最早引起科学界甚至公众的关注,也是因为其对生殖轴的干扰作用引起的生殖毒性问题㊂据此筛选出的最经典的EDCs有双酚类化合物[12-13]㊁除草剂阿特拉津[14]㊁有机氯农药滴滴涕[15]㊁邻苯二甲酸盐及其代谢物[15-16]㊁多氯联苯[17-19]等㊂以双酚类化合物为例,围产期暴露于高剂量双酚A,导致其雌性子代成年期发情周期模式发生改变,血浆LH水平降低[12];早期发育(自妊娠第8天至产后第19天)暴露于200μg㊃kg-1㊃d-1双酚S改变了小鼠子宫和卵巢中雌激素反应答基因的表达,并促进雌性后代在出生22d后卵巢卵泡的发育[13]㊂在甲状腺轴中,下丘脑分泌促肾上腺皮质素释放激素(corticotropin releasing hormone,CRH)或促甲状腺激素释放激素(thryotropin releasing hormone, TRH),促进垂体合成和分泌促甲状腺激素(thyroid stimulating hormone,TSH),TSH通过与其特异性受体结合,加速细胞对碘的摄取,进一步促进甲状腺合成和分泌甲状腺激素(thyroid hormones,THs),主要是甲状腺素(tetraiodothyronine,T4),T4在外周组织脱碘转化为活性更高的(triiodothyronine,T3)㊂EDCs 对甲状腺的干扰作用成为研究的新热点[20-27],发现了三丁基锡[21]㊁双酚A[22]㊁重金属镉[23]㊁二乙基羟胺(N,N-diethylhydroxylamine,DEHA)[24]㊁多溴二苯醚等溴化阻燃剂[25]具有甲状腺干扰作用㊂然而,由于甲状腺轴在哺乳动物的新陈代谢㊁能量消耗㊁生长发育㊁免疫等多个生理过程中发挥着广泛的调节作用,目前尚不能将EDCs的甲状腺干扰机制与毒性效应建立一一对应关系㊂2㊀EDCs与哺乳动物核受体的相互作用(Interac-tion of EDCs with mammalian nuclear receptors)尽管EDCs可以对哺乳动物内分泌系统生殖轴164㊀生态毒理学报第18卷和/或甲状腺轴中一个或多个调控位点进行干扰,影响内源激素的合成㊁分泌㊁转化㊁转运㊁代谢㊁结合等过程进而发挥其内分泌干扰效应,干扰激素与受体结合这一过程仍是EDCs发挥内分泌干扰作用的最重要方式,主要有生殖轴的ER㊁AR和甲状腺轴的TR(表1)㊂2.1㊀EDCs竞争结合ER/AR通常,经典的核受体介导途径是性激素发挥生物学功能的重要途径㊂E2㊁T/DHT进入靶细胞后分别与雌激素核受体(nuclear estrogen receptor,nER)㊁雄激素核受体(nuclear androgen receptor,nAR)结合形成激素-受体复合物,然后复合物作用于靶基因上游的雌激素应答元件(estrogen response element, ERE)㊁雄激素应答元件(androgen response element, ARE)进而调控下游靶基因的转录㊂ER㊁AR均为配体依赖性转录因子㊂在没有配体存在时,受体与共抑制蛋白结合而处于抑制状态;天然配体与受体结合,使其构象发生改变,共抑制蛋白解离,共激活蛋白结合而处于激活状态[28-29]㊂ER受体介导途径是EDCs发挥雌激素干扰效应的重要作用途径㊂目前已有很多研究表明雌激素类EDCs具有与E2相似的化学结构,可以模拟或阻断E2与哺乳动物nER的结合,激活或无法激活下游靶基因的转录,从而发挥类E2活性(ER激动剂效应)或抗E2活性(ER拮抗剂效应),并引起生殖毒性[2,6,30]㊂例如,邻苯二甲酸酯类[31]㊁双酚类化合物(如双酚A㊁双酚S㊁双酚F㊁双酚AF)[28,32-33]㊁滴滴涕及滴滴伊[34-35]㊁十氯酮(chlordecone)[35]等可以结合ER,发挥ER激动剂效应;而他莫昔芬[35-36]㊁雷洛昔芬[37]㊁叔丁基苯酚(tertiary butyl phenol,TBP)[38]及重金属Cd㊁Cu㊁Zn[39]等能够与E2竞争结合ER,阻断E2与ERs结合,发挥抗雌激素效应㊂此外,EDCs对不同的nER亚型有不同的亲和力㊂例如,染料木黄酮㊁香豆雌酚㊁特辛基苯酚(4-tert-octylphenol,PTOP)对ERβ的亲和力较强;药物类乙炔基雌二醇对ERα的亲和力较强[39-42]㊂AR受体介导途径是EDCs发挥雄激素干扰作用的重要作用途径㊂一些与雄激素具有相似化学结构的EDCs,可以模拟或阻断雄激素与nAR的结合,激活或无法激活下游靶基因的转录,从而发挥类雄激素活性(AR激动剂效应)或抗雄激素活性(AR拮抗剂效应),并引起生殖毒性㊂例如4-羟基睾酮和4-羟基雄烯二酮[43]及BPS[27]可以结合AR,发挥AR激动效应;邻叔丁基苯酚(2-TBP)和2,4-二叔丁基苯酚(2,4-DTBP)[44]㊁BPA和BPF[28]及3β,4β-二羟基-5α-雄甾烷-17-酮[43]可以与T或者DHT竞争结合AR,发挥抗雄激素效应㊂2.2㊀EDCs竞争结合TR核受体介导的转录调节是T3发挥生物学作用的重要机制[45]㊂TR与维甲酸受体(retinoid