CN105132446A - 一种启动子筛选系统 - Google Patents
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Abstract
本发明公开了一种启动子筛选系统,其是将半乳糖苷酶编码序列<i>LacS</i>插入到切除了阿拉伯糖启动子的质粒pSeSD中构建得到硫化叶菌启动子筛选系统,其中半乳糖苷酶编码序列<i>LacS</i>的核酸序列如SEQ?ID?NO:2所示;通过将潜在的启动子序列插入pSeSD-LacS的<i>Sph</i>?I和<i>Nde</i>?I酶切位点之间,构建重组载体,将重组载体导入硫化叶菌E233S中进行功能验证,如果转化子表达一种半乳糖苷酶,能在含有X-gal培养基中显蓝色,表明该潜在的启动子序列具有启动子活性。
Description
技术领域
本发明属于基因工程和遗传工程领域,主要涉及将质粒pSSR-LacS上的半乳糖苷酶编码序列LacS插入到切除了阿拉伯糖启动子(arapromoter)的质粒pSeSD中,构建出硫化叶菌启动子筛选质粒pSeSD-LacS。如果将硫化叶菌及其病毒的具有潜在启动子功能的序列插入到pSeSD-LacS中并转化到硫化叶菌E233S中启动LacS基因的表达,则表明该序列是一个启动子。
背景技术
古菌是一类单细胞的微生物,由于形态上与细菌相似,在很长一段时间里(Achaea)被划归为细菌(Bacteria)。20世纪70年代末,Woese根据SSUrRNA系统发育树揭示了古菌在进化树上与细菌距离很大,并将被其命名为古细菌,即Archaebacteria(Woese&Fox,1977),随后将其简称为古菌(Archaea),并将古菌与细菌、真核生物列为平等的3个域。古菌的细胞膜含由分支碳氢链与D型磷酸甘油,以醚键相连的脂类;而细菌和真核生物的细胞膜则含有不分枝脂肪酸和L型磷酸甘油,以醋键相连接而成脂类。细菌细胞壁的主要成分是肤聚糖,而古细菌细胞壁不含肤聚糖(Kandler&Hippe,1977)。在上世纪90年代,随着高通量测序技术的应用,大量古菌基因组序列在短时间内测定完成。基因组分析表明古菌具有细菌和真核生物杂合的特性:与细菌相似,古菌染色体成闭合环状,基因也排列成操纵子模式;但在DNA复制。转录和翻译方面,古菌却具有明显的真核特征:启动子、转录因子、DNA聚合酶、RNA聚合酶等均与真核生物的相似(Langeretal.,1995;Brenneisetal.,2007)。
泉古菌门(Crenarchaea)包含3大分支:除硫球菌目Desulfuroeoeeales(Huber&Stetter,2001),热变形菌目Thermoproteales(Zilligetal.,1981)和硫化叶菌目。硫化叶菌细胞直径从1到5μm,所有硫化叶菌种类都是嗜热菌或者超嗜热菌,最适生长温度从65到90℃。这一种类微生物的另一个共同特点是生长最适pH值在2.0-3.0左右。硫化叶菌生长好氧、兼性厌氧或者厌氧。在自养生长条件(autotrophiccondition)下,硫化叶菌靠氧化硫元素、硫代硫酸盐、硫磺矿或者氢分子获取能量,并以二氧化碳为碳源(有趣的是S.solfataricus和S.acidocaldarius不能氧化硫元素)。异养生长条件下,硫化叶菌进行好氧呼吸、厌氧硫呼吸(anaerobicsulfurresPiration)或者有机底物发酵。
硫化叶菌目(order)包含l个科(Family)即Sulfolobaceae(Stetter,1989),下含6个属(Genera),大约20个种(Speeies)。另外,核酸杂交和SSUrRNA分析表明硫化叶菌属(Sulfolobus)内各菌之间尽管表型相似,但是遗传联系并不紧密。硫化叶菌属内各菌之间可以按照基因组DNA的GC含量,好氧/厌氧生长或使用不同的电子受体进行区分。与其他古菌相比,硫化叶菌比较容易培养,因此,即使在其遗传操作体系未建立前,古菌学家己经进行了大量的研究。到目前为止,测序完成的硫化叶菌有S.solfataricus(Sheetal.,2001),S.tokodaii(Kawarabayasietal.,2001),S.acidocaldarius(Chenetal.,2005)和S.islandicus(Renoetal.,2009)。并且建立起完善的遗传操作体系的菌株包括S.acidocaldarius(wagneretal.,2009;Berkneretal.,2007)和S.islandicus(Sheetal.,2009;Dengetal.,2009)。
