CN111979257A - 一种重组dna及其应用 - Google Patents

一种重组dna及其应用 Download PDF

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CN111979257A
CN111979257A CN201910430555.2A CN201910430555A CN111979257A CN 111979257 A CN111979257 A CN 111979257A CN 201910430555 A CN201910430555 A CN 201910430555A CN 111979257 A CN111979257 A CN 111979257A
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陈玲
周豪宏
雷云凤
刘修才
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Abstract

本发明提供一种重组DNA及其应用。所述重组DNA至少包括稳定期特异性启动子和带有促溶标签的赖氨酸脱羧酶融合蛋白基因；其中，所述促溶标签选自荧光蛋白、麦芽糖结合蛋白、谷胱甘肽转移酶或它们的组合。本发明为提高重组菌株发酵生产多胺的产量提供了新策略。

Description

一种重组DNA及其应用
技术领域
本发明属于生物技术领域，具体地说，涉及一种重组DNA及其应用，特别是在生产多胺中的 应用。
背景技术
1,5-戊二胺(也称为1,5-二氨基戊烷、尸胺)是化学工业中重要的五碳化合物，用途相当广泛， 可以用于制造聚酰胺、聚氨酯、异氰酸酯、吡啶、哌啶等重要化工原料。目前，微生物法1,5-戊二 胺生产主要采用以下两种方法：微生物发酵生产和微生物体外酶催化生产。酶催化生产使用的赖氨 酸脱羧酶(L-lysine decarboxylase,简称LDC,EC4.1.1.18)广泛存在于微生物、昆虫、动物和高 等植物中，其可以将L-赖氨酸脱去一个羧基生成1,5-戊二胺和CO2。
赖氨酸脱羧酶催化赖氨酸生成1,5-戊二胺的工艺中通常使用游离状态的赖氨酸脱羧酶或赖氨 酸脱羧酶细胞，或者使用可同时生产赖氨酸和赖氨酸脱羧酶的菌株发酵生产1,5-戊二胺，但是以上 这些生产方式导致酶或细胞的重复利用效率低、产物回收困难、生产成本高，不利于1,5-戊二胺的 工业化生产。而且，由于菌体本身耐受的1,5-戊二胺浓度有限，如果发酵体系中前期表达了赖氨酸 脱羧酶转化生成的1,5-戊二胺过多，就会对菌体造成毒害，从而抑制菌体生长及利用葡萄糖生产L- 赖氨酸的过程(Qian,et al.,Biotechnol.Bioeng.2011；108:93–103)。因此，需要获得更高1,5-戊二胺产 量的新技术。
发明内容
本发明的目的是提供一种重组DNA及其应用，特别是在生产多胺如1,5-戊二胺中的应用。
本发明构思如下：通过筛选来源于嗜热菌株的赖氨酸脱羧酶，利用荧光蛋白与其融合表达， 可以通过升高温度使赖氨酸脱羧酶发挥脱羧功能；另外，具有颜色的荧光蛋白的使用还可以明确地 指示表达融合蛋白的细胞。进一步借助于稳定期的启动子，在稳定期时才会启动赖氨酸脱羧酶的表 达，大大降低了因宿主细胞受到1,5-戊二胺毒害而导致减产，显著提高了重组菌株发酵生产1,5-戊 二胺的产量，并显著降低了赖氨酸的残留，从而简化后续提取1,5-戊二胺的工艺。
为了实现本发明目的，第一方面，本发明提供一种重组DNA，所述重组DNA至少包括稳定期 特异性启动子和带有促溶标签的赖氨酸脱羧酶融合蛋白基因；所述融合蛋白包含促溶标签和来自嗜 热菌的赖氨酸脱羧酶，二者之间通过Linker连接；
其中，所述促溶标签选自荧光蛋白、麦芽糖结合蛋白、谷胱甘肽转移酶或它们的组合。
在原核生物如大肠杆菌中进行异源蛋白表达时，麦芽糖结合蛋白MBP，谷胱甘肽转移酶GST， 以及组氨酸His7等可以促进蛋白的可溶性以及蛋白的正确折叠。MBP(麦芽糖结合蛋白)标签蛋白 大小为42kDa，等电点pI＝5.03，MBP可增加在细菌中过量表达的融合蛋白的溶解性，但该标签较大 对蛋白的结构或功能会有一定的影响。GST(谷胱甘肽巯基转移酶)标签蛋白，它的天然大小为26KD， 等电点pI＝6.10。
本发明中，所述荧光蛋白选自红色荧光蛋白、蓝色荧光蛋白、蓝绿色荧光蛋白、绿色荧光蛋白、 黄色荧光蛋白、橙色荧光蛋白或光学加亮荧光蛋白；优选RedStar、tdtomato或mCherry中的至少一 种。研究中发明人意外地发现，上述荧光蛋白可以作为促溶标签用于异源蛋白表达中，并且其荧光 特性还可以方便地指示正确表达融合蛋白的细胞。
在一些实施方案中，所述mCherry是来自于珊瑚中的红色荧光蛋白，从DsRed演化来的性能最 好的一个单体红色荧光蛋白(Graewe,et al.,Biotechnology Journal,2009,4(6))，大小为26KDa，等电 点pI＝5.62。
在一些实施方案中，所述mCherry的氨基酸序列如SEQ ID NO:17所示，mCherry编码基因的核 苷酸序列如SEQ ID NO:18所示，其经密码子优化。
在一些实施方案中，所述赖氨酸脱羧酶选自以下①～④中任一种：①来源于嗜热菌 Thermosynechoccus elongatus的赖氨酸脱羧酶TeLDC，其氨基酸序列如SEQ ID NO：1所示，其编码 基因为Teldc(GenBank ID BAC09418.1,SEQ ID NO:2)，；②来源于Tepidanaerobacter syntrophicus的 赖氨酸脱羧酶TsLDC，其氨基酸序列如SEQ ID NO:3所示，编码基因为Tsldc(GenBank ID GAQ24853.1,SEQ ID NO:4)；③来源于Geobacilluskaustophilus的赖氨酸脱羧酶GkLDC，其氨基酸 序列如SEQ ID NO:5所示，编码基因为Gkldc(GenBankID BAD75350.1,SEQ ID NO:6)；④来源于 Thermomicrobium roseum的赖氨酸脱羧酶TrLDC，其氨基酸序列如SEQ ID NO:7所示，编码基因为 Trldc(GenBank IDACM05730.1，SEQ ID NO:8)。优选地，其中全长核苷酸序列根据大肠杆菌密码 子偏好性进行了优化。
在一些实施方案中，所述赖氨酸脱羧酶与SEQ ID NO：1,3,5或7中的任一个具有至少70％的氨 基酸序列同一性，或至少80％，至少85％，至少90％，至少95％的序列同一性。
在一些实施方案中，所述Linker包括螺旋形式的Linker或低疏水性、低电荷效应的氨基酸组成 的柔性Linker，所述Linker的长度应至少为10个氨基酸。优选地，所述的Linker为柔性Linker，例如 (GGGGS)3或(SG)5-8；更优选地，所述Linker为(SG)5-8；最优选地，所述Linker为SGSGSGSGSG。
在一些实施方案中，所述融合蛋白选自荧光蛋白-Linker-TeLDC、荧光蛋白-Linker-TsLDC、荧 光蛋白-Linker-GkLDC、荧光蛋白-Linker-TrLDC、TeLDC-Linker-荧光蛋白、TsLDC-Linker-荧光蛋 白、TsLDC-Linker-荧光蛋白或TsLDC-Linker-荧光蛋白中的至少一种。
在一些实施方案中，所述融合蛋白为mCherry-Linker-TeLDC，TeLDC-Linker-mCherry， mCherry-Linker-TsLDC，mCherry-Linker-GkLDC或mCherry-Linker-TrLDC。
在一些实施方案中，所述融合蛋白为SEQ ID No:10，SEQ ID No:12，SEQ ID No:14，SEQ ID No:16所示的重组核苷酸序列中任一种，其从5’方向至3’方向包含编码荧光蛋白的核苷酸序列(SEQ ID NO:5)和编码嗜热菌来源的赖氨酸脱羧酶的核苷酸序列。
在一些实施方案中，所述稳定期特异性启动子选自pcsiE(SEQ ID NO:20)，pbolA(SEQ ID NO: 21)，posmY(SEQ ID NO:22)，pkatE(SEQ ID NO:23)，P1(SEQ ID NO:24)，P2(SEQ ID NO: 25)，P3(SEQ ID NO:26)或P4(SEQ ID NO:27)中的任意一种。
在一些实施方案中，所述重组DNA至少包括如下3个元件：a、稳定期特异性启动子；b、红色 荧光蛋白基因；以及c、嗜热菌来源的赖氨酸脱羧酶基因；其中，各元件按照a-b-c或a-c-b顺次可操 作地连接。
第二方面，本发明提供含有上述重组DNA的生物材料，所述生物材料为表达盒、转座子、质 粒载体、噬菌体载体、病毒载体或工程菌等。
第三方面，本发明提供一种重组质粒，所述重组质粒携带有上述重组DNA。优选地，所述出 发质粒为pUC或pBR322质粒或其衍生质粒，更优选pUC18、pUC19、pBR322、pACYC、pET、pSC101 和它们的任何衍生质粒。
第四方面，本发明提供产1,5-戊二胺的基因工程菌，所述基因工程菌为具有生产L-赖氨酸能力 的菌株，并携带有上述重组DNA、生物材料或重组质粒。
其中，所述基因工程菌的出发菌株选自埃希氏菌属(Escherichia)、哈夫尼菌属(Hafnia)中 的菌种；优选地，所述出发菌株为大肠杆菌(Escherichia.coli)、蜂房哈夫尼菌(Hafnia alvei)，或经 过诱变或随机突变之后的菌株或基因工程菌。
第五方面，本发明提供1,5-戊二胺的生产方法，包括发酵培养上述工程菌以生产1,5-戊二胺。
第六方面，本发明提供所述重组DNA在生产1,5-戊二胺中的应用，包括(a)将所述重组DNA 构建到具有生产L-赖氨酸能力的工程菌中，发酵培养重组菌并进行赖氨酸积累，发酵初期培养温度 控制在20-50℃，进行菌体的快速生长以及赖氨酸积累；(b)在余下的发酵阶段温度控制在50-110℃， 使赖氨酸脱羧酶具有活性，转化生产1,5-戊二胺。
如本文所用，术语“约”在用于修改温度范围内的数值时表示该数值与该值的合理偏差，例如， 在该范围内所述的值之下或之上1℃或2℃以内，是在所述值或范围的预期含义内。
在一些实施方案中，步骤(a)在约25℃至约45℃的温度下进行。在其他实施方案中，步骤(a) 在约30℃至约40℃的温度下进行。在进一步的实施方案中，步骤(a)在约35℃至约39℃的温度下 进行。在一些实施方案中，步骤(b)在约55℃至约90℃的温度下进行。在其他实施方案中，步骤 (b)在约60℃至约75℃的温度下进行。在进一步的实施方案中，步骤(b)在约60℃至约70℃的温 度下进行。
在一些实施方案中，1,5-戊二胺的生产方法，包括将所述重组DNA构建至具有生产L-赖氨酸能 力的工程菌，优选对编码基因进行密码子优化后再构建至工程菌中，优选为大肠杆菌或蜂房哈夫尼 菌中，发酵培养重组菌，发酵初期培养温度控制在20-50℃，例如37℃±2℃，进行菌体的快速生长 以及积累赖氨酸；余下的发酵阶段将温度控制在50-110℃，例如55℃±2℃，使赖氨酸脱羧酶具有 活性或活性提高，转化赖氨酸生产1,5-戊二胺。其中，优选地，所述融合蛋白为荧光蛋白-Linker- 赖氨酸脱羧酶(TeLDC、TsLDC、GkLDC或TrLDC)。优选地，当发酵培养至赖氨酸的含量不再增 加时，再升高发酵体系的温度，并控制温度为50-110℃。优选地，所述构建至具有生产L-赖氨酸能 力的工程菌时，可以使用重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程 菌等材料进行构建。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明通过筛选来源于嗜热菌株的赖氨酸脱羧酶，利用促溶标签与其融合表达，当来源于嗜 热菌株的赖氨酸脱羧酶与促溶标签连接时能够帮助其发挥赖氨酸脱羧功能，与来源于大肠杆菌的赖 氨酸脱羧酶CadA相比，可以简单地通过升高温度使其发挥功能；另外，促溶标签的使用还可以明 确地指示表达融合蛋白的细胞，尤其是荧光蛋白作为促溶标签更易于明确指示。将其应用于1,5-戊 二胺生产，可以显著降低细胞生长及L-赖氨酸生产阶段产生的1,5-戊二胺的细胞毒性，少量的1,5- 戊二胺还可以解除赖氨酸的反馈抑制效应，提高L-赖氨酸产量；升高温度后，可以将L-赖氨酸几乎 全部转化为1,5-戊二胺，从而实现了1,5-戊二胺产量的增加。同时使用了稳定期的启动子，当菌体 生长到稳定期后才会启动下游赖氨酸脱羧酶基因的表达，综合显著地提高重组菌株发酵生产1,5-戊 二胺的产量。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规 实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中涉及到的PCR扩增、纯化、质粒提取、酶切、酶切产物连接、转化等的具体步骤、 条件参数等均按所购相关酶和试剂的说明书建议的条件进行。其中PCR扩增所用的DNA聚合酶、 酶切所用的限制性内切酶、酶切产物连接所用的连接酶均购自宝生物工程(大连)有限公司。质粒提 取试剂盒、DNA胶回收试剂盒、PCR纯化试剂盒均购自康宁生命科学(吴江)有限公司，商标Axygen； 引物均购自赛默飞世尔科技(中国)有限公司，商标INVITROGEN。
以下实施例中涉及到的质粒转化方法如下：将连接产物加至100μl的大肠杆菌E.coli BL21(DE3) 感受态细胞中，冰浴30min后，42℃热激90s。冰上孵育5min后加入1ml的LB。涂布至相对应的抗性 平板上。
本发明中，培养基中L-赖氨酸和1,5-戊二胺的量可以通过核磁共振方法检测。
以下实施例中所用的引物如表1所示：
表1引物信息
Figure BDA0002068857380000041
Figure BDA0002068857380000051
实施例1赖氨酸脱羧酶基因cadA的克隆
使用引物cadA-SacI-F(SEQ ID No:28)和cadA-XbaI-R(SEQ ID No:29)将赖氨酸脱羧酶(SEQ ID No:30)编码基因cadA(SEQ ID No:31)从大肠杆菌MG1655K12(E.coliMG1655K12，购自北京天恩 泽生物技术有限公司)的基因组中扩增出来，通过SacI和XbaI双酶切后，连入同样双酶切的pUC18 质粒(购自宝生物工程(大连)有限公司)中。利用CaCl2法制备感受态，并用热激法将连接产物 转化至大肠杆菌E.coli BL21(购自宝生物工程(大连)有限公司)细胞中，利用LB培养基中添加 氨苄抗生素进行筛选，克隆PCR和测序验证正确后，抽提质粒即得到pCIB60质粒。
以质粒pCIB60为模板，进一步利用引物cadA-F2(SEQ ID No:32)和cadA-R2(SEQID No:33) 对pCIB60质粒中cadA基因的5’序列进行优化，使CadA可以顺利在大肠杆菌E.coli BL21中翻译成 蛋白，cadA基因的5’序列由5’- tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC-3’(SEQ ID No:34)替换为5’-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTGAGCTC-3’(SEQ ID No:35)。PCR扩增后，利用DpnI限制性内切酶对PCR产物进行消化，同样利用热激法转化至E.coli BL21中，测序验证后得到质粒pCIB71。
实施例2嗜热菌Thermosynechoccus elongatus的赖氨酸脱羧酶Teldc的克隆