X recep-tor,RXR)形成异源二聚体(TR-RXR);在没有配体存在时,TR-RXR与共抑制蛋白如核受体辅抑制因子(nuclear receptor corepressor,NCOR)㊁维甲酸和甲状腺激素受体的沉默介质(silencing mediator of retinoic acid and thyroid hormone receptor,SMRT)结合,TR-RXR处于抑制状态;T3进入靶组织细胞后,与TR-RXR结合,使得二聚体的构象发生改变,共抑制蛋白解离,共激活蛋白结合,配体㊁TR-RXR㊁共激活蛋白形成的复合物与下游靶基因DNA上的甲状腺激素应答元件(thyroid hormone response element,TRE)结合,调控下游靶基因的转录[44-48]㊂TR介导途径是甲状腺干扰物(thyroid disrupting chemicals,TDCs)发挥甲状腺干扰效应的最重要作用途径㊂TDCs与TRβ结合后可能激活下游靶基因的转录,发挥类T3活性(TR激动剂效应),亦或无法激活下游靶基因的转录却因占据了有限的T3与TR的结合位点而发挥抗T3活性(TR拮抗剂效应)[20,25,49-51]㊂例如,羟基化的多氯联苯㊁阻燃剂磷酸三(1,3-二氯丙基)酯及其主要代谢物双(1,3-二氯-2-丙基)磷酸酯㊁全氟辛酸替代物氯化聚氟醚磺酸盐可以结合TRβ,发挥TR激动剂效应[49,51];而双酚S㊁四溴双酚A㊁羟基化多溴联苯(OH-BB80)和多溴联苯则与T3竞争结合TRβ,发挥拮抗作用,抑制下游基因的转录[20,25,50,52-53]㊂2.3㊀EDCs与多种核受体的相互作用由于直接或者间接作用,很多EDCs能够通过2种甚至2种以上受体介导途径发挥内分泌干扰作用㊂一方面,目前许多已知的EDCs发挥内分泌干扰作用的直接靶受体并非是单一的㊂例如,双酚类化合物能与ER结合,从而影响体质量和雌激素依赖性肿瘤的发生发展,也能与TR结合,进而上调多个与甲状腺细胞增殖和活性相关基因的表达,影响甲状腺功能[20,28,32-33,56-57]㊂TR㊁ER及AR被证明均是多溴联苯醚的作用靶点[57-59]㊂另一方面,鉴于甲状腺轴和生殖轴之间存在着复杂的交互作用,污染物对某一内分泌轴线的直接影响也可能会造成对另第4期史一姣等:内分泌干扰物与哺乳动物核受体的相互作用165㊀表1㊀内分泌干扰物(E D C s )通过受体介导途径发挥内分泌干扰作用T a b l e 1㊀E n d o c r i n e d i s r u p t i n g c h e m i c a l s (E D C s )e x e r t e n d o c r i n e d i s r u p t i n g e f f e c t s t h r o u g h r e c e p t o r -m e d i a t e d p a t h w a y轴线A x i s 化合物C h e m i c a l受体R e c e p t o r 作用方式M o d e o f a c t i o n 实验方法E x p e r i m e n t 参考文献R e f e r e n c e下丘脑-垂体-性腺轴(H P G )H y p o t h a l a m u s -p i t u i t a r y -g o n a d (H P G )双酚A (B P A )㊁双酚F (B P F )㊁双酚S (B P S )B i s p h e n o l A (B P A ),b i s p h e n o l F (B P F ),b i s p h e n o l S (B P S )雌激素受体α(E R α)㊁雌激素受体β(E R β)E s t r o g e n r e c e p t o r α(E R α),e s t r o g e n r e c e p t o r β(E R β)激动活性A g o n i s t 荧光素酶报告基因(M E L N )L u c if e r a s e r e p o r t e rg e n e e x p e r i m e n t (M E L N )[28]2-叔丁基苯酚(2-T B P )㊁2,4-叔丁基苯酚(2,4-T B P )㊁2,6-叔丁基苯酚(2,6-T B P )2-t e r t -b u t y l p h e n o l (2-T B P ),2,4-t e r t -b u t y l p h e n o l (2,4-T B P ),2,6-t e r t -b u t y l p h e n o l (2,6-T B P )E R α拮抗活性A n t a g o n i s t 重组人E R α双杂交酵母R e c o m b i n a n t h u m a n E R αy e a s t b i o a s s a y e x p e r i m e n t[44]2,4,6-三氯苯酚2,3,6-t r i c h l o r o p h e n