CN105132446A - 一种启动子筛选系统 - Google Patents
一种启动子筛选系统 Download PDFInfo
- Publication number
- CN105132446A CN105132446A CN201510608035.8A CN201510608035A CN105132446A CN 105132446 A CN105132446 A CN 105132446A CN 201510608035 A CN201510608035 A CN 201510608035A CN 105132446 A CN105132446 A CN 105132446A
- Authority
- CN
- China
- Prior art keywords
- lacs
- psesd
- promoter
- plasmid
- screening system
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000012216 screening Methods 0.000 title claims abstract description 20
- 239000013612 plasmid Substances 0.000 claims abstract description 35
- 241000205091 Sulfolobus solfataricus Species 0.000 claims description 30
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 229960002203 tilactase Drugs 0.000 claims description 6
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 6
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract description 5
- 108010060845 lactose permease Proteins 0.000 abstract description 5
- 108010093031 Galactosidases Proteins 0.000 abstract description 4
- 238000012795 verification Methods 0.000 abstract description 2
- 102000002464 Galactosidases Human genes 0.000 abstract 3
- 108091026890 Coding region Proteins 0.000 abstract 2
- 241000205101 Sulfolobus Species 0.000 abstract 2
- 238000001976 enzyme digestion Methods 0.000 abstract 2
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 150000007523 nucleic acids Chemical group 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 35
- 230000029087 digestion Effects 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 11
- 108700026226 TATA Box Proteins 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000013613 expression plasmid Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000003259 recombinant expression Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000009182 swimming Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000005498 polishing Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000206602 Eukaryota Species 0.000 description 3
- 102100040296 TATA-box-binding protein Human genes 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 241000203069 Archaea Species 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241000205098 Sulfolobus acidocaldarius Species 0.000 description 2
- 241000167564 Sulfolobus islandicus Species 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 108700009124 Transcription Initiation Site Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- TXKMVPPZCYKFAC-UHFFFAOYSA-N disulfur monoxide Inorganic materials O=S=S TXKMVPPZCYKFAC-UHFFFAOYSA-N 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- AWUCVROLDVIAJX-UHFFFAOYSA-N glycerol 1-phosphate Chemical compound OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- XTQHKBHJIVJGKJ-UHFFFAOYSA-N sulfur monoxide Chemical compound S=O XTQHKBHJIVJGKJ-UHFFFAOYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000196835 Achaea Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001074903 Methanobacteria Species 0.000 description 1
- 241000249496 Sulfolobaceae Species 0.000 description 1
- 241000160715 Sulfolobus tokodaii Species 0.000 description 1
- 102000006467 TATA-Box Binding Protein Human genes 0.000 description 1
- 108010044281 TATA-Box Binding Protein Proteins 0.000 description 1
- 241000205177 Thermoproteales Species 0.000 description 1
- 241001314279 Zoopagales Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000004103 aerobic respiration Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000009569 heterotrophic growth Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 102200082920 rs33993004 Human genes 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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