来自于嗜热菌株Thermosynechoccus elongatus中的赖氨酸脱羧酶TeLDC(SEQ IDNO:1,GenBank ID BAC09418.1)，通过对其基因全长密码子进行优化后，利用引物拼接的方法合成该基因(SEQ ID NO:2)，密码子优化及基因合成的方法参考Hoover DM&LubkowskiJ,Nucleic Acids Research 30:10, 2002，利用TeLDC-SacI-F(SEQ ID No:36)和TeLDC-XbaI-R(SEQ ID No:37)对拼接的Teldc进行 扩增，利用SacI和XbaI双酶切后，连入同样双酶切的pCIB71质粒中。利用CaCl2法制备感受态，并 用热激法将连接产物转化至大肠杆菌E.coli BL21(购自宝生物工程(大连)有限公司)感受态细 胞中，利用LB培养基中添加氨苄抗生素进行筛选，克隆PCR和测序验证正确后，抽提质粒即得到 pCIB90质粒。
以质粒pCIB90为模板，进一步利用引物TeLDC-SacI-F2(SEQ ID No:38)和TeLDC-SacI-R2(SEQ ID No:39)对pCIB90质粒中Teldc基因的5’序列进行优化，使TeLDC可以顺利在大肠杆菌E.coli BL21 中翻译成蛋白，Teldc基因起始密码子ATG上游的序列由 5’-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTGAGCTC-3’(SEQ ID No:40)替换为 5’-tgtggaattgtgagctcATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACAT-3’(SEQ ID No:41)。PCR扩增后，利用DpnI限制性内切酶对PCR产物进行消化，同样利用热激法转化至E.coli BL21中，测序验证后得到质粒pCIB91。
实施例3构建红色荧光蛋白与嗜热菌来源的赖氨酸脱羧酶进行融合表达的质粒
如下密码子优化及基因合成的方法参考Hoover DM&Lubkowski J,Nucleic AcidsResearch 30:10, 2002。
(1)对来自于珊瑚中的红色荧光蛋白MRFP(mCherry,SEQ ID No:17)，进行密码子优化后， 利用引物拼接的方法合成该基因(mCherry,SEQ ID No:18)。
(2)构建红色荧光蛋(MRFP)-TeLDC融合表达质粒：以拼接的MRFP为模板，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和linker-MRFP-R(SEQ ID No:43)进行扩增，以质粒pCIB91为模 板，利用引物linker-TeLDC-F(SEQ ID No:44)和TeLDC-XbaI-R(SEQ ID No:37)进行扩增，分别对目 的片段切胶回收后，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和TeLDC-XbaI-R(SEQ ID No:37) 进行融合PCR，目的片段纯化后，利用SacI和XbaI双酶切，连接入同样双酶切的质粒pCIB91中，得 到MRFP-TeLDC融合蛋白(SEQ ID No:9)表达质粒pCIB92，其中TeLDC编码基因的5’端通过linker (SGSGSGSGSG)与红色荧光蛋白MRFP的3’相连(SEQ ID No:10)。
(3)构建红色荧光蛋白(MRFP)-TsLDC融合表达质粒：利用引物拼接的方法合成来源于 Tepidanaerobacter syntrophicus的赖氨酸脱羧酶TsLDC，其氨基酸序列如SEQ IDNO:3所示，编码基 因为Tsldc(GenBank ID GAQ24853.1,SEQ ID NO:4)。以拼接的MRFP为模板，利用引物 SacI-MRFP-TeLDC-F(SEQ ID No:42)和linker-MRFP-R(SEQ ID No:43)进行扩增，以拼接的基因Tsldc 为模板，利用引物linker-TsLDC-F(SEQ ID No:45)和TsLDC-XbaI-R(SEQ ID No:46)进行扩增，分别 对目的片段切胶回收后，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和TsLDC-XbaI-R(SEQ ID No:46)进行融合PCR，目的片段纯化后，利用SacI和XbaI双酶切，连接入同样双酶切的质粒pCIB91 中，得到MRFP-TeLDC融合蛋白(SEQ ID No:11)表达质粒pCIB96，其中TsLDC编码基因的5’端通 过linker(SGSGSGSGSG)与红色荧光蛋白MRFP的3’相连(SEQ ID No:12)。
(4)构建红色荧光蛋白(MRFP)-TrLDC融合表达质粒：利用引物拼接的合成来源于Geobacillus kaustophilus的赖氨酸脱羧酶GkLDC，其氨基酸序列如SEQ ID NO:5所示,编码基因为Gkldc (GenBank ID BAD75350.1,SEQ ID NO:6)。以拼接的MRFP为模板，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和linker-MRFP-R(SEQ ID No:43)进行扩增，以拼接的基因 Gkldc为模板，利用引物linker-GkLDC-F(SEQ ID No:47)和GkLDC-XbaI-R(SEQ IDNo:48)进行扩 增，分别对目的片段切胶回收后，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和 GkLDC-XbaI-R(SEQ ID No:48)进行融合PCR，目的片段纯化后，利用SacI和XbaI双酶切，连接入 同样双酶切的质粒pCIB91中，得到MRFP-GkLDC融合蛋白(SEQ ID No:13)表达质粒pCIB97，其中 GkLDC编码基因的5’端通过linker(SGSGSGSGSG)与红色荧光蛋白MRFP的3’相连(SEQ ID No: 14)。
(5)构建红色荧光蛋白(MRFP)-GkLDC融合表达质粒：利用引物拼接的合成来源于Thermomicrobium roseum的赖氨酸脱羧酶TrLDC，其氨基酸序列如SEQ ID NO:7所示，编码基因为 Trldc(GenBank ID ACM05730.1，SEQ ID NO:8)。以拼接的MRFP为模板，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和linker-MRFP-R(SEQ ID No:43)进行扩增，以拼接的基因Trldc 为模板，利用引物linker-TrLDC-F(SEQ ID No:49)和TrLDC-XbaI-R(SEQ IDNo:50)进行扩增，分别 对目的片段切胶回收后，利用引物SacI-MRFP-TeLDC-F(SEQ ID No:42)和TrLDC-XbaI-R(SEQ ID No: 50)进行融合PCR，目的片段纯化后，利用SacI和XbaI双酶切，连接入同样双酶切的质粒pCIB91中， 得到MRFP-TrLDC融合蛋白(SEQ ID No:15)表达质粒pCIB98，其中TrLDC编码基因的5’端通过 linker(SGSGSGSGSG)与红色荧光蛋白MRFP的3’相连(SEQ ID No:16)。
实施例4构建嗜热菌来源的赖氨酸脱羧酶与红色荧光蛋白融合表达的菌株
分别将构建的MRFP-TeLDC，MRFP-TsLDC，MRFP-GkLDC和MRFP-TrLDC融合蛋白表达质 粒pCIB92，pCIB96，pCIB97，pCIB98转化至E.coli BL21(购自宝生物工程(大连)有限公司)感 受态细胞中，涂布于含有终浓度为100μg/ml的氨苄青霉素抗性的LB平板中，并在37℃倒置培养 过夜，得到MRFP-TeLDC，MRFP-TsLDC，MRFP-GkLDC和MRFP-TrLDC融合蛋白表达的菌株 CIB92，CIB96，CIB97和CIB98。分别各挑取3个单克隆接种至5ml的含有终浓度为100μg/ml的氨 苄青霉素抗性的LB液体试管中，37℃，200rpm培养过夜。
实施例5不同温度条件下融合蛋白MRFP-TeLDC、MRFP-TsLDC、MRFP-TrLDC和 MRFP-GkLDC的赖氨酸脱羧酶活性检测
各菌株的菌液OD600经测定未表现出明显的差异，分别取等体积的各菌株的菌液，于不同温度 (37℃、55℃、65℃和75℃)条件下进行Lys-HCl的转化反应，取各菌液600μl，各加入400g/L Lys-HCl (L-赖氨酸盐酸盐)400μl和20mM PLP(磷酸吡哆醛)5μl，反应时间为4h。结果如表2所示，与 表达MRFP-TeLDC融合蛋白的CIB92菌株相比，在37℃反应时赖氨酸转化率无显著差异；55℃反应 时，表达融合蛋白MRFP-TsLDC，MRFP-GkLDC和MRFP-TrLDC的菌株CIB96，CIB97，CIB98的 赖氨酸转化率出现显著提高，CIB96菌株的赖氨酸转化率为83.7％，CIB97菌株的赖氨酸转化率为 89.9％，CIB98菌株的赖氨酸转化率为85.1％，它们的最适温度都在55℃附近。
表2测定表达各赖氨酸脱羧酶的重组细胞的赖氨酸转化率
Figure BDA0002068857380000081
实施例6构建稳定期启动子诱导赖氨酸脱羧酶表达的菌株
以大肠杆菌MG1655K12(E.coli MG1655K12，购自北京天恩泽生物技术有限公司)的基因组 为模板，分别利用引物pcsiE-F(SEQ ID No：51)/pcsiE-R(SEQ ID No：52),pbolA-F(SEQ ID No： 53)/pbolA-R(SEQ ID No：54),posmY-F(SEQ ID No：55)/posmY-R(SEQ ID No：56),pkatE-F(SEQ ID No：57)/pkatE-R(SEQ ID No：58)扩增得到稳定期启动子pcsiE(SEQ ID No：20)，pbolA(SEQ ID No：21)，posmY(SEQ ID No：22)，pkatE(SEQ IDNo：23)，分别利用KpnI和ClaI双酶切后，与 同样双酶切的质粒pCIB92,pCIB96,pCIB97,pCIB98质粒连接，得到pCIB92-101，pCIB92-102， pCIB92-103,pCIB92-104,pCIB96-101，pCIB96-102，pCIB96-103,pCIB96-104,pCIB97-101， pCIB97-102，pCIB97-103,pCIB97-104,pCIB98-101，pCIB98-102，pCIB98-103,pCIB98-104。
使用本领域常用基因序列合成方法分别合成如表3所列的4条启动子(P1，P2，P3和P4) 的双链DNA序列(5’-3’)，并在序列5’和3’端分别连接有KpnI和ClaI的酶切位点，利用KpnI 和ClaI双酶切后，分别连接至KpnI和ClaI双酶切的质粒pCIB92,pCIB96,pCIB97,pCIB98中， 得到含有这4个启动子的质粒(表3)，pCIB92-P1，pCIB96-P1，pCIB97-P1，pCIB98-P1，pCIB92-P2， pCIB96-P2，pCIB97-P2，pCIB98-P2，pCIB92-P3，pCIB96-P3，pCIB97-P3，pCIB98-P3，pCIB92-P4， pCIB96-P4，pCIB97-P4和pCIB98-P4。
表3稳定期启动子和赖氨酸脱羧酶组合及其表达质粒
Figure BDA0002068857380000091
实施例7比较不同启动子诱导赖氨酸脱羧酶表达的菌株的赖氨酸和1,5-戊二胺产量
本实施例中采用可生产L-赖氨酸的大肠杆菌Ela6116(Escherichia coliEla6116)菌株，该菌株现 已保藏于中国典型培养物保藏中心，地址：中国武汉，武汉大学，邮编430072，保藏编号CCTCC No： M2018736，保藏日期2018年11月1日。
分别将质粒pCIB71，pCIB92，pCIB96，pCIB97，pCIB98和表3所列32个质粒转化至大肠 杆菌Ela611b菌株中，分别得到对应的37个重组菌株。以大肠杆菌Ela611b菌株作为对照，分别 挑取三个单克隆至5mL液体培养基中(含有4％葡萄糖,0.1％KH2PO4,0.1％MgSO4,1.6％ (NH4)2SO4,0.001％FeSO4,0.001％MnSO4,0.2％酵母提取物,0.01％L-苏氨酸,0.005％L-异亮氨酸, 10μg/mL四环素,100μg/mL氨苄青霉素)，于37℃培养过夜。次日，每个菌株再分别转接至100ml 新鲜的含有30g/L葡萄糖,0.7％Ca(HCO3)2,10μg/mL四环素和100μg/mL氨苄青霉素，0.1％ KH2PO4，0.1％MgSO4,1.6％(NH4)2SO4,0.001％FeSO4,0.001％MnSO4,0.2％酵母提取物,0.01％L- 苏氨酸,0.005％L-异亮氨酸的培养基中于37℃继续培养68h。培养结束后，取样，利用核磁检测 并计算各培养基中的L-赖氨酸和1,5-戊二胺的含量(表4)，之后对照菌株Ela611b和重组菌株 Ela611b-71继续37℃反应4h；其他的重组菌株升温至55℃，反应4h，利用核磁检测并计算各培 养基中的最终的L-赖氨酸和1,5-戊二胺的含量(表4)。
从表4可以看出，表达CadA的重组菌株Ela611b-71发酵68h后，检测到2.8g/L的L-赖氨酸 和3.6g/L的1,5-戊二胺，并且随着发酵时间的延长，发酵液中检测到的L-赖氨酸和1,5-戊二胺的 量并没有显著增加，最后积累了4.0g/L的1,5-戊二胺，还有2.0g/L的L-赖氨酸残留，这可能是 由于CadA蛋白在赖氨酸生长的温度37℃就有很高的活性，较早地将菌株合成的L-赖氨酸过多的 催化为1,5-戊二胺，1,5-戊二胺在细胞内的积累对细胞产生了毒性，在一定程度上抑制细胞的代谢， 包括L-赖氨酸合成和1,5-戊二胺的转化。
另外，从表4可以看出，组成型表达嗜热菌株来源的赖氨酸脱羧酶的重组菌株Ela611b-92， Ela611b-96，Ela611b-97，Ela611b-98在发酵68h生产了5.9-6.8g/L的L-赖氨酸和1.8-2.2g/L的 1,5-戊二胺，说明这些嗜热菌来源的赖氨酸脱羧酶在37℃时有较低的活性，微弱地将L-赖氨酸转 化为1,5-戊二胺，会在一定程度上解除L-赖氨酸的反馈抑制，同时少量的1,5-戊二胺不会造成细 胞毒性；当温度升高至55℃时，赖氨酸脱羧酶的活性升高，可以将剩余的L-赖氨酸全部转化为 1,5-戊二胺，最后积累了5.1-5.8g/L的1,5-戊二胺，仅有0.02-0.09g/L的L-赖氨酸残留。
而使用稳定期的启动子，理论上只有当菌体在进入稳定期后才开始启动下游基因的转录，如 此可以进一步降低其在赖氨酸生产过程中的1,5-戊二胺的积累，通过测试发现在发酵68h的时候， 使用了稳定期启动子的各重组菌株(菌株编号7～38)与使用组成型启动子的4个重组菌株(菌株 编号3～6)相比，L-赖氨酸水平有进一步的提高，同时降低了转化生成的1,5-戊二胺的产量。当 温度升高至55℃时，红色蛋白与嗜热菌来源的脱羧酶融合表达蛋白的活性升高，继续将剩余的 L-赖氨酸近乎全部转化为1,5-戊二胺，最后积累了5g/L以上的1,5-戊二胺，且几乎没有L-赖氨酸 残留。
表4检测能同时表达赖氨酸合成蛋白及赖氨酸脱羧酶的菌株L-赖氨酸和1,5-戊二胺的水平
Figure BDA0002068857380000101
Figure BDA0002068857380000111
注：n.d.表示未检测到。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上， 可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神 的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 上海凯赛生物技术研发中心有限公司