古菌编码蛋白的基因和编码RNA的基因共用一类启动子结构和RNA聚合酶系统。古菌基本启动子包括2个主要的启动子元件:TALA-box和BRE(转录因子B识别位点,TranscriptionfactorBRecognition),以及研究较少的另外两个位点:IE(InitiationElement)和近启动子元件ProximalPromoterElement,PPE;Reeveetal.,1997;Soppa,1999;图l-3)。TATA-box和BRE主要作为真核生物同源的TATA-box结合蛋白(TATA-boxbindingprotein)和TFllB(在古菌中成为TFB)的结合位点(Hausneretal.,1996)。在古菌中,TATA-box作为一个A/T富集的序列位于转录起始位点(Transcriptionstartsite,TSS)的上游24-28个碱基左右(Reiteretal.,1990)。大部分古菌的TATA-box代表一个高度保守的8碱基序列(TTTAWAtr,withW=A/T,R=A/G)用于结合TBP。TBP/TFB/Promoter的晶体结构分析表明TATA-box这8bp与TBP是直接相互作用的(Littlefieldetal.,1999)。但是在盐古菌启动子的生物信息学分析表明,TATA-box的前6个碱基才是必需的并足以起始转录(Brenneisetal.,2007)。不同的古菌分支中TATA-box的保守序列有所区别:泉古菌(主要是硫化叶菌)中是“TTTTTAAA”,甲烷古菌(Methanogens)是“TTTATAT”,而在盐古菌(Halophiles)中是“NTTTWWWN(W=AorT;N=anynueleotide)”(Reeve,2003)。在古菌基本启动子的基础上,不同启动子一般还具有特异性的调控位点,如阻遏蛋白结合位点和转录激活蛋白结合位点。阻遏蛋白结合位点一般位于TATA-box和BRE附近或者其下游,而激活蛋白结合位点一般位于BRE上游。
发明内容
本发明的目的是提供一种启动子筛选系统,其是将半乳糖苷酶编码序列LacS插入到切除了阿拉伯糖启动子的质粒pSeSD中构建得到的,其中半乳糖苷酶编码序列LacS的核酸序列如SEQIDNO:2所示;该筛选系统的质粒模型为pSeSD-LacS,质粒图谱如图1所示。
上述硫化叶菌启动子筛选系统的核酸序列如SEQIDNO:1所示。
本发明提供的质粒模型pSeSD-LacS是一种有效的研究古细菌启动子活性的质粒模型,利用硫化叶菌作为古细菌生化和遗传研究的模式生物,该质粒模型有助于作为深入研究DNA转录、复制和修复、微生物多样性、病毒与宿主相互作用等研究领域的新切入点;研究硫化叶菌对于揭示生命活动的基本规律具有重要意义;该基因报告系统和基因表达系统,可用于其它问题的研究;本启动子筛选系统适用于硫化叶菌及其病毒启动子的筛选,通过将潜在的启动子序列插入pSeSD-LacS的SphI和NdeI酶切位点之间,构建重组载体,将重组载体导入硫化叶菌E233S中进行功能验证,如果转化子表达一种半乳糖苷酶,能在含有X-gal培养基中显蓝色,表明该潜在的启动子序列具有启动子活性。
附图说明
图1为本发明筛选系统的结构图谱示意图;
图2为本发明筛选系统pSeSD-LacS的构建策略示意图;
图3为本发明重组质粒pSeSD-LacS双酶切验证电泳图;
图4为本发明重组质粒模型pSeSD-LacS-P37构建策略示意图;
图5为本发明中重组质粒pSeSD-LacS-P37双酶切验证电泳图;
图6为本发明中X-gal显色反应图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂和使用常规方法。
实施例1:半乳糖苷酶基因LacS片段(带有酶切位点序列NdeI/SalI)的获取
取2μL质粒pSSR-LacS为模板进行聚合酶链式反应,设计引物(引物P1和引物P2)进行PCR扩增,反应所用引物、组分和扩增条件如下:
引物P1:LacSF:5’-GGAATTCCATATGTACTCATTTCCAAATAGCT-3’(SEQIDNO:7)
引物P2:LacSR:5’-GCGTCGACCTAGTGTTGCAAGGCAGAT-3’(SEQIDNO:8)
PCR扩增体系(50μL)组成如下:
模板(pSSR-LacS):2μL
2×phantamaxBuffer:25μL
dNTP(10mM):4μL
引物P1:2μL
引物P2:2μL
phantaMaxsuper-FidelityDNApolymerase:2μL
ddH2O:13μL
扩增条件:95℃变性3min,再用95℃30s、60℃30s、72℃1min进行30个循环,最后72℃5min,反应完后取产物1μL,然后在浓度为1%的琼脂糖凝胶中,进行电泳分析,经凝胶成像系统成像确认片段大小正确。
实施例2:重组表达质粒pSeSD-LacS的构建