o lE R α拮抗活性A n t a g o n i s t荧光素酶报告基因,分子对接L u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t ,m o l e c u l a r d o c k i n g[49]五氯苯酚P e n t a c h l o r o p h e n o lE R α激动/拮抗活性A g o n i s t /A n t a g o n i s t荧光素酶报告基因,分子对接L u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t ,m o l e c u l a r d o c k i n g[49]4-羟基睾酮㊁4-羟基雄烯二酮4-h y d r o x y t e s t o s t e r o n e ,4-h y d r o x y a n d r o s t e n e d i o n e雄激素受体(A R )A n d r o g e n r e c e p t o r (A R )激动活性A g o n i s t重组人A R 双杂交酵母R e c o m b i n a n t h u m a n A R y e a s t b i o a s s a y e x p e r i m e n t[43]B P S A R激动活性A g o n i s t荧光素酶报告基因(P A L M )L u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t (P A L M )[28]2-T B P ,2,4-T B P A R拮抗活性A n t a g o n i s t重组人A R 双杂交酵母R e c o m b i n a n t h u m a n A R y e a s t b i o a s s a y e x p e r i m e n t[44]B P A ,B P F A R拮抗活性A n t a g o n i s t荧光素酶报告基因(P A L M )L u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t (P A L M )[28]3β,4β-二羟基-5α-雄甾烷-17-酮3β,4β-d i h y d r o x y -5α-a n d r o s t a n e -17-o n eA R拮抗活性A n t a g o n i s t重组人A R 双杂交酵母R e c o m b i n a n t h u m a n A R y e a s t b i o a s s a y e x p e r i m e n t[43]166㊀生态毒理学报第18卷续表1轴线A x i s化合物C h e m i c a l 受体R e c e p t o r 作用方式M o d e o f a c t i o n 实验方法E x p e r i m e n t 参考文献R e f e r e n c eH P T羟基化多溴联苯醚H y d r o x y l a t e d p o l y b r o m i n a t e dd i p he n y l e t h e r s羟基化四溴联苯H y d r o x y l a t e d t e t r a b r o m o b i p h e n y lB P AB P SB P F有机磷酸酯O r g a n i c p h o s p h a t e甲状腺激素受体β(T R β)T h y r o i d h o r m o n er e c e p t o r β(T R β)激动活性A g o n i s t 重组人T R β双杂交酵母,分子对接R e c o m b i n a n t T R βy e a s t b i o a s s a y e x p e r i m e n t ,m o l e c u l a r d o c k i n g [54]T R β拮抗活性A n t a g o n i s t重组人T R β双杂交酵母,分子对接R e c o m b i n a n t T R βy e a s t b i o a s s a y e x p e r i m e n t ,m o l e c u l a r d o c k i n g[25]T R β拮抗活性A n t a g o n i s t 荧光素酶报告基因(Z F L )L u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t (Z F L )[50]T R β激动/拮抗活性A g o n i s t /A n t a g o n i s t 分子对接,荧光偏振,荧光素酶报告基因(g h 3),g h 3细胞增殖M o l e c u l a r d o c k i n g ,f l u o r e s c e n c e p o l a r