CIBT美国公司
<120> 一种重组DNA及其应用
<130> KHP181116615.0
<160> 58
<170> SIPOSequenceListing 1.0
<210> 1
<211> 437
<212> PRT
<213> 嗜热菌(Thermosynechoccus elongatus)
<400> 1
Met Glu Pro Leu Leu Arg Ala Leu Trp Gly Thr Ala Leu Glu Gln Asp
1 5 10 15
Leu Ser Glu Leu Pro Gly Leu Asp Asn Leu Ala Gln Pro Thr Gly Val
20 25 30
Leu Ala Glu Ala Gln Ala Val Val Ala Ala Thr Val Gly Ser Asp Arg
35 40 45
Ala Trp Phe Leu Val Asn Gly Ala Thr Gly Gly Leu Leu Ala Ala Leu
50 55 60
Leu Ala Thr Val Gly Pro Gly Asp Arg Val Leu Val Gly Arg Asn Val
65 70 75 80
His Arg Ser Val Ile Ala Gly Leu Val Leu Ala Gly Ala Lys Pro Val
85 90 95
Tyr Leu Gly Val Gly Val Asp Pro Gln Trp Gly Leu Pro Trp Pro Val
100 105 110
Thr Arg Asp Val Val Ala Ala Gly Leu Ala Ala Tyr Pro Asp Thr Lys
115 120 125
Ala Val Val Leu Val Ser Pro Thr Tyr Glu Gly Leu Cys Ser Pro Leu
130 135 140
Leu Glu Ile Ala Gln Cys Val His Asn His Gly Val Pro Leu Ile Val
145 150 155 160
Asp Glu Ala His Gly Ser His Phe Ala Tyr His Pro Ala Phe Pro Val
165 170 175
Thr Ala Leu Ala Ala Gly Ala Asp Val Val Val Gln Ser Trp His Lys
180 185 190
Thr Leu Gly Thr Leu Thr Gln Thr Ala Val Leu His Leu Lys Gly Glu
195 200 205
Arg Val Ser Ala Glu Arg Leu Ser Gln Ala Leu Asn Leu Val Gln Thr
210 215 220
Ser Ser Pro Asn Tyr Trp Leu Leu Ala Ala Leu Glu Gly Ala Gly Val
225 230 235 240
Gln Met Ala Gln Gln Gly Glu Gln Ile Tyr Gly Arg Leu Leu Gln Trp
245 250 255
Val Lys Thr Phe Glu Trp Pro Leu Pro Arg Trp Gln Pro Pro Gly Ile
260 265 270
Pro Gln Asp Pro Leu Arg Leu Thr Leu Gly Thr Trp Pro Ile Gly Leu
275 280 285
Thr Gly Phe Ala Leu Asp Glu Leu Leu Gln Pro Gln Ile Ile Ala Glu
290 295 300
Phe Pro Ser Gly Arg Ser Leu Thr Phe Cys Leu Gly Leu Gly Thr Thr
305 310 315 320
Gln Thr Met Leu Glu Thr Leu Ala Asp Arg Leu Lys Ser Val Tyr Thr
325 330 335
Glu Tyr Cys His Asn Ala Pro Leu Pro Pro Leu Ala Ile Pro Ser Ile
340 345 350
Pro Ser Cys Gln Glu Pro Ala Leu Ser Pro Arg Glu Ala Tyr Phe Cys
355 360 365
Pro Gln Arg Ser Ile Pro Leu Arg Ala Ala Leu Asn Glu Ile Ser Ala
370 375 380
Glu Thr Ile Ala Pro Tyr Pro Pro Gly Ile Pro Thr Val Ile Ala Gly
385 390 395 400
Glu Arg Phe Thr Glu Ser Val Ile Ala Thr Leu Gln Thr Leu Gln Glu
405 410 415
Leu Gly Ala Glu Met Val Gly Ala Ser Asp Pro Thr Leu Gln Thr Leu
420 425 430
Arg Ile Cys Lys Val
435
<210> 2
<211> 1314
<212> DNA
<213> 嗜热菌(Thermosynechoccus elongatus)
<400> 2
atggaaccat tacttcgcgc actgtggggg accgcgctgg aacaggacct tagcgaactt 60
ccgggtcttg acaatttagc gcaaccaacc ggcgtgttag ccgaagcgca agctgtggtc 120
gctgcgacgg tcggctctga tcgtgcgtgg tttctggtga acggcgctac tggcggcctg 180
cttgcggctt tacttgcgac cgtaggtccc ggcgaccggg tgctggttgg ccgtaatgtg 240
catcgtagcg tgattgcggg cttggtactg gctggcgcaa aaccggtgta tcttggcgtc 300
ggcgtcgatc cacaatgggg tctgccgtgg cccgtgaccc gggacgttgt cgcggcaggc 360
ttggctgcgt accccgacac caaggcggtc gtacttgtaa gtcctaccta tgaaggcctg 420
tgctcgccgc tgttagaaat cgcgcagtgc gtgcataatc atggcgtacc gctgattgtc 480
gacgaagcac atggcagtca tttcgcgtat catccggcat ttcctgtgac cgcgttagct 540
gctggggctg acgtcgtcgt tcagtcatgg cacaaaacgt tgggcacgct gacccaaacg 600
gcggtgctgc atctgaaagg cgaacgcgtg tcggcagagc ggctgagcca ggcgttgaat 660
ctggtgcaga cctcgagccc gaactattgg cttctggccg cacttgaagg tgccggggtc 720
cagatggcgc agcagggcga acagatttat ggccggctgc tgcagtgggt aaaaacattt 780
gagtggcctt tgccgcggtg gcagcctcca ggaatccccc aagatcctct gcgtttgacc 840
ctggggacgt ggccgattgg tttaaccgga tttgcactgg atgaactttt acaacctcag 900
ataattgcgg aatttccaag cgggcgtagc ctgacctttt gtctgggtct gggcacaaca 960
cagactatgc tggagacgct tgcagatcgc ctgaagagcg tctataccga atattgccat 1020
aatgcgccct tgcctccgtt ggcgataccg tctattccga gctgtcagga acccgcgctt 1080
tcgccgcgtg aagcgtactt ttgcccgcag cgtagcatac cgcttcgtgc agctcttaat 1140
gaaatctcgg ctgaaaccat tgccccgtac cctcccggca tacctaccgt gatcgctggg 1200
gagcgcttta ccgaaagtgt tattgcgact ctgcaaacgc tgcaggaatt aggtgcggaa 1260
atggtagggg caagcgatcc gaccttacaa accctgcgga tatgtaaagt gtaa 1314
<210> 3
<211> 482
<212> PRT
<213> 嗜热菌(Tepidanaerobacter syntrophicus)
<400> 3
Met Glu Lys Gln Glu Ile Asn Lys Phe Ser Lys Thr Pro Leu Ile Gln
1 5 10 15
Ala Leu Lys Glu Tyr Glu Lys Lys Asp Ser Leu Arg Phe His Met Pro
20 25 30
Gly His Lys Gly Arg Cys Pro Lys Gly Val Phe Cys Asp Ile Lys Glu
35 40 45
Asn Leu Phe Gly Trp Asp Val Thr Glu Ile Pro Gly Leu Asp Asp Phe
50 55 60
Ala Gln Pro Glu Gly Pro Ile Lys Glu Ala Gln Glu Lys Leu Ser Ala
65 70 75 80
Leu Tyr Gly Ala Asp Thr Ser Tyr Phe Leu Val Asn Gly Ala Thr Ser
85 90 95
Gly Ile Ile Ser Met Met Ala Gly Ala Leu Ser Glu Lys Asp Lys Ile
100 105 110
Leu Ile Pro Arg Thr Ser His Lys Ser Val Leu Ser Gly Leu Ile Leu
115 120 125
Thr Gly Ala Ser Ala Ala Tyr Ile Met Pro Glu Arg Cys Glu Glu Leu
130 135 140
Gly Val Tyr Ala Gln Val Glu Pro Cys Ala Ile Thr Asn Lys Leu Ile
145 150 155 160
Glu Asn Pro Asp Ile Lys Ala Ile Leu Val Thr Asn Pro Val Tyr Gln
165 170 175
Gly Phe Cys Pro Asp Ile Ala Arg Val Ala Glu Ile Ala Lys Glu Arg
180 185 190
Gly Thr Thr Leu Leu Ala Asp Glu Ala Gln Gly Pro His Phe Gly Phe
195 200 205
Ser Lys Lys Val Pro Gln Ser Ala Gly Lys Phe Ala Asp Ala Trp Val
210 215 220
Gln Ser Pro His Lys Met Leu Thr Ser Leu Thr Gln Ser Ala Trp Leu
225 230 235 240
His Ile Lys Gly Asn Arg Ile Asp Lys Glu Arg Leu Glu Asp Phe Leu
245 250 255
His Ile Val Thr Thr Ser Ser Pro Ser Tyr Ile Leu Met Ala Ser Leu
260 265 270
Asp Gly Thr Arg Glu Leu Ile Glu Glu Asn Gly Asn Ser Tyr Ile Glu
275 280 285
Lys Ala Val Glu Leu Ala Gln Lys Ala Arg Tyr Glu Ile Asn Asn Ser
290 295 300
Thr Val Phe Tyr Ala Pro Gly Gln Glu Ile Leu Gly Lys Tyr Gly Ile
305 310 315 320
Ser Ser Gln Asp Pro Leu His Leu Met Val Asn Val Ser Cys Ala Gly
325 330 335
Tyr Thr Gly Tyr Asp Ile Glu Lys Ala Leu Arg Glu Asp Phe Ser Ile
340 345 350
Tyr Ala Glu Tyr Ala Asp Leu Cys Asn Val Tyr Phe Leu Ile Thr Phe
355 360 365
Ser Asn Thr Leu Glu Asp Ile Lys Gly Leu Leu Ala Val Leu Ser His
370 375 380
Phe Lys Pro Leu Lys Asn Lys Val Lys Pro Cys Phe Trp Ile Lys Asp
385 390 395 400
Leu Pro Lys Val Ala Leu Glu Pro Lys Lys Ala Phe Lys Leu Pro Ala
405 410 415
Lys Ser Val Pro Phe Lys Asp Ser Ala Gly Ser Val Ser Lys Arg Pro
420 425 430
Leu Val Pro Tyr Pro Pro Gly Ala Pro Leu Val Met Pro Gly Glu Ile
435 440 445
Ile Glu Lys Glu His Ile Glu Met Ile Asn Glu Ile Leu Asn Ser Gly
450 455 460
Gly Tyr Cys Gln Gly Val Thr Ser Glu Lys Phe Ile Gln Val Val Thr
465 470 475 480
Asp Phe
<210> 4
<211> 1449
<212> DNA
<213> 嗜热菌(Tepidanaerobacter syntrophicus)
<400> 4
atggagaagc aagagattaa caagttctct aagaccccgc tcatccaagc gctgaaagaa 60
tacgagaaaa aggattctct gcgtttccac atgccaggtc acaaaggccg ttgtccaaaa 120
ggtgtttttt gcgatattaa ggagaacctg ttcggttggg atgttaccga aatcccgggt 180
ctggatgact tcgctcaacc ggaaggtccg atcaaggaag cacaggagaa actgtctgcg 240
ctgtacggtg ccgacacctc ctatttcctc gttaatggtg caacctctgg tatcatttct 300