取实施例1中LacS基因的PCR产物和质粒pSeSD分别用SalⅠ和NdeⅠ酶切过夜,50μLPCR产物双酶切体系:PCR产物25μl,10×BufferH5μl,SalⅠ1.5μl和NdeⅠ1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜。50μL质粒pSeSD双酶切体系:质粒pSeSD15μL,10×BufferH5μl,SalⅠ1.5μL和NdeⅠ1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜。用凝胶回收试剂盒对酶切产物进行纯化和回收。再将回收片段进行连接,连接体系(10μL):纯化的PCR产物和表达载体pSeSD按7:1的比例用0.5μL的T4DNA连接酶,T4Buffer1μL,16℃连接过夜。连接产物转入大肠杆菌DH5α感受态细胞中。37℃振荡培养1h后,涂布在含氨苄的LB培养基平板,37℃培养箱中培养12h,挑取平板上的转化子进行菌落PCR,筛选阳性克隆,构建获得重组表达质粒命名为pSeSD-LacS,构建流程见图2,pSeSD-LacS质粒图谱如图1所示。进一步进行双酶切分析鉴定,如图3第3泳道所示,用SalⅠ和NdeⅡ双酶切,重组质粒产生两条带,小分子条带与泳道4中该基因的PCR产物大小一致,大分子条带与泳道2中用相同两个内切酶酶切pSeSD-LacS(+)产生的条带大小一致,表明所构建的重组表达质粒正确。
实施例3:STSV2启动子P37基因潜在启动子序列(带有酶切位点序列SphⅠ/NdeⅠ)的克隆
取10μL携带病毒STSV2的硫化叶菌H5-2裂解,取其中1μL裂解液为模板进行聚合酶链式反应,设计引物(引物P3和引物P4)进行PCR扩增,反应所用引物、组分和扩增条件如下:
引物P3:P37F:5’-ACATGCATGCGGCCGGCCCATTATTCATTA-3’(SEQIDNO:9)
引物P4:P37R:5’-CATATGAGCTTCACCTCATTCTCTTTTTACCCCTAAATACCTATTT-3’(SEQIDNO:10);
PCR扩增体系(50μL)组成如下:
5×FastPfuBuffer10μL
dNTP(2.5μmol/L)5μL
H5-2(STSV2+)1μL
引物P3(10μmol/L)1.5μL
引物P4(10μmol/L)1.5μL
FastPfuDNApolymerase(5U/μL)2μL
无菌ddH2O补足至50μL;
扩增条件:94℃变性4min,再用94℃30s、58℃30s、72℃1min进行30个循环,最后72℃5min,反应完后取产物1μL,然后在浓度为1%的琼脂糖凝胶中,进行电泳分析;经凝胶成像系统成像确认片段大小正确。
实施例4:重组表达质粒pSeSD-LacS-P37的构建
取实施例3中P37基因的PCR产物和实施例2中构建的重组质粒pSeSD-LacS分别用SphⅠ和NdeⅠ酶切过夜,50μLPCR产物双酶切体系:PCR产物25μl,10×BufferH5μl,SphI1.5μl和NdeI1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜;50μL质粒pSeSD-LacS双酶切体系:质粒pSeSD-LacS15μL、10×BufferH5μl、SphI1.5μL和NdeI1.5μL,用灭菌的双蒸水补齐,37℃酶切过夜;用凝胶回收试剂盒对酶切产物进行纯化和回收;再将回收片段进行连接,连接体系(10μL):纯化的PCR产物和表达载体pSeSD-LacS按7:1的比例用0.5μL的T4DNA连接酶,T4Buffer1μL,16℃连接过夜。连接产物转入大肠杆菌DH5α感受态细胞中。37℃振荡培养1h后,涂布在含氨苄的LB培养基平板,37℃培养箱中培养12h,挑取平板上的转化子进行菌落PCR,筛选阳性克隆,构建获得重组表达质粒命名为pSeSD-LacS-P37,其构建流程如图4所示。进一步进行双酶切分析鉴定,如图5第3泳道所示,用SphⅠ和NdeⅠ双酶切,重组质粒产生两条带,小分子条带与泳道4中该基因的PCR产物大小一致,大分子条带与泳道2中用相同两个内切酶酶切pSeSD-LacS(+)产生的条带大小一致,表明所构建的重组表达质粒正确。
实施例5:STSV2启动子基因P37在硫化叶菌E233S中启动LacS基因表达,并与X-gal发生显色反应。
为了验证该启动子的活性,分别将1μg质粒pSeSD(阴性对照)、质粒pSeSD-LacS(阳性对照)、以及重组质粒pSeSD-LacS-P37加入50μl硫化叶菌E233S感受态细胞中充分混匀,另外,取50μl硫化叶菌E233S感受态细胞,不加质粒,作为空白对照,将整个体系常温放置10min。将重组质粒与感受态细胞加入到电转杯(0.2cm)中,使用:1.2KV,600Ω,1mm,25μF(Bio-RadGenepulserⅡ)条件电转化。然后加入800μL预热的FY培养基中,75℃、100rpm振荡孵育1h。孵育结束后,取50μL上述FY培养基与75℃含0.7%Gel的MT培养基混匀,迅速平铺到MTSV平板培养基上,75℃倒置培养10天。挑取阳性转化子阳性克隆验证,并划线转移到新的MTSV平板培养基上,75℃培养7天左右。然后,在形成菌落的平板上均匀喷洒少量5mg/mL的X-gal,75℃培养1d。结果如图6,转有质粒pSeSD-LacS与pSeSD-LacS-P37的E233S菌液均出现蓝色沉淀,说明该启动子具有活性,其核苷酸序列如SEQIDNO:3所示。