caattgtgctgcatgccatatgtactcatttccaaatagctttaggtttggttggtccca60
ggccggatttcaatcagaaatgggaacaccagggtcagaagatccaaatactgactggta120
taaatgggttcatgatccagaaaacatggcagcgggattagtaagtggagatctaccaga180
aaatgggccaggctactggggaaactataagacatttcacgataatgcacaaaaaatggg240
attaaaaatagctagactaaatgtggaatggtctaggatatttcctaatccattaccaag300
gccacaaaactttgatgaatcaaaacaagatgtgacagaggttgagataaacgaaaacga360
gttaaagagacttgacgagtacgctaataaagacgcattaaaccattacagggaaatatt420
caaggatcttaaaagtagaggactttactttatactaaacatgtatcattggccattacc480
tctatggttacacgacccaataagagtaagaagaggagattttactggaccaagtggttg540
gctaagtactagaacagtttacgaattcgctagattctcagcttatatagcttggaaatt600
cgatgatctagtggatgagtactcaacaatgaatgaacctaacgttgttggaggtttagg660
atacgttggtgttaagtccggttttcccccaggatacctaagctttgaactttcccgtag720
ggcaatgtataacatcattcaagctcacgcaagagcgtatgatgggataaagagtgtttc780
taaaaaaccagttggaattatttacgctaatagctcattccagccgttaacggataaaga840
tatggaagcggtagagatggctgaaaatgataatagatggtggttctttgatgctataat900
aagaggtgagatcaccagaggaaacgagaagattgtaagagatgacctaaagggtagatt960
ggattggattggagttaattattacactaggactgttgtgaagaggactgaaaagggata1020
cgttagcttaggaggttacggtcacggatgtgagaggaattctgtaagtttagcgggatt1080
accaaccagcgacttcggctgggagttcttcccagaaggtttatatgacgttttgacgaa1140
atactggaatagatatcatctctatatgtacgttactgaaaatggtattgcggatgatgc1200
cgattatcaaaggccctattatttagtatctcacgtttatcaagttcatagagcaataaa1260
tagtggtgcagatgttagagggtatttacattggtctctagctgataattacgaatgggc1320
ttcaggattctctatgaggtttggtctgttaaaggtcgattacaacactaagagactata1380
ctggagaccctcagcactagtatatagggaaatcgccacaaatggcgcaataactgatga1440
aatagagcacttaaatagcgtacctccagtaaagccattaaggcactaaactttctcaag1500
tctcactataccaaatgagttttcttttaatcttattctaatctcattttcattagattg1560
caatactttcataccttctatattatttattttgtaccttttgggatctacacttaatgt1620
tagcctaattggaaagtcatttagatttaatactgttaccagtccatcccttttaattat1680
taatgaaaataagaagggataagtagcgatagcccttattccgatatggtctccaacaat1740
atcccttattatctgcatgcaacactaggtcgacgctgatcaagcggccgcacatcatca1800
tcaccaccattaagcaaaatcttttttttcccgggtacctactggcgtagtttaacataa1860
ggttttgtaagaaattcatataaatcctcaatatctcatcataaattacaaaatactcat1920
acacacttctaccctttttgactaaatcgttttcgagatcaaattcataatctctatcta1980
tataaatataagttaaatctgttgttggaattacgccatattcctcgtgtagattttccc2040
ctattaatgttagtttataaagaccggctattttttcactaatttcattttttcctaaaa2100
attgctcctttacatttcatcaccttatcctcgataatcttatttatagttcttaatgcg2160
ttaatggattccctgcattataaatacttcttccaatgatttcataatccgctccagcac2220
atactgcatcgccataacttccaccttgactacccatacccggagagactatggtcattt2280
tttcgaagtctctcctatactgcgttatatgatctaatttagtccctccaactactattc2340
cttttgggcttatctctcttataacgtttttaatatagtctgcgaataacgtactccatc2400
cttcatgtgacattacggcaactaagtataaatttttagagtttgcatcaagatatcttt2460
ttaattcatctagagatcccttaacgcctataaaggaatgtgctatgaacgagttggcga2520
aagataatctttcaactatgcttttcattatgtatccgatatctgcaagcttaaaatcaa2580
caataatttcctccacgtctaaaccaattaagagctctctagttttatccactcctagat2640
ctaaaactaaaggtaaaccaacttttatcccatataactcattttccatctctttaagaa2700
cttgatatgagagaggtttatccattgctaatattactctacttttcaacattcttcacc2760
aaataatctagaattgacttcttttcattatccttaagtttatcactcttcaacaattca2820
tctagaatttctgaaattttaaatagagagtgtaatttgactcctagtttttccaatctt2880
tgtgaagccccttcttgtctatctatgattactagtgcgtctgaaactttacctccaccg2940
ttaagaatctccaatgttgctttctctatggatactcctgtagttgcaacgtcatctact3000
aacaatactctttttccttttacatcgagttctaatgtacgattagttccatgacctttc3060
ttttctattctaatatatcccataggctctttaaggttacaagctatgaatgccgataag3120
ggaactcctccagtggctattcctactattatatcatggggtatatcttttgctttcttt3180
atagcttgattaactatatcgtaaaattctggataatttggtaaaggtcttaagtctaag3240
taatatggactaaccttacctgatgttaaaacgaaacttcctattaataataatttcctt3300