atgatggcgg gtgctctgtc cgaaaaggac aaaatcctga tcccgcgtac cagccataag 360
agcgtactct ctggtctgat tctcactggc gcctctgcgg cgtacatcat gccggagcgt 420
tgcgaagagc tgggtgttta cgcacaggtg gaaccttgtg ccatcaccaa caaactgatc 480
gagaacccgg atatcaaagc gattctggtt accaacccag tgtaccaggg tttctgcccg 540
gacatcgcgc gtgttgcgga aatcgcgaaa gaacgcggta ccaccctgct cgcagacgaa 600
gcgcaaggcc cacatttcgg cttttccaag aaagttccgc agtctgcggg taagttcgcg 660
gatgcgtggg ttcagtcccc tcacaaaatg ctgacgagcc tgacccaatc tgcgtggctg 720
cacatcaagg gcaatcgtat cgacaaggaa cgtctggaag actttctcca catcgttacc 780
acctcttctc cgtcttacat cctcatggcg tctctggacg gtacccgcga gctgattgaa 840
gaaaacggta actcctacat tgaaaaggcg gttgaactgg ctcagaaagc gcgttatgaa 900
atcaacaact ctactgtttt ctacgcgcca ggccaggaga ttctcggtaa atacggtatt 960
tcttctcagg acccgctgca tctgatggtt aatgtttctt gcgcgggtta cacgggctac 1020
gacatcgaaa aagccctgcg tgaggacttt tctatctacg ccgaatacgc ggacctgtgt 1080
aacgtttact tcctcattac gtttagcaat accctggagg acattaaagg tctcctcgcg 1140
gttctgtctc acttcaaacc gctcaaaaac aaagttaaac cgtgcttctg gatcaaagac 1200
ctgccgaaag ttgcgctgga gccaaagaag gcgttcaaac tgccggcgaa atctgtgcct 1260
ttcaaagatt ctgctggtag cgtttctaaa cgcccgctgg ttccgtatcc gccaggtgcg 1320
ccactcgtga tgccgggtga gatcattgag aaagagcaca tcgagatgat taatgaaatt 1380
ctcaactctg gcggctactg ccagggtgtt acgtctgaaa agttcattca ggttgtaacc 1440
gatttctaa 1449
<210> 5
<211> 490
<212> PRT
<213> 嗜热菌(Geobacillus kaustophilus)
<400> 5
Met Ser Gln Leu Glu Thr Pro Leu Phe Thr Gly Leu Leu Glu His Met
1 5 10 15
Lys Lys Asn Pro Val Gln Phe His Ile Pro Gly His Lys Lys Gly Ala
20 25 30
Gly Met Asp Pro Glu Phe Arg Ala Phe Ile Gly Asp Asn Ala Leu Ala
35 40 45
Ile Asp Leu Ile Asn Ile Ser Pro Leu Asp Asp Leu His His Pro Lys
50 55 60
Gly Met Ile Lys Arg Ala Gln Glu Leu Ala Ala Glu Ala Phe Gly Ala
65 70 75 80
Asp Tyr Thr Phe Phe Ser Val Gln Gly Thr Ser Gly Ala Ile Met Thr
85 90 95
Met Val Met Ser Val Ala Gly Pro Gly Asp Lys Ile Ile Val Pro Arg
100 105 110
Asn Val His Lys Ser Val Met Ser Ala Ile Val Phe Ser Gly Ala Thr
115 120 125
Pro Ile Phe Ile His Pro Glu Ile Asp Lys Glu Leu Gly Ile Ser His
130 135 140
Gly Ile Thr Pro Gln Ala Val Glu Lys Ala Leu Arg Gln His Pro Asp
145 150 155 160
Ala Lys Gly Val Leu Val Ile Asn Pro Thr Tyr Phe Gly Ile Ala Gly
165 170 175
Asp Leu Lys Lys Ile Val Asp Ile Ala His Ser Tyr Asn Val Pro Val
180 185 190
Leu Val Asp Glu Ala His Gly Val His Ile His Phe His Glu Asp Leu
195 200 205
Pro Leu Ser Ala Met Gln Ala Gly Ala Asp Met Ala Ala Thr Ser Val
210 215 220
His Lys Leu Gly Gly Ser Leu Thr Gln Ser Ser Ile Leu Asn Val Arg
225 230 235 240
Glu Gly Leu Val Ser Ala Lys His Val Gln Ala Ile Leu Ser Met Leu
245 250 255
Thr Thr Thr Ser Thr Ser Tyr Leu Leu Leu Ala Ser Leu Asp Val Ala
260 265 270
Arg Lys Gln Leu Ala Thr Lys Gly Arg Glu Leu Ile Asp Lys Ala Ile
275 280 285
Arg Leu Ala Asp Trp Thr Arg Arg Gln Ile Asn Glu Ile Pro Tyr Leu
290 295 300
Tyr Cys Val Gly Glu Glu Ile Leu Gly Thr Glu Ala Thr Tyr Asp Tyr
305 310 315 320
Asp Pro Thr Lys Leu Ile Ile Ser Val Lys Glu Leu Gly Leu Thr Gly
325 330 335
His Asp Val Glu Arg Trp Leu Arg Glu Thr Tyr Asn Ile Glu Val Glu
340 345 350
Leu Ser Asp Leu Tyr Asn Ile Leu Cys Ile Ile Thr Pro Gly Asp Thr
355 360 365
Glu Arg Glu Ala Ser Leu Leu Val Glu Ala Leu Arg Arg Leu Ser Lys
370 375 380
Gln Phe Ser His Gln Ala Glu Lys Gly Ile Lys Pro Lys Val Leu Leu
385 390 395 400
Pro Asp Ile Pro Ala Leu Ala Leu Thr Pro Arg Asp Ala Phe Tyr Ala
405 410 415
Glu Thr Glu Val Val Pro Phe His Glu Ser Ala Gly Arg Ile Ile Ala
420 425 430
Glu Phe Val Met Val Tyr Pro Pro Gly Ile Pro Ile Phe Ile Pro Gly
435 440 445
Glu Ile Ile Thr Glu Glu Asn Leu Lys Tyr Ile Glu Thr Asn Leu Ala
450 455 460
Ala Gly Leu Pro Val Gln Gly Pro Glu Asp Asp Thr Leu Gln Thr Leu
465 470 475 480
Arg Val Ile Lys Glu Tyr Lys Pro Ile Arg
485 490
<210> 6
<211> 1473
<212> DNA
<213> 嗜热菌(Geobacillus kaustophilus)
<400> 6
atgtctcagc tcgagacccc tctgttcacc ggtctgctcg aacacatgaa gaaaaacccg 60
gtccagtttc acattccagg tcacaagaaa ggtgctggta tggaccctga gttccgtgcg 120
tttatcggtg ataacgcgct cgcgatcgac ctgatcaaca tctcccctct cgacgacctc 180
caccacccga aaggcatgat caaacgtgcg caggaactgg ctgcggaagc gtttggcgcg 240
gactacacgt tcttcagcgt tcaaggcacc agcggtgcca tcatgacgat ggtaatgtct 300
gttgcgggtc cgggcgataa gatcatcgtc cctcgtaacg ttcacaaatc tgttatgtct 360
gccatcgttt tctctggcgc gacccctatt ttcatccacc cggaaatcga taaggagctg 420
ggtattagcc acggtattac cccgcaggcc gtggagaaag ccctgcgtca acaccctgat 480
gctaaaggcg ttctggtaat caacccgact tatttcggta tcgcgggtga cctcaaaaag 540
atcgttgaca tcgcgcactc ttataatgtg ccggtcctgg tagatgaagc gcacggtgtt 600
catattcact tccacgagga cctcccactc agcgcaatgc aggcgggtgc ggatatggcg 660
gcgacgtccg tgcacaagct gggcggtagc ctgactcagt cttccattct gaacgtacgc 720
gaaggtctgg tttctgctaa acacgtgcaa gcgattctct ctatgctgac caccacttct 780
acctcttatc tgctgctggc ttccctggac gtagcgcgta aacagctggc aaccaaaggt 840
cgtgaactca tcgacaaagc catccgcctc gcggattgga cccgtcgcca gattaacgag 900
atcccgtacc tctactgcgt gggtgaagag atcctgggta ccgaagcaac ctacgactac 960
gatccgacta aactgatcat cagcgtaaaa gaactcggtc tcactggcca tgacgttgag 1020
cgttggctcc gtgaaaccta caatatcgaa gttgaactgt ctgacctcta taacatcctc 1080
tgcatcatca ccccgggtga tactgagcgc gaagcgtctc tcctggtgga agcactgcgc 1140
cgtctgtcta aacaattctc ccatcaggcc gaaaagggta tcaaacctaa ggttctcctg 1200
ccggatattc ctgccctcgc cctgacgcct cgtgacgcgt tctatgcgga aaccgaagtc 1260
gttccgttcc atgagtccgc cggtcgtatc atcgcggagt ttgtaatggt ttacccaccg 1320
ggcatcccaa tcttcatccc tggcgagatt atcactgagg aaaacctgaa atacatcgaa 1380
accaacctgg cggctggcct cccggttcag ggcccagaag acgacacgct gcagaccctc 1440
cgtgtcatta aagaatacaa accaattcgt taa 1473
<210> 7
<211> 495
<212> PRT
<213> Thermomicrobium roseum
<400> 7
Met Ser Glu Glu Gln Gln Arg Ala Pro Tyr Leu Glu Gln Trp Leu Ala
1 5 10 15
Tyr Val Asp Glu Cys Val Ile Pro Phe Thr Thr Pro Gly His Lys Gln
20 25 30
Gly Arg Gly Ala Pro Pro Glu Phe Val Ala Ala Phe Gly Glu Arg Ala
35 40 45
Leu Ala Leu Asp Ile Pro His Asp Gly Gly Thr Phe Asp Ala His Leu
50 55 60
Glu His Asp Pro Leu Val Ala Ala Glu Arg Leu Ala Ala Ala Leu Trp
65 70 75 80
Gly Ala Arg Asp Ala Val Phe Leu Val Asn Gly Ser Thr Thr Gly Asn
85 90 95
Leu Ala Ala Leu Leu Thr Leu Gly Arg Pro Gly Gln Pro Ile Val Val
100 105 110
Thr Arg Ala Met His Lys Ser Leu Leu Ala Gly Leu Val Leu Ser Gly
115 120 125
Ala Arg Pro Val Tyr Val Val Pro Ala Val His Pro Glu Ser Gly Ile
130 135 140
Leu Leu Asp Leu Pro Pro Glu Ser Val Ala Gln Ala Leu Ala Ala Trp
145 150 155 160
Pro Asp Ala Thr Ala Val Ala Leu Val Ser Pro Thr Tyr Thr Gly Val
165 170 175
Thr Ser Asp Thr Ala Glu Leu Ala Ala Leu Cys His Ala His Gly Val
180 185 190
Pro Leu Phe Val Asp Glu Ala Trp Gly Pro His Leu Pro Phe His Pro
195 200 205
Ala Leu Pro Ala Ala Ala Ile Pro Ser Gly Ala Asp Leu Ala Val Thr
210 215 220
Ser Leu His Lys Leu Ala Gly Ser Leu Thr Gln Thr Ala Leu Leu Leu
225 230 235 240
Met Ala Gly Asn Leu Val Asp Gln Ala Gln Leu Arg Ala Ala Thr Ala