实施例6:本实施例方法同实施例4、5中方法,分别将具有潜在启动子功能的序列P2(其核酸序列如SEQIDNO:4)、P41(其核酸序列如SEQIDNO:5)以及NP(其核酸序列如SEQIDNO:6)构建到质粒pSeSD-LacS上,分别得到质粒pSeSD-LacS-P2、pSeSD-LacS-P41、pSeSD-LacS-NP,并转入E233S中表达;实验结果显示:转化了质粒pSeSD-LacS-P2、pSeSD-LacS-P41的E233S菌体均出现蓝色沉淀,而转有质粒pSeSD-LacS-NP的E233S菌液无明显变化,说明该报告系统有效果,可用于各类硫化叶菌及其病毒基因启动子活性研究。
序列表
<110>昆明理工大学
<120>一种启动子筛选系统
<160>10
<170>PatentInversion3.5
<210>1
<211>10004
<212>DNA
<213>人工构建
<400>1
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aaggatccgaatggtaaagaaattaggagaaggtgttttgtggggatcacgattaactat9240
ggggcgttagagttagaaaaattaggaaaaccacaagacatcacgccttctgtaccacag9300
gagattaggagatttgaagaatgtttgggtttttcggtaggaaatatagcatatcaactt9360
tccacaagaactgttgtgagagaagaagaattattgaaactcttcaaagatcaacacggt9420
gttgttaagaaagtagaggaatgtctgcgtgaattgaattttgttgataatggagatggg9480
acatttagaaaacccactccagaggaactatataaagactataaatgcacaacaatatgg9540
tatacaagtcaatataatgagttaaagggcaaaacggtgaaagtcaaagatctggatcca9600
gattttctgatgtgtctaaagaaattcgggcaaatggaacaggtagatgatgaaacaatt9660
cgaataaaataacaacacgcggggttttacagttttttgaaattcaatttttaatctata9720
atgaccaattatcggaccgataggacaggaaatcatatattttttaacacggtttttggg9780
tgagttttataggggtaaaaaatagtgtgtttcctgtcctatcggtccgttttttgcgta9840
agccgtgttaatatctgtatagtaatggcgtttttcgaatttttttagtaagaatatttg9900
aatttatctatcgcttttctctctctagagccgtgctataatcaaattaatatataataa9960
tatgtataaaataaactggcggtacatagtggtacattaaagta10004
<210>2
<211>1746
<212>DNA
<213>质粒pSSR-LacS
<400>2
tactcatttccaaatagctttaggtttggttggtcccaggccggatttcaatcagaaatg60
ggaacaccagggtcagaagatccaaatactgactggtataaatgggttcatgatccagaa120
aacatggcagcgggattagtaagtggagatctaccagaaaatgggccaggctactgggga180
aactataagacatttcacgataatgcacaaaaaatgggattaaaaatagctagactaaat240
gtggaatggtctaggatatttcctaatccattaccaaggccacaaaactttgatgaatca300
aaacaagatgtgacagaggttgagataaacgaaaacgagttaaagagacttgacgagtac360
gctaataaagacgcattaaaccattacagggaaatattcaaggatcttaaaagtagagga420
ctttactttatactaaacatgtatcattggccattacctctatggttacacgacccaata480
agagtaagaagaggagattttactggaccaagtggttggctaagtactagaacagtttac540