i z a t i o nd e t e c t i o n ,l u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t (g h 3),g h 3c e l l p r o l i f e r a t i o n e x p e r i m e n t [55]T R β拮抗活性A n t a g o n i s t 分子动力学模拟,重组人T R β双杂交酵母,基因转录(斑马鱼)M o l e c u l a r d y n a m i c s s i m u l a t i o n ,r e c o m b i n a n t h u m a n T R βy e a s tb i o a s s a y e x p e r i m e n t ,t r a n sc r i p t i o n o f g e n e (D a n i o r e r i o )[20]T R β激动/拮抗活性A g o n i s t /A n t a g o n i s t 分子对接,荧光偏振,荧光素酶报告基因(g h 3),g h 3细胞增殖M o l e c u l a r d o c k i n g ,f l u o r e s c e n c e p o l a r i z a t i o nd e t e c t i o n ,l u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t (g h 3),g h 3c e l l p r o l i f e r a t i o n e x p e r i m e n t[55]T R β激动/拮抗活性A g o n i s t /A n t a g o n i s t分子对接,荧光偏振检测,荧光素酶报告基因(g h 3),g h 3细胞增殖M o l e c u l a r d o c k i n g ,f l u o r e s c e n c e p o l a r i z a t i o nd e t e c t i o n ,l u c i f e r a s e r e p o r t e r g e n e e x p e r i m e n t (g h 3),g h 3c e l l p r o l i f e r a t i o n e x p e r i m e n t[55]T R β分子对接M o l e c u l a r d o c k i n g[49]第4期史一姣等:内分泌干扰物与哺乳动物核受体的相互作用167㊀一轴线的间接影响㊂除了协同作用,2个轴线之间某些受体也存在呈竞争关系的相互作用㊂在CV-1细胞系中,TRα与配体结合后能够抑制ERα所介导的含有ERE的下游靶基因转录,TRβ没有类似作用;但TR却不能抑制同样受E2-ER调控的含有孕酮应答元件(progesterone response element,PRE)的下游靶基因转录[60]㊂在GH3细胞和JEG-3绒毛膜癌细胞中,TR与TRE结合,调控垂体糖蛋白激素α亚基的合成,E2可以抑制此过程,且体外合成的ERα可以与TRE结合[61]㊂这表明TR和ER之间存在竞争的可能性,且这2个核受体之间的相互作用取决于细胞类型㊁受体亚型㊁反应元件类型等因素[62]㊂目前尚缺乏EDCs对受体之间相互作用的影响研究㊂3㊀EDCs与哺乳动物核受体相互作用的研究方法(Methods for studying the interaction of EDCs with mammalian nuclear receptors)目前研究EDCs与哺乳动物核受体相互作用的主要方法包括理论计算模拟㊁体外结合实验㊁细胞增殖实验㊁报告基因实验等㊂与受体的相互作用是EDCs通过受体介导途径发挥内分泌干扰效应的前提,首先通过理论模拟㊁体外结合实验判断化合物能否与受体相结合㊂然后,通过细胞增殖实验㊁报告基因实验等进一步确定EDCs的具体作用方式(类天然激素活性或抗天然激素活性)㊂3.1㊀理论计算模拟和体外结合实验研究EDCs与受体的结合情况通过理论计算模拟可以在分子水平上预测化合物与受体之间的相互作用模式㊂常用的理论模拟方式有分子对接㊁分子动力学模拟等㊂其中,分子对接是依据配体与受体作用的 锁-钥原理 ,模拟配体小分子与受体生物大分子相互作用的一种技术方法,对接过程中,将小分子配体放置于受体蛋白的活性残基处,寻找两者的最佳结合形态,通过软件来获得结合位点的具体信息与结合能[63]㊂有研究发现羟基化多溴联苯可以与TRβ结合,且占据TRβ的活性口袋,通过氢键及疏水作用形成稳定的复合物[25]㊂有研究将4-羟基雄烯二酮的降解产物与ERα进行分子对接,发现其与ERα在His524㊁Arg394和Glu353处形成氢键[43]㊂分子动力学模拟则通过构建近乎生理状态的受体蛋白溶剂化模型㊁计算机建立动态实验数据,进而达到分析受体与配体结合的自由能㊁配体与受体的反应过程及受体的构象变化的目的[64]㊂有研究通过分子动力学模拟发现BPS能与TRβ结合,导致TRβ配体结合域第11号α螺旋(Helix11, H11)His435残基和第12号α螺旋(Helix12,H12) Phe459残基之间的距离发生显著变化,H12相对重新定位,扰乱TRβ的构象[20]㊂表面等离子共振实验及荧光偏振检测实验等体外结合实验可以用于验证化合物与受体的结合㊂表面等离子共振实验是将受体蛋白固定在生物传感器表面,将EDCs溶液流经生物传感器表面㊂受体与化合物间的结合引起生物传感器表面质量的增加,导致折射指数按比例改变㊂有研究用表面等离子共振实验检测到乙炔雌酚及BPA能与ER结合,推测这是它们发挥雌激素效应的机制[65]㊂此外,前列腺癌治疗药物卡鲁胺能与T竞争结合AR[66]㊂荧光偏振检测实验采用荧光标记的天然配体作为探针,将受体㊁荧光标记配体及不同浓度的EDCs混合, EDCs与配体竞争结合会导致体系荧光强度发生变化㊂有研究以荧光素标记的共激活因子SRC为探针,将探针㊁TR和一系列浓度BPA㊁BPF㊁BPS孵育,然后用荧光偏振法检测发现这3种双酚类化合物均可以诱导TRβ募集共激活因子[55]㊂3.2㊀细胞增殖实验和报告基因实验确定EDCs的具体作用方式通过细胞增殖实验㊁报告基因实验可以明确EDCs作为配体对受体的激动或/和拮抗活性㊂前者选用激素依赖型细胞,检测指标为细胞增殖率,目前最常用的细胞系为人乳腺癌细胞系MCF-7㊁大鼠垂体瘤细胞系GH3㊂MCF-7富含ER,其增殖具有雌激素依赖性[67],常作为体外模型被广泛用于检测环境化合物的类/抗E2活性或检测环境样本中是否存在雌激素类EDCs[68]㊂十氯酮(chlordecone)[34]㊁有机氯农药滴滴涕和滴滴伊[35]㊁双酚类[49]均通过与ERα结合,促进MCF-7的增殖,被认为是ERα激动剂㊂检测抗雌激素效应通常采用与类雌激素效应相似的方法,不同的是检测抗雌激素效应时需要添加E2同时暴露,通过是否能降低E2介导的效应来测试其是否具有抗雌激素效应㊂GH3内TR大量表达,作为一种THs依赖性生长细胞,其增殖可受TDCs与TR 相互作用的影响,因此该细胞株常被用于筛选TDCs[69-71]㊂几种羟基化的多氯联苯在1ˑ10-7~1ˑ10-4mol㊃L-1浓度范围内促进GH3细胞增殖[51],常用的阻燃剂磷酸三(1,3-二氯丙基)酯及其主要代谢物双(1,3-二氯-2-丙基)磷酸酯分别在20mmol㊃L-1168㊀生态毒理学报第18卷和200mmol ㊃L -1时促进GH3细胞增殖并调节细胞周期[49];此外,有研究发现饮用水消毒副产物碘乙酸与T 3联合暴露GH3细胞可以显著降低T 3激活的GH3细胞增殖,认为碘乙酸在体外具有T 3拮抗活性[72]㊂报告基因实验中,反应元件被剪接到报告基因上游,这些反应元件可以调控宿主细胞中报告基因表达,报告基因的表达产物通常易被检测㊂若化合物能够与受体结合,则会促使共激活因子结合至应答元件上,诱导下游报告基因的转录,通过测定报告基因的表达产物可以判断反应化合物与受体结合后对下游基因的调控作用㊂目前常用于验证EDCs 受体介导途径的报告基因实验为构建重组受体基因双杂交酵母和构建荧光素酶报告基因系统㊂重组基因酵母选用LacZ 为报告基因,通过测定其表达产物β-半乳糖苷酶的活性,即可表征化合物的类激素活性;而当内源激素与可拮抗其活性的EDCs 同时存在时,内源激素诱导的β-半乳糖苷酶活性将受到抑制[73]㊂TBP (5ˑ10-6~50μmol ㊃L -1)单独暴露重组ER α基因双杂交酵母,与溶剂对照相比,β-半乳糖苷酶活性没有显著变化;但叔丁基酚与2.5ˑ10-4mol ㊃L -1E 2共同暴露时,与E 2单独暴露组相比,则显著抑制了β-半乳糖苷酶活性[44]㊂环境相关浓度(5ˑ10-7~50μmol ㊃L -1)BPS 暴露重组TR β基因双杂交酵母,与溶剂对照相比,β-半乳糖苷酶活性没有显著变化;但BPS 与T 3共同暴露时,与T 3单独暴露组相比,BPS 则显著抑制了β-半乳糖苷酶活性,且具有剂量依赖性[20]㊂有研究利用双杂交酵母评估羟基化多溴联苯(OH -BB80)的甲状腺干扰效应,结果显示OH -BB80(0.005nmol ㊃L -1~5μmol ㊃L -1)在单独暴露时未出现类T 3活性,但是当OH -BB80与0.5μmol ㊃L -1T 3共同暴露时则表现出拮抗作用,其拮抗能力呈浓度依赖性[25]㊂双荧光素酶报告基因实验则在细胞中用绿色荧光蛋白作为报告基因检测EDCs 对靶基因表达的调控㊂有研究构建含有ERE 的MELN 细胞,发现BPA ㊁BPF 及BPS 均以浓度依赖方式诱导荧光素酶表达,诱导能力BPA>BPF>BPS ,且BPA 和BPF 可以与E 2竞争结合ER [28]㊂有研究用人骨肉瘤细胞U2-OS 构建荧光素酶报告基因体系,发现甲基三烯酮化合物能显著增强荧光信号,认为其具有雄激素效应[74]㊂4㊀总结与展望(Summary and prospect )采用理论计算模拟㊁表面等离子共振㊁荧光偏振㊁细胞增殖㊁报告基因等技术方法,目前已经明确了邻苯二甲酸酯类㊁双酚类㊁有机氯农药等EDCs 