245 250 255
Met Val Gln Thr Thr Ser Pro Ala Ala Phe Leu Tyr Ala Ser Leu Asp
260 265 270
Ala Ala Arg Arg Arg Leu Ala Leu Glu Gly Glu Gln Leu Leu Ala Arg
275 280 285
Thr Leu Glu Leu Ala Glu His Ala Arg Arg Glu Leu Ala Ala Ile Pro
290 295 300
Gly Leu Glu Val Val Gly Pro Glu Ile Val Ala Gly Arg Pro Gly Ala
305 310 315 320
Gly Phe Asp Arg Thr Arg Leu Val Val Asp Val Gln Gly Phe Gly Leu
325 330 335
Thr Gly Leu Glu Val Lys Arg Ile Leu Arg Arg Asp Phe Arg Ile Ala
340 345 350
Ala Glu Met Ala Asp Leu Val Ser Val Val Phe Leu Ile Thr Ile Gly
355 360 365
Asp Thr Pro Glu Thr Ile Ala Ala Leu Val Ala Ala Phe Arg Ala Leu
370 375 380
Ala Ala Asp Arg Thr Arg Pro Asp Cys Ala Ala Gly Arg Arg Ala Val
385 390 395 400
Arg Ala Leu Leu Arg Ser Thr Gly Pro Ile Val Ala Gly Ala Pro Gln
405 410 415
Ala Met Thr Pro Arg Glu Ala Phe Phe Ala Pro Ala Glu Arg Val Pro
420 425 430
Leu Ala Asp Ala Val Gly Arg Val Ala Ala Glu Pro Val Thr Pro Tyr
435 440 445
Pro Pro Gly Ile Pro Val Leu Ala Pro Gly Glu Val Val Arg Pro Glu
450 455 460
Val Val Glu Phe Leu Gln Ala Gly Arg Ala Ala Gly Met Arg Phe Asn
465 470 475 480
Gly Ala Ser Asp Pro Thr Leu Ala Thr Leu Arg Val Val Arg Ala
485 490 495
<210> 8
<211> 1488
<212> DNA
<213> Thermomicrobium roseum
<400> 8
atgtctgaag aacagcaacg tgctccgtac ctggagcaat ggctggcgta cgttgacgag 60
tgcgttatcc cgtttaccac tccgggtcac aaacaaggtc gcggtgcgcc accggagttc 120
gttgcggcgt tcggtgaacg tgcgctcgct ctggacattc cgcatgacgg tggcaccttt 180
gacgcgcatc tggaacatga cccgctcgtt gccgccgaac gtctggctgc cgcactgtgg 240
ggtgcacgcg atgcggtgtt tctggttaac ggttccacca ctggtaacct ggcggctctg 300
ctcactctcg gtcgcccagg tcagccgatt gttgttactc gtgccatgca taagagcctg 360
ctggcaggtc tggtcctgag cggtgctcgc cctgtctacg ttgtaccggc cgtacaccca 420
gaatccggta tcctcctcga tctccctccg gaatctgttg cgcaggcgct ggccgcgtgg 480
cctgatgcga cggctgtagc tctggtgtcc ccgacctaca ctggcgttac ctctgacact 540
gctgaactgg cagccctctg tcacgctcat ggtgttccac tgtttgttga tgaagcgtgg 600
ggtccgcacc tcccgttcca tccagcactc ccagcagcag ctattccgtc tggtgccgat 660
ctggcggtta cttctctgca caaactggcg ggttccctca cccaaaccgc tctcctcctg 720
atggcaggca acctcgtaga ccaagcccag ctgcgtgcag ccacggcaat ggtgcaaacc 780
accagccctg cagccttcct gtacgcgtcc ctggatgctg cccgtcgccg tctcgcgctc 840
gaaggtgaac agctcctcgc acgtactctc gagctggctg agcacgctcg ccgtgaactc 900
gccgccatcc cgggtctgga ggtggtcggt ccagaaattg ttgcgggtcg tccgggtgcc 960
ggcttcgatc gtactcgcct cgttgttgac gttcagggtt tcggtctgac tggcctcgaa 1020
gtaaagcgta tcctgcgtcg tgacttccgt attgcagctg aaatggcaga tctcgtctct 1080
gttgttttcc tcatcaccat cggtgacacc ccagagacca tcgctgccct ggtagcagct 1140
ttccgtgcac tcgctgctga ccgtacccgt ccagactgtg ctgccggtcg tcgtgcagta 1200
cgcgccctcc tccgttctac cggtccgatc gtcgcgggtg ctcctcaggc gatgaccccg 1260
cgtgaagctt tcttcgctcc agctgagcgc gttccgctcg cggatgccgt cggtcgtgtt 1320
gcagccgagc cggttacccc atatccgcct ggtattccgg tactggcccc aggtgaagtg 1380
gttcgcccgg aggtagttga attcctccag gcaggccgtg ccgctggtat gcgtttcaat 1440
ggcgcgtctg acccgactct ggcgaccctc cgtgtcgttc gtgcctaa 1488
<210> 9
<211> 683
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Glu Pro Leu Leu Arg Ala Leu Trp Gly
245 250 255
Thr Ala Leu Glu Gln Asp Leu Ser Glu Leu Pro Gly Leu Asp Asn Leu
260 265 270
Ala Gln Pro Thr Gly Val Leu Ala Glu Ala Gln Ala Val Val Ala Ala
275 280 285
Thr Val Gly Ser Asp Arg Ala Trp Phe Leu Val Asn Gly Ala Thr Gly
290 295 300
Gly Leu Leu Ala Ala Leu Leu Ala Thr Val Gly Pro Gly Asp Arg Val
305 310 315 320
Leu Val Gly Arg Asn Val His Arg Ser Val Ile Ala Gly Leu Val Leu
325 330 335
Ala Gly Ala Lys Pro Val Tyr Leu Gly Val Gly Val Asp Pro Gln Trp
340 345 350
Gly Leu Pro Trp Pro Val Thr Arg Asp Val Val Ala Ala Gly Leu Ala
355 360 365
Ala Tyr Pro Asp Thr Lys Ala Val Val Leu Val Ser Pro Thr Tyr Glu
370 375 380
Gly Leu Cys Ser Pro Leu Leu Glu Ile Ala Gln Cys Val His Asn His
385 390 395 400
Gly Val Pro Leu Ile Val Asp Glu Ala His Gly Ser His Phe Ala Tyr
405 410 415
His Pro Ala Phe Pro Val Thr Ala Leu Ala Ala Gly Ala Asp Val Val
420 425 430
Val Gln Ser Trp His Lys Thr Leu Gly Thr Leu Thr Gln Thr Ala Val
435 440 445
Leu His Leu Lys Gly Glu Arg Val Ser Ala Glu Arg Leu Ser Gln Ala
450 455 460
Leu Asn Leu Val Gln Thr Ser Ser Pro Asn Tyr Trp Leu Leu Ala Ala
465 470 475 480
Leu Glu Gly Ala Gly Val Gln Met Ala Gln Gln Gly Glu Gln Ile Tyr
485 490 495
Gly Arg Leu Leu Gln Trp Val Lys Thr Phe Glu Trp Pro Leu Pro Arg
500 505 510
Trp Gln Pro Pro Gly Ile Pro Gln Asp Pro Leu Arg Leu Thr Leu Gly
515 520 525
Thr Trp Pro Ile Gly Leu Thr Gly Phe Ala Leu Asp Glu Leu Leu Gln
530 535 540
Pro Gln Ile Ile Ala Glu Phe Pro Ser Gly Arg Ser Leu Thr Phe Cys
545 550 555 560
Leu Gly Leu Gly Thr Thr Gln Thr Met Leu Glu Thr Leu Ala Asp Arg
565 570 575
Leu Lys Ser Val Tyr Thr Glu Tyr Cys His Asn Ala Pro Leu Pro Pro
580 585 590
Leu Ala Ile Pro Ser Ile Pro Ser Cys Gln Glu Pro Ala Leu Ser Pro
595 600 605
Arg Glu Ala Tyr Phe Cys Pro Gln Arg Ser Ile Pro Leu Arg Ala Ala
610 615 620
Leu Asn Glu Ile Ser Ala Glu Thr Ile Ala Pro Tyr Pro Pro Gly Ile
625 630 635 640
Pro Thr Val Ile Ala Gly Glu Arg Phe Thr Glu Ser Val Ile Ala Thr
645 650 655
Leu Gln Thr Leu Gln Glu Leu Gly Ala Glu Met Val Gly Ala Ser Asp
660 665 670
Pro Thr Leu Gln Thr Leu Arg Ile Cys Lys Val
675 680
<210> 10
<211> 2052
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat ggaaccatta cttcgcgcac tgtgggggac cgcgctggaa 780
caggacctta gcgaacttcc gggtcttgac aatttagcgc aaccaaccgg cgtgttagcc 840
gaagcgcaag ctgtggtcgc tgcgacggtc ggctctgatc gtgcgtggtt tctggtgaac 900
ggcgctactg gcggcctgct tgcggcttta cttgcgaccg taggtcccgg cgaccgggtg 960
ctggttggcc gtaatgtgca tcgtagcgtg attgcgggct tggtactggc tggcgcaaaa 1020
ccggtgtatc ttggcgtcgg cgtcgatcca caatggggtc tgccgtggcc cgtgacccgg 1080
gacgttgtcg cggcaggctt ggctgcgtac cccgacacca aggcggtcgt acttgtaagt 1140
cctacctatg aaggcctgtg ctcgccgctg ttagaaatcg cgcagtgcgt gcataatcat 1200
ggcgtaccgc tgattgtcga cgaagcacat ggcagtcatt tcgcgtatca tccggcattt 1260
cctgtgaccg cgttagctgc tggggctgac gtcgtcgttc agtcatggca caaaacgttg 1320
ggcacgctga cccaaacggc ggtgctgcat ctgaaaggcg aacgcgtgtc ggcagagcgg 1380
ctgagccagg cgttgaatct ggtgcagacc tcgagcccga actattggct tctggccgca 1440
cttgaaggtg ccggggtcca gatggcgcag cagggcgaac agatttatgg ccggctgctg 1500
cagtgggtaa aaacatttga gtggcctttg ccgcggtggc agcctccagg aatcccccaa 1560
gatcctctgc gtttgaccct ggggacgtgg ccgattggtt taaccggatt tgcactggat 1620
gaacttttac aacctcagat aattgcggaa tttccaagcg ggcgtagcct gaccttttgt 1680
ctgggtctgg gcacaacaca gactatgctg gagacgcttg cagatcgcct gaagagcgtc 1740
tataccgaat attgccataa tgcgcccttg cctccgttgg cgataccgtc tattccgagc 1800
tgtcaggaac ccgcgctttc gccgcgtgaa gcgtactttt gcccgcagcg tagcataccg 1860
cttcgtgcag ctcttaatga aatctcggct gaaaccattg ccccgtaccc tcccggcata 1920
cctaccgtga tcgctgggga gcgctttacc gaaagtgtta ttgcgactct gcaaacgctg 1980
caggaattag gtgcggaaat ggtaggggca agcgatccga ccttacaaac cctgcggata 2040
tgtaaagtgt aa 2052
<210> 11