tcgagtaagacttctgcgaaattcatacgtagagactctgtgaaaaagaatttaaatata3360
cttctatcataaccagttataagggctttgtgagattaagacacgtagtttcgtcgcttg3420
acttgaccagagatgactactttagaatattcgaacttgcagacaagttctatgatgtaa3480
aaaaactaaattatctatcagggaaagtagtttcattagcattctttgagccaagtacta3540
gaactgctcaaagctttcatactgcagcaataaaattaggtgctgatgtgataggatttg3600
catccgaggagtctacttcgatagcaaaaggtgaaaatttggctgataccattaggatgc3660
taaacaactattcaaactgtattgtaatgagacataagtttgatggggtaccgagctcga3720
attcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgact3780
gggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccaggt3840
caggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatac3900
attcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaa3960
aaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcat4020
tttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatc4080
agttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgaga4140
gttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcg4200
cggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctc4260
agaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacag4320
taagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttc4380
tgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatg4440
taactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtg4500
acaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactac4560
ttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggac4620
cacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtg4680
agcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcg4740
tagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctg4800
agataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatac4860
tttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttg4920
ataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccg4980
tagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc5040
aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactc5100
tttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgt5160
agccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgc5220
taatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggact5280
caagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacac5340
agcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgag5400
aaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcg5460
gaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctg5520
tcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgga5580
gcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctt5640
ttgctcacatgttctttcctgcgttatccctgattctgtggataaccgtattaccgcctt5700
tgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcga5760
ggaagctctcttatacattagtacaagttcgccgttattttgttcaacacttaacaagaa5820
ttcgtctccttttttcagacctaacgcttttacaatttctcgtgggacattcagataaaa5880
cgtaagttcaccacgcccgcccttttttgcggatagtcttaccataattatattttccgt5940
tcaccattttttaaaacttttccgttcaccaaaagctttaaatatcacggggggcaaatc6000
taattttggtgaccggaaagtgaacggaaaaataaacaaaataaacaccctaaataagat6060
ttttttagaccctgaccccgaaaacgtgaaaaacaaaaccacggtgagggaactatgaat6120
gcagatgaaatattcagtaacctagaagagatattgaactataacagcctggtgttcatt6180
aatagaaagaatattgaggtagtgtgggctataaggtctgatacagacacggttcaggga6240
ttcgtgagagttgataacaaagtattcccattcaaagcgtgggtagaatttgaaggagag6300
ttaagagtgcaaatcggcaatttaatccacttcataatagactccaaaaccgtggaaaaa6360
gcaatacagagggagtctgaataacacgcggtaataccatgtttcaaggtactacttttt6420
cccaatctctacgcgggtaatacgcaaaattaacaccaagaggaatttaacttatttcca6480
ctatattgtccatggtggttaaatggtaagcacgaaaccaaatgtgcatattagattaag6540
agaagaggaaaggaaattgttgaaggaaatagcacaaaagtatgatatttccgaatctga6600
tgtggtaaagatagcactgaagaagttagcacgtgagttaggaatggataattcaccatg6660