能够竞争结合核受体(ER ㊁AR 和/或TR),以此为作用靶点,通过受体介导途径发挥内分泌干扰效应,因而产生发育毒性㊁生殖毒性㊁免疫毒性㊂基于目前的研究现状,我们认为未来的研究应更加注重EDCs 与其他核受体的相互作用㊁膜受体介导途径以及体内实验与体外实验的有机结合㊂4.1㊀EDCs 对孕激素受体㊁维甲酸受体的相互作用仍需进一步探究除ER ㊁AR 及TR ,哺乳动物生殖轴和甲状腺轴中还存在其他核受体㊂例如,除了ER 和AR ,生殖轴中还有孕激素受体(progesterone receptor,PR)[75-76];除了TR ,甲状腺轴中还有维甲酸受体(retinoic acid re -ceptor RAR)[77-78]㊂这些核受体同样属于配体依赖型受体,在内分泌系统中发挥重要作用㊂研究证明,EDCs 可以与这些核受体竞争结合发挥内分泌干扰作用㊂例如BPA 及其衍生物四氯BPA 和四溴BPA 发挥PR 激动剂效应[28];多溴联苯醚除了与ER ㊁AR 及TR 结合发挥内分泌干扰作用外,还可以与PR 结合[58-59,79]㊂但相较于ER ㊁AR 及TR ,EDCs 与PR 及RAR 等核受体相互作用的研究较少,需要进一步探究㊂4.2㊀膜受体介导途径有望成为探究EDCs 作用机制的新切入点近年来,多种在细胞膜上表达的膜受体相继被发现,核受体与膜受体的同源性较低[80]㊂与核受体相比,尽管膜受体表达量低,但是其介导的非基因组调控途径发挥着重要的生物学功能㊂例如,E 2㊁睾酮分别与位于细胞膜上的雌激素膜受体(membrane es -trogen receptor,mER)㊁雄激素膜受体(membrane an -drogen receptor,mAR)结合后,诱导胞内产生多种第二信使,激活钙离子流㊁PKA 和蛋白激酶C ,从而调节其他转录因子活性,产生一系列生物学效应[81-83];T 3也可以通过非基因组途径发挥作用,即不涉及nTR ,而是直接由特异性整合素αv/β3受体组成的甲状腺激素膜受体(membrane thyroid hormone re -ceptor,mTR)发挥转录调控作用[59,84-85]㊂最新的研究表明,EDCs 可以与膜受体相互作用而发挥内分泌干扰作用[49,86-87]㊂例如,mTR 介导途径在邻苯二甲酸二正丁酯㊁磷酸三(1,3-二氯丙基)酯诱导的甲状腺干扰效应中也发挥了重要作用[49,87]㊂BPA 可以通过非基因组调控的方式对胰岛㊁内皮㊁乳腺和垂体产生第4期史一姣等:内分泌干扰物与哺乳动物核受体的相互作用169㊀快速激活效应,但是其通过何种膜受体介导这种非基因组调控效应目前尚不清楚[88]㊂目前对EDCs的受体介导途径研究主要集中于其竞争结合nER㊁nAR和nTR,膜受体介导途径或许可以成为探究EDCs作用机制的新切入点㊂4.3㊀有必要采取体外㊁体内实验相结合的方式来研究EDCs的受体介导途径除了理论模拟及体外实验,体内实验也可以用于验证EDCs干扰受体介导途径㊂目前,体内实验多通过受体调控的下游基因表达水平表征EDCs是否能够通过受体介导途径发挥作用㊂相较于操作简单㊁快速且干扰因素较少的体外实验,尽管体内实验中存在多种内源性因子可能对调控过程产生干扰,但却可以更真实反映地EDCs对生物体的内分泌干扰作用,减少假阴性/阳性结果㊂因此,有必要采取体外㊁体内实验相结合的方式来研究化合物的受体介导途径㊂例如,通过生物体暴露实验研究化合物对于下游靶基因的调控及生物学效应,然后结合体外实验验证是否是通过某些受体介导途径进行调控㊂这样既能够排除干扰因素,确定分子靶点,又能将分子靶点与生物学效应相结合,更好地表征化合物的内分泌干扰作用㊂通信作者简介:田华(1983 ),女,博士,副教授,主要研究方向为生态毒理学㊂参考文献(References):[1]㊀Mattheij J A,Swarts J J,Lokerse P,et al.Effect of hypot-hyroidism on the pituitary-gonadal axis in the adult fe-male rat[J].The Journal of Endocrinology,1995,146(1):87-94[2]㊀Bleak T C,Calaf G M.Breast and prostate glands affectedby environmental substances(Review)[J].Oncology Re-ports,2021,45(4):20[3]㊀Diamanti-Kandarakis E,Bourguignon J P,Giudice L C,etal.Endocrine-disrupting chemicals:An Endocrine 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收稿日期：2011-10-31；接受日期：2011-12-29基金项目：中国科学院基础前沿研究专项项目(KSCX2-EW-R-11, KSCX2-EW-J-23); 中国科学院和云南省动物模型与人类疾病机理重点实验室资助项目∗通信作者(Corresponding author)，E-mail: rlai@动 物 学 研 究 2012，Feb. 33(1): 75−78 CN 53-1040/Q ISSN 0254-5853 Zoological Research DOI ：10.3724/SP.J.1141.2012.01075树鼩神经肽Y 的分子克隆及其灵长类类似物的同源性比较董 丽1,2, 吕龙宝1, 赖 仞1,*(1. 