<211> 728
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Glu Lys Gln Glu Ile Asn Lys Phe Ser
245 250 255
Lys Thr Pro Leu Ile Gln Ala Leu Lys Glu Tyr Glu Lys Lys Asp Ser
260 265 270
Leu Arg Phe His Met Pro Gly His Lys Gly Arg Cys Pro Lys Gly Val
275 280 285
Phe Cys Asp Ile Lys Glu Asn Leu Phe Gly Trp Asp Val Thr Glu Ile
290 295 300
Pro Gly Leu Asp Asp Phe Ala Gln Pro Glu Gly Pro Ile Lys Glu Ala
305 310 315 320
Gln Glu Lys Leu Ser Ala Leu Tyr Gly Ala Asp Thr Ser Tyr Phe Leu
325 330 335
Val Asn Gly Ala Thr Ser Gly Ile Ile Ser Met Met Ala Gly Ala Leu
340 345 350
Ser Glu Lys Asp Lys Ile Leu Ile Pro Arg Thr Ser His Lys Ser Val
355 360 365
Leu Ser Gly Leu Ile Leu Thr Gly Ala Ser Ala Ala Tyr Ile Met Pro
370 375 380
Glu Arg Cys Glu Glu Leu Gly Val Tyr Ala Gln Val Glu Pro Cys Ala
385 390 395 400
Ile Thr Asn Lys Leu Ile Glu Asn Pro Asp Ile Lys Ala Ile Leu Val
405 410 415
Thr Asn Pro Val Tyr Gln Gly Phe Cys Pro Asp Ile Ala Arg Val Ala
420 425 430
Glu Ile Ala Lys Glu Arg Gly Thr Thr Leu Leu Ala Asp Glu Ala Gln
435 440 445
Gly Pro His Phe Gly Phe Ser Lys Lys Val Pro Gln Ser Ala Gly Lys
450 455 460
Phe Ala Asp Ala Trp Val Gln Ser Pro His Lys Met Leu Thr Ser Leu
465 470 475 480
Thr Gln Ser Ala Trp Leu His Ile Lys Gly Asn Arg Ile Asp Lys Glu
485 490 495
Arg Leu Glu Asp Phe Leu His Ile Val Thr Thr Ser Ser Pro Ser Tyr
500 505 510
Ile Leu Met Ala Ser Leu Asp Gly Thr Arg Glu Leu Ile Glu Glu Asn
515 520 525
Gly Asn Ser Tyr Ile Glu Lys Ala Val Glu Leu Ala Gln Lys Ala Arg
530 535 540
Tyr Glu Ile Asn Asn Ser Thr Val Phe Tyr Ala Pro Gly Gln Glu Ile
545 550 555 560
Leu Gly Lys Tyr Gly Ile Ser Ser Gln Asp Pro Leu His Leu Met Val
565 570 575
Asn Val Ser Cys Ala Gly Tyr Thr Gly Tyr Asp Ile Glu Lys Ala Leu
580 585 590
Arg Glu Asp Phe Ser Ile Tyr Ala Glu Tyr Ala Asp Leu Cys Asn Val
595 600 605
Tyr Phe Leu Ile Thr Phe Ser Asn Thr Leu Glu Asp Ile Lys Gly Leu
610 615 620
Leu Ala Val Leu Ser His Phe Lys Pro Leu Lys Asn Lys Val Lys Pro
625 630 635 640
Cys Phe Trp Ile Lys Asp Leu Pro Lys Val Ala Leu Glu Pro Lys Lys
645 650 655
Ala Phe Lys Leu Pro Ala Lys Ser Val Pro Phe Lys Asp Ser Ala Gly
660 665 670
Ser Val Ser Lys Arg Pro Leu Val Pro Tyr Pro Pro Gly Ala Pro Leu
675 680 685
Val Met Pro Gly Glu Ile Ile Glu Lys Glu His Ile Glu Met Ile Asn
690 695 700
Glu Ile Leu Asn Ser Gly Gly Tyr Cys Gln Gly Val Thr Ser Glu Lys
705 710 715 720
Phe Ile Gln Val Val Thr Asp Phe
725
<210> 12
<211> 2187
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat ggagaagcaa gagattaaca agttctctaa gaccccgctc 780
atccaagcgc tgaaagaata cgagaaaaag gattctctgc gtttccacat gccaggtcac 840
aaaggccgtt gtccaaaagg tgttttttgc gatattaagg agaacctgtt cggttgggat 900
gttaccgaaa tcccgggtct ggatgacttc gctcaaccgg aaggtccgat caaggaagca 960
caggagaaac tgtctgcgct gtacggtgcc gacacctcct atttcctcgt taatggtgca 1020
acctctggta tcatttctat gatggcgggt gctctgtccg aaaaggacaa aatcctgatc 1080
ccgcgtacca gccataagag cgtactctct ggtctgattc tcactggcgc ctctgcggcg 1140
tacatcatgc cggagcgttg cgaagagctg ggtgtttacg cacaggtgga accttgtgcc 1200
atcaccaaca aactgatcga gaacccggat atcaaagcga ttctggttac caacccagtg 1260
taccagggtt tctgcccgga catcgcgcgt gttgcggaaa tcgcgaaaga acgcggtacc 1320
accctgctcg cagacgaagc gcaaggccca catttcggct tttccaagaa agttccgcag 1380
tctgcgggta agttcgcgga tgcgtgggtt cagtcccctc acaaaatgct gacgagcctg 1440
acccaatctg cgtggctgca catcaagggc aatcgtatcg acaaggaacg tctggaagac 1500
tttctccaca tcgttaccac ctcttctccg tcttacatcc tcatggcgtc tctggacggt 1560
acccgcgagc tgattgaaga aaacggtaac tcctacattg aaaaggcggt tgaactggct 1620
cagaaagcgc gttatgaaat caacaactct actgttttct acgcgccagg ccaggagatt 1680
ctcggtaaat acggtatttc ttctcaggac ccgctgcatc tgatggttaa tgtttcttgc 1740
gcgggttaca cgggctacga catcgaaaaa gccctgcgtg aggacttttc tatctacgcc 1800
gaatacgcgg acctgtgtaa cgtttacttc ctcattacgt ttagcaatac cctggaggac 1860
attaaaggtc tcctcgcggt tctgtctcac ttcaaaccgc tcaaaaacaa agttaaaccg 1920
tgcttctgga tcaaagacct gccgaaagtt gcgctggagc caaagaaggc gttcaaactg 1980
ccggcgaaat ctgtgccttt caaagattct gctggtagcg tttctaaacg cccgctggtt 2040
ccgtatccgc caggtgcgcc actcgtgatg ccgggtgaga tcattgagaa agagcacatc 2100
gagatgatta atgaaattct caactctggc ggctactgcc agggtgttac gtctgaaaag 2160
ttcattcagg ttgtaaccga tttctaa 2187
<210> 13
<211> 736
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Ser Gln Leu Glu Thr Pro Leu Phe Thr
245 250 255
Gly Leu Leu Glu His Met Lys Lys Asn Pro Val Gln Phe His Ile Pro
260 265 270
Gly His Lys Lys Gly Ala Gly Met Asp Pro Glu Phe Arg Ala Phe Ile
275 280 285
Gly Asp Asn Ala Leu Ala Ile Asp Leu Ile Asn Ile Ser Pro Leu Asp
290 295 300
Asp Leu His His Pro Lys Gly Met Ile Lys Arg Ala Gln Glu Leu Ala
305 310 315 320
Ala Glu Ala Phe Gly Ala Asp Tyr Thr Phe Phe Ser Val Gln Gly Thr
325 330 335
Ser Gly Ala Ile Met Thr Met Val Met Ser Val Ala Gly Pro Gly Asp
340 345 350
Lys Ile Ile Val Pro Arg Asn Val His Lys Ser Val Met Ser Ala Ile
355 360 365
Val Phe Ser Gly Ala Thr Pro Ile Phe Ile His Pro Glu Ile Asp Lys
370 375 380
Glu Leu Gly Ile Ser His Gly Ile Thr Pro Gln Ala Val Glu Lys Ala
385 390 395 400
Leu Arg Gln His Pro Asp Ala Lys Gly Val Leu Val Ile Asn Pro Thr
405 410 415
Tyr Phe Gly Ile Ala Gly Asp Leu Lys Lys Ile Val Asp Ile Ala His
420 425 430
Ser Tyr Asn Val Pro Val Leu Val Asp Glu Ala His Gly Val His Ile
435 440 445
His Phe His Glu Asp Leu Pro Leu Ser Ala Met Gln Ala Gly Ala Asp
450 455 460
Met Ala Ala Thr Ser Val His Lys Leu Gly Gly Ser Leu Thr Gln Ser
465 470 475 480
Ser Ile Leu Asn Val Arg Glu Gly Leu Val Ser Ala Lys His Val Gln
485 490 495
Ala Ile Leu Ser Met Leu Thr Thr Thr Ser Thr Ser Tyr Leu Leu Leu
500 505 510
Ala Ser Leu Asp Val Ala Arg Lys Gln Leu Ala Thr Lys Gly Arg Glu
515 520 525
Leu Ile Asp Lys Ala Ile Arg Leu Ala Asp Trp Thr Arg Arg Gln Ile
530 535 540
Asn Glu Ile Pro Tyr Leu Tyr Cys Val Gly Glu Glu Ile Leu Gly Thr
545 550 555 560
Glu Ala Thr Tyr Asp Tyr Asp Pro Thr Lys Leu Ile Ile Ser Val Lys
565 570 575
Glu Leu Gly Leu Thr Gly His Asp Val Glu Arg Trp Leu Arg Glu Thr
580 585 590
Tyr Asn Ile Glu Val Glu Leu Ser Asp Leu Tyr Asn Ile Leu Cys Ile
595 600 605
Ile Thr Pro Gly Asp Thr Glu Arg Glu Ala Ser Leu Leu Val Glu Ala
610 615 620
Leu Arg Arg Leu Ser Lys Gln Phe Ser His Gln Ala Glu Lys Gly Ile
625 630 635 640
Lys Pro Lys Val Leu Leu Pro Asp Ile Pro Ala Leu Ala Leu Thr Pro
645 650 655
Arg Asp Ala Phe Tyr Ala Glu Thr Glu Val Val Pro Phe His Glu Ser
660 665 670
Ala Gly Arg Ile Ile Ala Glu Phe Val Met Val Tyr Pro Pro Gly Ile
675 680 685
Pro Ile Phe Ile Pro Gly Glu Ile Ile Thr Glu Glu Asn Leu Lys Tyr
690 695 700
Ile Glu Thr Asn Leu Ala Ala Gly Leu Pro Val Gln Gly Pro Glu Asp
705 710 715 720
Asp Thr Leu Gln Thr Leu Arg Val Ile Lys Glu Tyr Lys Pro Ile Arg
725 730 735
<210> 14
<211> 2211
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat gtctcagctc gagacccctc tgttcaccgg tctgctcgaa 780
cacatgaaga aaaacccggt ccagtttcac attccaggtc acaagaaagg tgctggtatg 840