gaattcgctagattctcagcttatatagcttggaaattcgatgatctagtggatgagtac600
tcaacaatgaatgaacctaacgttgttggaggtttaggatacgttggtgttaagtccggt660
tttcccccaggatacctaagctttgaactttcccgtagggcaatgtataacatcattcaa720
gctcacgcaagagcgtatgatgggataaagagtgtttctaaaaaaccagttggaattatt780
tacgctaatagctcattccagccgttaacggataaagatatggaagcggtagagatggct840
gaaaatgataatagatggtggttctttgatgctataataagaggtgagatcaccagagga900
aacgagaagattgtaagagatgacctaaagggtagattggattggattggagttaattat960
tacactaggactgttgtgaagaggactgaaaagggatacgttagcttaggaggttacggt1020
cacggatgtgagaggaattctgtaagtttagcgggattaccaaccagcgacttcggctgg1080
gagttcttcccagaaggtttatatgacgttttgacgaaatactggaatagatatcatctc1140
tatatgtacgttactgaaaatggtattgcggatgatgccgattatcaaaggccctattat1200
ttagtatctcacgtttatcaagttcatagagcaataaatagtggtgcagatgttagaggg1260
tatttacattggtctctagctgataattacgaatgggcttcaggattctctatgaggttt1320
ggtctgttaaaggtcgattacaacactaagagactatactggagaccctcagcactagta1380
tatagggaaatcgccacaaatggcgcaataactgatgaaatagagcacttaaatagcgta1440
cctccagtaaagccattaaggcactaaactttctcaagtctcactataccaaatgagttt1500
tcttttaatcttattctaatctcattttcattagattgcaatactttcataccttctata1560
ttatttattttgtaccttttgggatctacacttaatgttagcctaattggaaagtcattt1620
agatttaatactgttaccagtccatcccttttaattattaatgaaaataagaagggataa1680
gtagcgatagcccttattccgatatggtctccaacaatatcccttattatctgcatgcaa1740
cactag1746
<210>3
<211>500
<212>DNA
<213>硫化叶菌H5-2病毒STSV2
<400>3
aaatcaaaaatatcttattttattgtttatgatttgaagtagcctagtagatactttatt60
gctacaccgattgatgctattattaatactatcattacaatctcaaagactgtagatgat120
aagccaagtgtgtagttaataccgcctgtaactttcgtagttatgttattattttgtggt180
aatgatgggattaatgttcctccaagtatacctactatgaaatatgctactataattaga240
actataacaaatactgcagcctttgcaagatttatgaaaacatctgtactaaagtctccg300
ccacctgctgatttatagaaccttcctagcttctttgctacctttgctcttgcgttatta360
ttggcagttatgagccatcccgccatcttttctcgttactttttatatggattggagtat420
aaaaagactccttaaacatattaacaatggcttaacgagtctttttatatctcattttga480
aaaaaggttattgagcaaaa500
<210>4
<211>498
<212>DNA
<213>硫化叶菌H5-2病毒STSV2
<400>4
ggccggcccattattcattattatattataattgccgcaggcctttatcattattataat60
aatgccgccgccggcctgccgccgcattaatcattatattataataataaaatgtataac120