中国科学院和云南省动物模型与人类疾病机理重点实验室, 中国科学院昆明动物研究所, 云南 昆明 650223;2. 中国科学院研究生院, 北京 100049)摘要：树鼩由于与灵长类动物有较密切的亲缘关系和其个体小，以及繁殖周期短等特性而倍受关注, 尤其是作为医用实验动物的研究，近年来已受到越来越多的重视, 但树鼩的分类地位还一直有所争论。
该研究从树鼩脑cDNA 文库中克隆得到编码树鼩神经肽Y(neuropeptide Y, NPY)前体序列, 序列比对发现该序列与灵长类NPY 序列同源性高达96.9%。
将该序列与GenBank 数据库中其他物种的NPY 序列构建系统进化树, 发现树鼩与灵长类处于同一分支。
该研究结果揭示了树鼩与灵长类较近的亲缘关系。
关键词：神经肽Y; 树鼩; 灵长类 中图分类号：Q959.832; Q951.3; Q347 文献标志码：A 文章编号：0254-5853-(2012)01-0075-04Molecular cloning of Tupaia belangeri chinensis neuropeptide Y and homology comparison with other analogues from primatesDONG Li 1,2，LÜ Long-Bao 1, LAI Ren 1,*(1. Key Laboratory of Animal Models and Human Diseases Mechanisms of the Chinese Academy of Sciences and YunnanProvince , Kunming Institute of Zoology , the Chinese Academy of Sciences , Kunming 650223, China ;2. Graduate University of the Chinese Academy Sciences , Beijing 100049, China )Abstract: Much attention has been payed to tree shrews for their close phylogenetic relationship with primates, small size, and short reproductive cycle. Especially, they are considered as excellent experiential animals for medicine or/and disease research. A nucleotide sequence encoding neuropeptide Y(NPY) precursor has been cloned from the cDNA library of Tupaia belangeri chinensis . Sequence alignment revealed that the sequence homology with primate NPY was up to 96.9%. The phylogenetic analysis based on NPY precursor sequence revealed that the tree shrew has a close relationship with primates.Key words: Neuropeptide Y; Tupaia belangeri chinensis ; Primates神经肽Y(neuropeptide Y, NPY)是Tatemoto et al (1982)首次从猪脑中提取纯化出的一种具有生物活性的内源性信息物质, 由36个氨基酸组成, 相对分子质量为 4.2×103。
在结构和功能上与胰多肽(avian pancreatic polypeptide, APP)和肽YY(peptide YY, PYY)非常相似, 所以将其划分为胰多肽家族(Tatemoto et al,1982)。
NPY 序列N 端和C 端各有一个酪氨酸残基, C 端酪氨酸残基存在酰胺化修饰(Allen et al,1987), 这与生物体内许多活性肽(如促性腺激素GnRH)相同(Cerdá-Reverter & Larhammar, 2000)。
这一修饰不仅可保护C 端免受羧肽酶降解, 同时与生物活性密切相关。