gaccctgagt tccgtgcgtt tatcggtgat aacgcgctcg cgatcgacct gatcaacatc 900
tcccctctcg acgacctcca ccacccgaaa ggcatgatca aacgtgcgca ggaactggct 960
gcggaagcgt ttggcgcgga ctacacgttc ttcagcgttc aaggcaccag cggtgccatc 1020
atgacgatgg taatgtctgt tgcgggtccg ggcgataaga tcatcgtccc tcgtaacgtt 1080
cacaaatctg ttatgtctgc catcgttttc tctggcgcga cccctatttt catccacccg 1140
gaaatcgata aggagctggg tattagccac ggtattaccc cgcaggccgt ggagaaagcc 1200
ctgcgtcaac accctgatgc taaaggcgtt ctggtaatca acccgactta tttcggtatc 1260
gcgggtgacc tcaaaaagat cgttgacatc gcgcactctt ataatgtgcc ggtcctggta 1320
gatgaagcgc acggtgttca tattcacttc cacgaggacc tcccactcag cgcaatgcag 1380
gcgggtgcgg atatggcggc gacgtccgtg cacaagctgg gcggtagcct gactcagtct 1440
tccattctga acgtacgcga aggtctggtt tctgctaaac acgtgcaagc gattctctct 1500
atgctgacca ccacttctac ctcttatctg ctgctggctt ccctggacgt agcgcgtaaa 1560
cagctggcaa ccaaaggtcg tgaactcatc gacaaagcca tccgcctcgc ggattggacc 1620
cgtcgccaga ttaacgagat cccgtacctc tactgcgtgg gtgaagagat cctgggtacc 1680
gaagcaacct acgactacga tccgactaaa ctgatcatca gcgtaaaaga actcggtctc 1740
actggccatg acgttgagcg ttggctccgt gaaacctaca atatcgaagt tgaactgtct 1800
gacctctata acatcctctg catcatcacc ccgggtgata ctgagcgcga agcgtctctc 1860
ctggtggaag cactgcgccg tctgtctaaa caattctccc atcaggccga aaagggtatc 1920
aaacctaagg ttctcctgcc ggatattcct gccctcgccc tgacgcctcg tgacgcgttc 1980
tatgcggaaa ccgaagtcgt tccgttccat gagtccgccg gtcgtatcat cgcggagttt 2040
gtaatggttt acccaccggg catcccaatc ttcatccctg gcgagattat cactgaggaa 2100
aacctgaaat acatcgaaac caacctggcg gctggcctcc cggttcaggg cccagaagac 2160
gacacgctgc agaccctccg tgtcattaaa gaatacaaac caattcgtta a 2211
<210> 15
<211> 741
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Ser Glu Glu Gln Gln Arg Ala Pro Tyr
245 250 255
Leu Glu Gln Trp Leu Ala Tyr Val Asp Glu Cys Val Ile Pro Phe Thr
260 265 270
Thr Pro Gly His Lys Gln Gly Arg Gly Ala Pro Pro Glu Phe Val Ala
275 280 285
Ala Phe Gly Glu Arg Ala Leu Ala Leu Asp Ile Pro His Asp Gly Gly
290 295 300
Thr Phe Asp Ala His Leu Glu His Asp Pro Leu Val Ala Ala Glu Arg
305 310 315 320
Leu Ala Ala Ala Leu Trp Gly Ala Arg Asp Ala Val Phe Leu Val Asn
325 330 335
Gly Ser Thr Thr Gly Asn Leu Ala Ala Leu Leu Thr Leu Gly Arg Pro
340 345 350
Gly Gln Pro Ile Val Val Thr Arg Ala Met His Lys Ser Leu Leu Ala
355 360 365
Gly Leu Val Leu Ser Gly Ala Arg Pro Val Tyr Val Val Pro Ala Val
370 375 380
His Pro Glu Ser Gly Ile Leu Leu Asp Leu Pro Pro Glu Ser Val Ala
385 390 395 400
Gln Ala Leu Ala Ala Trp Pro Asp Ala Thr Ala Val Ala Leu Val Ser
405 410 415
Pro Thr Tyr Thr Gly Val Thr Ser Asp Thr Ala Glu Leu Ala Ala Leu
420 425 430
Cys His Ala His Gly Val Pro Leu Phe Val Asp Glu Ala Trp Gly Pro
435 440 445
His Leu Pro Phe His Pro Ala Leu Pro Ala Ala Ala Ile Pro Ser Gly
450 455 460
Ala Asp Leu Ala Val Thr Ser Leu His Lys Leu Ala Gly Ser Leu Thr
465 470 475 480
Gln Thr Ala Leu Leu Leu Met Ala Gly Asn Leu Val Asp Gln Ala Gln
485 490 495
Leu Arg Ala Ala Thr Ala Met Val Gln Thr Thr Ser Pro Ala Ala Phe
500 505 510
Leu Tyr Ala Ser Leu Asp Ala Ala Arg Arg Arg Leu Ala Leu Glu Gly
515 520 525
Glu Gln Leu Leu Ala Arg Thr Leu Glu Leu Ala Glu His Ala Arg Arg
530 535 540
Glu Leu Ala Ala Ile Pro Gly Leu Glu Val Val Gly Pro Glu Ile Val
545 550 555 560
Ala Gly Arg Pro Gly Ala Gly Phe Asp Arg Thr Arg Leu Val Val Asp
565 570 575
Val Gln Gly Phe Gly Leu Thr Gly Leu Glu Val Lys Arg Ile Leu Arg
580 585 590
Arg Asp Phe Arg Ile Ala Ala Glu Met Ala Asp Leu Val Ser Val Val
595 600 605
Phe Leu Ile Thr Ile Gly Asp Thr Pro Glu Thr Ile Ala Ala Leu Val
610 615 620
Ala Ala Phe Arg Ala Leu Ala Ala Asp Arg Thr Arg Pro Asp Cys Ala
625 630 635 640
Ala Gly Arg Arg Ala Val Arg Ala Leu Leu Arg Ser Thr Gly Pro Ile
645 650 655
Val Ala Gly Ala Pro Gln Ala Met Thr Pro Arg Glu Ala Phe Phe Ala
660 665 670
Pro Ala Glu Arg Val Pro Leu Ala Asp Ala Val Gly Arg Val Ala Ala
675 680 685
Glu Pro Val Thr Pro Tyr Pro Pro Gly Ile Pro Val Leu Ala Pro Gly
690 695 700
Glu Val Val Arg Pro Glu Val Val Glu Phe Leu Gln Ala Gly Arg Ala
705 710 715 720
Ala Gly Met Arg Phe Asn Gly Ala Ser Asp Pro Thr Leu Ala Thr Leu
725 730 735
Arg Val Val Arg Ala
740
<210> 16
<211> 2226
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat gtctgaagaa cagcaacgtg ctccgtacct ggagcaatgg 780
ctggcgtacg ttgacgagtg cgttatcccg tttaccactc cgggtcacaa acaaggtcgc 840
ggtgcgccac cggagttcgt tgcggcgttc ggtgaacgtg cgctcgctct ggacattccg 900
catgacggtg gcacctttga cgcgcatctg gaacatgacc cgctcgttgc cgccgaacgt 960
ctggctgccg cactgtgggg tgcacgcgat gcggtgtttc tggttaacgg ttccaccact 1020
ggtaacctgg cggctctgct cactctcggt cgcccaggtc agccgattgt tgttactcgt 1080
gccatgcata agagcctgct ggcaggtctg gtcctgagcg gtgctcgccc tgtctacgtt 1140
gtaccggccg tacacccaga atccggtatc ctcctcgatc tccctccgga atctgttgcg 1200
caggcgctgg ccgcgtggcc tgatgcgacg gctgtagctc tggtgtcccc gacctacact 1260
ggcgttacct ctgacactgc tgaactggca gccctctgtc acgctcatgg tgttccactg 1320
tttgttgatg aagcgtgggg tccgcacctc ccgttccatc cagcactccc agcagcagct 1380
attccgtctg gtgccgatct ggcggttact tctctgcaca aactggcggg ttccctcacc 1440
caaaccgctc tcctcctgat ggcaggcaac ctcgtagacc aagcccagct gcgtgcagcc 1500
acggcaatgg tgcaaaccac cagccctgca gccttcctgt acgcgtccct ggatgctgcc 1560
cgtcgccgtc tcgcgctcga aggtgaacag ctcctcgcac gtactctcga gctggctgag 1620
cacgctcgcc gtgaactcgc cgccatcccg ggtctggagg tggtcggtcc agaaattgtt 1680
gcgggtcgtc cgggtgccgg cttcgatcgt actcgcctcg ttgttgacgt tcagggtttc 1740
ggtctgactg gcctcgaagt aaagcgtatc ctgcgtcgtg acttccgtat tgcagctgaa 1800
atggcagatc tcgtctctgt tgttttcctc atcaccatcg gtgacacccc agagaccatc 1860
gctgccctgg tagcagcttt ccgtgcactc gctgctgacc gtacccgtcc agactgtgct 1920
gccggtcgtc gtgcagtacg cgccctcctc cgttctaccg gtccgatcgt cgcgggtgct 1980
cctcaggcga tgaccccgcg tgaagctttc ttcgctccag ctgagcgcgt tccgctcgcg 2040
gatgccgtcg gtcgtgttgc agccgagccg gttaccccat atccgcctgg tattccggta 2100
ctggccccag gtgaagtggt tcgcccggag gtagttgaat tcctccaggc aggccgtgcc 2160
gctggtatgc gtttcaatgg cgcgtctgac ccgactctgg cgaccctccg tgtcgttcgt 2220
gcctaa 2226
<210> 17
<211> 711
<212> DNA
<213> 蘑菇珊瑚(mushroom coral)
<400> 17
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaata a 711
<210> 18
<211> 236
<212> PRT
<213> 蘑菇珊瑚(mushroom coral)
<400> 18
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 19
<211> 276
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 60
gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 120
cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 180
cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 240
ctttatgctt ccggctcgta tgttgtgtgg aattgt 276
<210> 20
<211> 235
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 20
tgctttttcc gatcgtcacg gcgatgttta tcgcgaacag atggtggact ttatccttag 60
cgcgttgaat ccgcagaact aacccatgat cgctagcacg ataatcattc acaaaaccac 120
cttaagacat gctaatccac tggtcagaac agtttaagat gagaaaaatt ctgtgacgct 180
tgccaacatt tctgatgatt agcattccct tcgccatttc cttgagcaaa cttta 235