taatattcattattatatattatatattgaatatgccgccgcgcctatgccgcaggcccg180
ccgccgccctaattacattattataatataatgaataactaatctacaatattcattata240
tcaattataaaattacatatatacacttaattcttttacttatactttttgtataatatt300
atcatgatataaatagtttataatatcacttattctataatgataaataaaaactcaaat360
ctagtattgatttgaattcccttaattcttttaatttacgctattgataatcacgtaaac420
tttacggtgataataccgttaatcattaacaaaagctttaaataccccttttcataaata480
ggtatttaggggtaaaaa498
<210>5
<211>500
<212>DNA
<213>硫化叶菌H5-2病毒STSV2
<400>5
cccttcgtcgttccgaacctcaccatcaaggaggtcctcggcgcgatcccggcgcactgc60
ttcgagcgttcggcgcttcgctcgtcgacctacgtcgtcgccgactttattatggtcgct120
gcgctgggctacgccgcgtaccatatcgaccccgcattctcgtacgagggcggcaagtac180
ctcagcggctgggccggcttcgctgccaagtgggcgtgctggtcgctgtactggacattg240
cagggatgggtcggcacagggatttggattctaggccacgagtgcggtcaccaggcgttc300
tcgacgtccaagacggtcaacaacacgatgggcctcttcctccactcgttcgtccttgtg360
ccgtaccactcgtggcgcatctcgcacgccaagcaccacgctgcgaccggccacctcacc420
cgcgacgaggtcttcgtcccccgcaccaagtcgttccgcaagccggcgccgacgggcaag480
aagctcgaggtcgcgcacaa500
<210>6
<211>500
<212>DNA
<213>人工合成
<400>6
ctcttgcgataaccacattctcagttcttcttgtggcttgttgttttttgttatcctttt60
tcgccatatttaaaagtaggaaagaaaaaataattaagctttatcatcttttcttgattg120
agaatagtgcgtagaaaattagcgcaataccaaacattattgctattgaatatggattag180
ttagtaagtatgaaaaatatgctactgttgctgctactggacctactgcattgttataac240
ttccattcaaatattgaacagtcacgttttcaacactataattctctaccttcatatttg300
tcacgtttgataataataccgctatttggttattggctttaaggagttgtggaaccatag360
tagagaagaagaagtaaagcagcagtgtgactattgggcctaagagaaggatgagtagat420
atgttgttattgaaaatggcggagttgatccgttagcttttagtcgcatatttaataata480
tgaatgcatatttttaaact500
<210>7
<211>32
<212>DNA
<213>人工合成
<400>7
ggaattccatatgtactcatttccaaatagct32
<210>8
<211>27
<212>DNA
<213>人工合成
<400>8
gcgtcgacctagtgttgcaaggcagat27
<210>9
<211>30
<212>DNA
<213>人工合成
<400>9
acatgcatgcggccggcccattattcatta30
<210>10
<211>46
<212>DNA
<213>人工合成
<400>10
catatgagcttcacctcattctctttttacccctaaatacctattt46
Claims (2)
1.一种启动子筛选系统,其特征在于:是将半乳糖苷酶编码序列LacS插入到切除了阿拉伯糖启动子的质粒pSeSD中构建得到硫化叶菌启动子筛选系统,其中半乳糖苷酶编码序列LacS的核酸序列如SEQIDNO:2所示。
2.根据权利要求1所述的启动子筛选系统,其特征在于:筛选系统的核酸序列如SEQIDNO:1所示。
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