N 端的酪氨酸残基对于NPY 三级结构的稳定及与NPY 受体的结合非常重要(Tatemoto et al,1982)。
在细胞内, NPY 以前体方式合成, 前体由97～98个氨基酸组成, N 端28～29个氨基酸为信号肽,76 动物学研究 33卷前体翻译后经过酶切、C端酪氨酸酰基化修饰后成为具有生物活性的36个氨基酸组成的活性肽(Higuchi et al,1988)。
NPY在进化过程中高度保守, 哺乳动物中, 人以及大鼠、豚鼠的成熟NPY氨基酸序列完全相同, 而猪的NPY也只是在第17位氨基酸有所不同，鱼类电鳐目黑斑电鳐(Torpedo marmorata)有33个氨基酸与哺乳动物NPY序列中一致，只有另外3个不同(Pedrazzini et al，2003)。
NPY最初从哺乳动物中分离得到, 之后的研究发现, NPY不仅在哺乳动物中表达, 也广泛分布于鸟类、鱼类和两栖类。
在体内, 神经肽作为一种神经递质参与体内多种生理调节过程。
NPY可直接对下丘脑活动产生作用, 影响多种因子与激素的合成和分泌(Danger et al,1990; Breton et al,1991); NPY可促进机体的摄食行为, 调节机体能量平衡(Kuo et al, 2007); 调节血管收缩, 具有增强血管紧张素II(AngII)、内皮素和交感缩血管物质(NE)等血管收缩物质而抑制舒血管物质的作用, 并且与免疫功能也有着密切关系(Zukowska et al, 2003)。
除成熟肽以外, 前体加工过程中C端切割产生的30个氨基酸组成的肽段(CPON)也非常保守。
有研究表明, CPON广泛分布于中枢和外周神经系统中, 并且与NPY在神经细胞中有相同的定位(Gulbenkian et al,1985; Marti et al,1990)。
这似乎表明，CPON并不仅仅是NPY加工过程中的一个“副产物”, 它可能还发挥着其他的生物学功能, 遗憾的是, 目前仍没有发现该肽段的生物学功能。
树鼩是一种小型哺乳动物, 由于与灵长类相近, 近年来受到越来越多科学工作者的关注。
该文采用树鼩脑组织为材料, 构建cDNA文库, 以PCR方法扩增得到一条编码树鼩NPY前体的cDNA序列。
1 材料与方法1.1 树鼩脑cDNA文库的构建取成年树鼩(Tupaia belangeri, 来自昆明动物研究所实验动物中心) 1只，麻醉剂麻醉后, 解剖头部取出整个脑组织, 立即放入液氮中保存备用。
总RNA提取采用Trizol试剂(invitrogen), 参照试剂说明书进行。
cDNA双链合成采用CLONTECH公司SMART TM cDNA Library Construction Kit试剂盒。
一链使用CDSIII引物5'-ATTCTAGAGGCCG AGGCGGCCGACATG-d(T)30N-1N-3' (N=A、G、C、T; N-1=A、G、C)和SmartV寡聚核苷酸5'-AAGCA GTGGTATCAACGCAGAGTACGCGGG-3'合成；二链使用5'PCR引物5'-AAGCAGTGGTATCAACG CAGAGT-3'合成。
1.2 克隆筛选对上一步构建的cDNA文库进行随机克隆筛选, 根据构建cDNA文库时5'端和3'端通用序列设计引物,上游引物和下游引物分别为：5'-AAGCAGTGGTATC-AACGCAGAGT-3'和5'-ATTCTAGAGGCCGAGGCGGCCGACATG-3', 引物由上海生物工程有限公司合成。
以上一步smart合成双链cDNA为模板进行PCR。
反应条件如下：首先95 °C变性5 min；接下来95 °C变性30 s, 55 °C 退火30 s, 72 °C延伸1 min, 30个循环；最后72 °C 延伸10 min。
PCR产物琼脂糖凝胶电泳检测, 切胶回收, 克隆到PMD19-T载体(TaKaRa公司), 选择103个阳性克隆测序。
1.3 克隆序列与其他物种神经肽Y前体序列比对克隆得到的序列, 采用美国国家生物技术信息中心(NCBI, National Center for Biotechnology Information, )网站的BLAST 软件, 将测序后获得的cDNA序列与GenBank数据库中已公布的序列进行同源性比对。
1.4 树鼩神经肽Y前体序列分子进化分析根据BLAST比对结果, 从GenBank中下载其他物种神经肽Y前体序列, 采用MEGA5.0软件(Tamura et al,2011)邻近归并法(neighbor joining method)构建系统进化树。
2结果2.1 树鼩脑cDNA文库构建提取的RNA经紫外分光光度计及1%琼脂糖凝胶电泳检测, 结果显示RNA浓度为882 ng/µL, OD260/OD280值为2.01, RIN值为8.28S/18S值1.7, 各指标均符合后续建库要求。
以所提RNA按照SMART TM cDNA Library Construction Kit试剂盒操作手册建立cDNA文库, 电泳检测显示250～2 000 bp位置有连续弥散状, 符合说明书要求, 可作为模板进行后续实验。