<210> 21
<211> 238
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 21
tgtttggtaa aaattcccgc catcataaca ttgccaacgg cgaggggaag tgggtaaggc 60
atgtaaattc atcatgttga cgaaataatc gcccctggta aaagaaacac tgatgcgagg 120
cctgtgtttc aatctttaaa tcagtaaact tcatacgctt gacggaaaaa ccaggacgaa 180
acctaaatat ttgttgttaa gctgcaatgg aaacggtaaa agcggctagt atttaaag 238
<210> 22
<211> 233
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 22
ctcgcttaca tcgctaccag catggtcaac ctgcgcctgg cacaggaacg ttatccggac 60
gttcagttcc accagacccg cgagcattaa ttcttgcctc cagggcgcgg tagccgctgc 120
gccctgtcaa tttcccttcc ttattagccg cttacggaat gttcttaaaa cattcacttt 180
tgcttatgtt ttcgctgata tcccgagcgg tttcaaaatt gtgatctata ttt 233
<210> 23
<211> 237
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 23
gcagaaatga ctctcccatc agtacaaacg caacatattt gccacgcagc atccagacat 60
cacgaaacga atccatcttt atcgcatgtt ctggcggcgc gggttccgtg cgtgggacat 120
agctaataat ctggcggttt tgctggcgga gcggtttctt cattactggc ttcactaaac 180
gcatattaaa aatcagaaaa actgtagttt agccgattta gcccctgtac gtcccgc 237
<210> 24
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcccgccaaa tccctaaaat tgttctatac tgtattg 37
<210> 25
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tcccgccaaa ttattaaaat tgttctatac tgtattg 37
<210> 26
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
cgcgttcccg cctttagggg caattgttct atactgtatt g 41
<210> 27
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tcccgccaaa tctgcaaaat tgttctatac tgtattg 37
<210> 28
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tccgagctca tgaacgttat tgcaatattg 30
<210> 29
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gcctctagac cacttccctt gtacgagc 28
<210> 30
<211> 715
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 30
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 31
<211> 2148
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 31
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 32
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat 44
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
agctgtttcc tgtgtgaaat 20
<210> 34
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa 60
ttcgagctc 69
<210> 35
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
tgtggaattg tgagcggata acaatttcac acaggaaaca gctgagctc 49
<210> 36
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ggcgagctca tggaaccatt acttcgcgc 29
<210> 37
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ggctctagat tacactttac atatccgcag gg 32
<210> 38
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
cttgatatcg aattcttaac tttaagaagg aatatacata tggaaccatt acttcgcgc 59
<210> 39
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gttaagaatt cgatatcaag cttatcgatg agctcacaat tccacacaac atacgag 57
<210> 40
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tgtggaattg tgagcggata acaatttcac acaggaaaca gctgagctc 49
<210> 41
<211> 65
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
tgtggaattg tgagctcatc gataagcttg atatcgaatt cttaacttta agaaggaata 60
tacat 65
<210> 42
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ggcgagctca tcgataagct tgatatcgaa ttcttaactt taagaaggaa tatacatatg 60
gtgtctaaag gc 72
<210> 43
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
accagaacca gaaccagaac cagaaccaga tttatacagt tcatccattc cgcc 54
<210> 44
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
tctggttctg gttctggttc tggttctggt atggaaccat tacttcgcgc actgtgg 57
<210> 45
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
tctggttctg gttctggttc tggttctggt atggagaagc aagagattaa caagttc 57
<210> 46
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ggctctagat tagaaatcgg ttacaacctg aatg 34
<210> 47
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
tctggttctg gttctggttc tggttctggt atgtctcagc tcgagacccc tctgttc 57
<210> 48
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
ggctctagat taacgaattg gtttgtattc tttaatg 37
<210> 49
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
tctggttctg gttctggttc tggttctggt atgtctgaag aacagcaacg tgctccg 57
<210> 50
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
ggctctagat taggcacgaa cgacacggag gg 32
<210> 51
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
ggggtacctg ctttttccga tcgtcacg 28
<210> 52
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
ccatcgatta aagtttgctc aaggaaatgg c 31
<210> 53
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
ggggtacctg tttggtaaaa attcccg 27
<210> 54
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
ccatcgatct ttaaatacta gccgctttta c 31
<210> 55
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ggggtaccct cgcttacatc gctaccagc 29
<210> 56
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
ccatcgataa atatagatca caattttgaa 30
<210> 57
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
ggggtaccgc agaaatgact ctcccatc 28
<210> 58
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ggatcgatgc gggacgtaca ggggc 25

Claims (10)

1.重组DNA，其特征在于，所述重组DNA至少包括稳定期特异性启动子和带有促溶标签的赖氨酸脱羧酶融合蛋白基因；所述融合蛋白包含促溶标签和来自嗜热菌的赖氨酸脱羧酶，二者之间通过Linker连接；
其中，所述促溶标签选自荧光蛋白、麦芽糖结合蛋白、谷胱甘肽转移酶或它们的组合。
2.根据权利要求1所述的重组DNA，其特征在于，所述荧光蛋白选自红色荧光蛋白、蓝色荧光蛋白、蓝绿色荧光蛋白、绿色荧光蛋白、黄色荧光蛋白、橙色荧光蛋白或光学加亮荧光蛋白；优选RedStar、tdtomato或mCherry中的至少一种；和/或，
所述赖氨酸脱羧酶选自以下①～④中任一种：①来源于嗜热菌Thermosynechoccuselongatus的赖氨酸脱羧酶TeLDC，其氨基酸序列如SEQ ID NO：1所示；②来源于Tepidanaerobacter syntrophicus的赖氨酸脱羧酶TsLDC，其氨基酸序列如SEQ ID NO:3所示；③来源于Geobacillus kaustophilus的赖氨酸脱羧酶GkLDC，其氨基酸序列如SEQ IDNO:5所示；④来源于Thermomicrobium roseum的赖氨酸脱羧酶TrLDC，其氨基酸序列如SEQID NO:7所示；
优选地，所述赖氨酸脱羧酶与SEQ ID NO：1,3,5或7中的任一个具有至少70％的氨基酸序列同一性，或至少80％，至少85％，至少90％，至少95％的序列同一性；更优选地，所述融合蛋白选自荧光蛋白-Linker-TeLDC、荧光蛋白-Linker-TsLDC、荧光蛋白-Linker-GkLDC、荧光蛋白-Linker-TrLDC、TeLDC-Linker-荧光蛋白、TsLDC-Linker-荧光蛋白、TsLDC-Linker-荧光蛋白或TsLDC-Linker-荧光蛋白中的至少一种。
3.根据权利要求1所述的重组DNA，其特征在于，所述Linker包括螺旋形式的Linker或低疏水性、低电荷效应的氨基酸组成的柔性Linker，所述Linker的长度至少为10个氨基酸；
优选地，所述Linker为柔性Linker，更优选为(GGGGS)3或(SG)5-8。
4.根据权利要求1所述的重组DNA，其特征在于，所述稳定期特异性启动子选自pcsiE、pbolA、posmY、pkatE、P1、P2、P3或P4中的任一种，它们的核苷酸序列分别如SEQ ID NO:20-27所示。
5.根据权利要求1-4任一项所述的重组DNA，其特征在于，所述重组DNA至少包括如下3个元件：a、稳定期特异性启动子；b、红色荧光蛋白基因；以及c、嗜热菌来源的赖氨酸脱羧酶基因；其中，各元件按照a-b-c或a-c-b顺次可操作地连接。
6.含有权利要求1-5任一项所述重组DNA的生物材料，所述生物材料为表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程菌。
7.重组质粒，其特征在于，所述重组质粒携带有权利要求1-5任一项所述的重组DNA；优选地，出发质粒为pUC或pBR322质粒或其衍生质粒，更优选pUC18、pUC19、pBR322、pACYC、pET、pSC101和它们的任何衍生质粒。
8.产1,5-戊二胺的基因工程菌，其特征在于，所述基因工程菌携带有权利要求1-5任一项所述的重组DNA、权利要求6所述的生物材料或权利要求7所述的重组质粒；
其中，所述基因工程菌的出发菌株选自埃希氏菌属(Escherichia)、哈夫尼菌属(Hafnia)中的菌种；优选地，所述出发菌株为大肠杆菌(E.coli)、枯草芽孢杆菌(B.subtilis)、天蓝色链霉菌(S.coelicolor)、蜂房哈夫尼菌(H.alvei)、谷氨酸棒状杆菌(C.glutamicum)，或经过诱变或随机突变之后的菌株或基因工程菌。
9.1,5-戊二胺的生产方法，其特征在于，包括发酵培养权利要求8所述的基因工程菌以生产1,5-戊二胺。
10.权利要求1-5任一项所述重组DNA在生产1,5-戊二胺中的应用，其特征在于，(a)将权利要求1-5任一项所述重组DNA构建到具有生产L-赖氨酸能力的工程菌中，发酵培养重组菌并进行赖氨酸积累，发酵初期培养温度控制在20-50℃，；(b)在余下的发酵阶段温度控制在50-110℃，使赖氨酸脱羧酶具有活性，转化生产1,5-戊二胺。
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