atttgaggtgaaaacaaggagaaattattcatgatttgacgaaaaaaacacagaaaaagg6720
ggggagggctctatgagtaacttctcaaatcaagataaaaagcgtttcgccgaatggttt6780
atcgaacaaggctttgccattttccctattgatccgcaatcgaaaaagcccgtgatttcc6840
aattggcaaaaatatagccatgagccacttaccgatgaagagaagaagagattcttagaa6900
caaattgaaaagggctataattatgctgtgccgggtggtcagcagaacctggtaatctta6960
gattttgagagtaaggagttattggaaaaatggataagcaaatcggcacttgatgaactg7020
tgcagaaagaccctatgtgttaacacgccccacggcgggatccatgtttatgtccaggct7080
gacaatataccagagcacaagtttaatcccgcctttacgctaaacggcaaaggaatagcg7140
gatttgcagagttacaatagttacgtgttgggcgtgggttcatgtataaaccacaagttc7200
tgtgatagtgacaaatgtaagtggcggggacagaatcataccacgtgttattcattatat7260
aataataatgaattaaagataggtagagctgatctaaagggactacttaagttcttagct7320
gaaaaaggaaaagagttagggatagaattatcatctaacgcacgggcgtggttagaagga7380
gagaaagaggagactgatgttgaaaaagacctggagaagttgaagaaggaactatccagg7440
ttcaacaagtttaaaggcaaaaccatagaggcgataagatctgaagtctgtcaaagcatt7500
aagaagagtctggaaaacgtaaagagtgacaaagcaaaaaccatgcttaacacggctttc7560
caggttgtttgtcagggtaagagttatggggagatacaattggataggagtaggggtgat7620
tggcacgtgttgaaagtactgttaagccatggagtcacggatgttgaagtattgaaacaa7680
ctcttaccacaggattccaaagtctttgccccaaaatgggaccgatactttttacacacg7740
ttaatgaaagcctggaatgaagtaaaaccgtttctccaaatcataaagaatgccaaaaac7800
aagaagaccaaagaactcaaacaagaattggctgaagttctctcacaatatatcataagg7860
aaatatcatattgtcacattcattcaaaaacatagtaatggtgaatcaattataggaatc7920
ttcagatggaataggaaaaagggaatctatgaaccaattgatgaaacacttaagaagatt7980
attaggcatgaaatcatgcgtgttattgagacattccctaaatcagaagatgagaaatca8040
cccatgttctatgaagtaagaaatgaacttgtcaaactagtgtatgatgaaattagggac8100
ctaacactcacagaatatgatgatgataacacgcctcttagaatagctttcgaaaactgc8160
acactggaatggacatcagacaagttcaaactgattcctgcagataaacgcacagaagaa8220
cattatgcgtttcattatgtcccgcacaagatccgtgttgaagtatttaacaacacgccg8280
taccaggtcccagaattagaagagttagcccgaaaattgtgcccaaaatcacttaatgcc8340
tttaaacagtgggtaggagagaaatgggtcactttgtttgagataataggatatacatta8400
taccccgccacaaaattcaagttagccttcatgctcttaggaccccgtgattccggcaaa8460
tcaacattcctacaactgttgaagaggattctaggtaaacaaaacacggttagtattagg8520
ctaagggaattgtttgacccaaataacaggtttgtggcggggtttctattccacaaactt8580
gtgaacttaacggcggaaactaaggaatacaccatagaggatattgacaggtttaagaca8640
ttaacgggcggggaccaaatcacaagtgatgttaagttcaaaggacctatcacgtttact8700
ccttacgctaaactaatcatagcctctaacaagttaccagatataagagataagaatgac8760
acggctttttggagaaggtggctaattatagagtttccaaatcaattcccgaatgatgat8820
aactggttcaggcaaacgtttacagaagaggaaatagagggcatattgacagtctcaata8880
ttggcgtttgcccgtgttatgagtagagggcaatttgactaccaacagactccagaagaa8940
gtaagggacctatggctctataatatagacagtgtatggagtttcgttagaacttatgag9000
aagaaaggttttatcacggttgacccacgaaacgctgatctatgggtacctagaatagaa9060
ctctataaattatataaggattattgcatggataatgggtttcctggagtatcactgaaa9120
acgtttgctaacaaactcaacaaatactttggaataacaagtatgaagaagtattttgga9180
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。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510608035.8A CN105132446A (zh) | 2015-09-23 | 2015-09-23 | 一种启动子筛选系统 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510608035.8A CN105132446A (zh) | 2015-09-23 | 2015-09-23 | 一种启动子筛选系统 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105132446A true CN105132446A (zh) | 2015-12-09 |
Family
ID=54718014
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510608035.8A Pending CN105132446A (zh) | 2015-09-23 | 2015-09-23 | 一种启动子筛选系统 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105132446A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441506A (zh) * | 2018-03-13 | 2018-08-24 | 南京理工大学 | 硫化叶菌蛋白复合物的表达载体的构建及表达纯化方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008138579A1 (en) * | 2007-05-11 | 2008-11-20 | Universität Bayreuth | Archaeal plasmid vector system |
| CN102174552A (zh) * | 2011-01-19 | 2011-09-07 | 山东大学 | 一种基于抗生素标记的超嗜热泉古菌表达载体及其应用 |
| CN102337254A (zh) * | 2011-08-26 | 2012-02-01 | 江南大学 | 一种β-半乳糖苷酶的突变体及其制备方法和应用 |
| CN103937767A (zh) * | 2014-04-08 | 2014-07-23 | 昆明理工大学 | 一种半乳糖苷酶和编码这种酶的多核苷酸 |
-
2015
- 2015-09-23 CN CN201510608035.8A patent/CN105132446A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008138579A1 (en) * | 2007-05-11 | 2008-11-20 | Universität Bayreuth | Archaeal plasmid vector system |
| CN102174552A (zh) * | 2011-01-19 | 2011-09-07 | 山东大学 | 一种基于抗生素标记的超嗜热泉古菌表达载体及其应用 |
| CN102337254A (zh) * | 2011-08-26 | 2012-02-01 | 江南大学 | 一种β-半乳糖苷酶的突变体及其制备方法和应用 |
| CN103937767A (zh) * | 2014-04-08 | 2014-07-23 | 昆明理工大学 | 一种半乳糖苷酶和编码这种酶的多核苷酸 |
Non-Patent Citations (6)
| Title |
|---|
| CUBELLIS,M.V.等: "S.solfataricus betaDgalactosidase，Accession No：M34696.1", 《GENBANK》 * |
| ERDMANN,S.等: "Sulfolobus virus STSV2, complete genome", 《GENBANK》 * |
| PENG-JUAN LIANG等: "Knockouts of RecA-Like Proteins RadC1 and RadC2 Have Distinct Responses to DNA Damage Agents in Sulfolobus islandicus", 《JOURNAL OF GENETICS AND GENOMICS》 * |
| 华南农业大学园艺生物技术研究所编: "《园艺植物生物技术》", 28 February 2002 * |
| 江冬青: "硫化叶菌超表达载体特性和阿拉伯糖操纵子表达调控的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 白俊等: "LacS 基因原核表达载体构建及重组蛋白表达", 《上海畜牧兽医通讯》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441506A (zh) * | 2018-03-13 | 2018-08-24 | 南京理工大学 | 硫化叶菌蛋白复合物的表达载体的构建及表达纯化方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zeldes et al. | Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals | |
| US10202619B2 (en) | Compositions and methods directed to CRISPR/Cas genomic engineering systems | |
| Barabote et al. | Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations | |
| CN102703424B (zh) | 一种重组工程介导的大肠杆菌基因组点突变的方法 | |
| AU2016260738B2 (en) | IrrE protein functional domain for improving anti-oxidation capability of cell and application thereof | |
| Jeong et al. | Genetic engineering system for syngas-utilizing acetogen, Eubacterium limosum KIST612 | |
| CN109706148A (zh) | 一种用于敲除BCL11A基因或者BCL11A基因增强子的gRNA、gRNA组合物以及电转方法 | |
| CN106947766A (zh) | 一种枯草芽孢杆菌具有启动子功能的dna片段及其应用 | |
| Heng et al. | Complete genome sequence of Halocella sp. strain SP3-1, an extremely halophilic, glycoside hydrolase-and bacteriocin-producing bacterium isolated from a salt evaporation pond | |
| CN106701707A (zh) | 新的7α‑羟基类固醇脱氢酶基因S1‑a‑1 | |
| CN105132446A (zh) | 一种启动子筛选系统 | |
| CN104212827B (zh) | 不依赖生物工程酶的快速分子克隆方法 | |
| US11555193B1 (en) | Gene ANSB knockout mutant of citrobacter werkmanii and application thereof | |
| CN101157934A (zh) | 基于Phi BT1整合酶及Phi C31整合酶的基因克隆质粒及其应用 | |
| EP2922957B1 (en) | Nucleic acids useful for integrating into and gene expression in hyperthermophilic acidophilic archaea | |
| CN103232994B (zh) | 一种筛选嗜酸性氧化硫硫杆菌无标记基因敲除菌株的方法 | |
| CN107760697A (zh) | 用于表达限制性内切酶FspI的甲基化保护菌株的筛选方法 | |
| CN103757048A (zh) | 无抗药基因的酵母-细菌穿梭载体的构建和应用 | |
| CN106701708A (zh) | 新的7α‑羟基类固醇脱氢酶基因Y1‑a‑1 | |
| CN105861485A (zh) | 一种提高基因置换效率的方法 | |
| CN104513830A (zh) | 一种适用于氧化葡萄糖酸杆菌的基因表达载体及其应用 | |
| CN104087606A (zh) | 一种胆盐水解酶基因bsh及其重组原核表达载体 | |
| Pold et al. | Genome sequence of Verrucomicrobium sp. strain GAS474, a novel bacterium isolated from soil. Genome Announc 6: e01451-17 | |
| Zhao et al. | Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus | |
| Heng et al. | Complete genome sequence of Halocella sp. Strain SP3-1, an extremely halophilic, glycoside hydrolase-and bacteriocin-producing bacterium isolated from a salt evaporation pond. Microbiol Resour Announc 8: e01696–e01618 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151209 |
|
| RJ01 | Rejection of invention patent application after publication |