本发明涉及生物
技术领域：
，特别涉及基因过表达及获得的菌株、应用。
背景技术：
：甲基萘醌-7是脂溶性维生素k2的一种，维生素k的天然存在形式包括植物来源的维生素k1(又称叶绿醌，pk)和细菌来源的维生素k2(又称甲基萘醌，mk)。根据侧链中异戊二烯单元数量的不同，总共有14种甲基萘醌，记为mk-n，常见的有mk-4和mk-7等。在原核生物中，mk-n参与呼吸链的电子传递。对于人类和其他哺乳动物，由于维生素k是血液和骨骼中特定蛋白的谷氨酸残基翻译转化为γ-羧基谷氨酸(gla)的重要辅因子，因此用来维持钙稳态、抑制血管壁钙化、支持内皮完整性、促进骨矿化，并参与组织更新和细胞生长控制。常见的维生素k依赖性蛋白包括凝血因子(ii、vii、ix、x和凝血酶原)、蛋白c和蛋白s、骨钙蛋白、基质gla蛋白(mgp)、骨膜素等。有研究表明，尽管食物中的pk含量较高，但与mk-n(尤其是mk-7)相比，生物活性较差；长链mk-n如mk-7对正常凝血的作用比pk和mk-4更持久。因此，由于其半衰期长、生物利用度好，mk-7在食品、制药和保健行业更受欢迎，被广泛用作膳食补充剂或药物，用于治疗骨质疏松症、动脉钙化、心血管疾病、癌症和帕金森精神病等。传统的mk-7生产方法是通过对纳豆芽孢杆菌(bacillusnatto)进行经典诱变或发酵优化。toshirosato等从日本食物纳豆中分离出菌株b.subtilis，通过传统诱变筛选出甲萘醌抗性菌株，在7l罐中发酵4天可产35mg/l的mk-7；筛选出的二苯胺抗性菌株，上罐发酵37℃1天，45℃5天可产60mg/l的mk-7。song等人筛选出的1-羟基-2-萘甲酸抗性突变株b.subtilisnatto，在500ml瓶中发酵72小时可产3.593±0.107mg/l的mk-7。miao-miaoluo等人对分离出的菌株bacillusnatto的发酵培养基及发酵条件和mk-7的萃取方法进行优化，在5l反应器中发酵72小时可产32.2mg/l的mk-7。最近也有研究通过代谢途径工程的方法，来提高解淀粉芽孢杆菌(bacillusamyloliquefaciens)中mk-7的产量。jian-zhongxu等人从中国豆豉中分离出6株产纤维蛋白原酶的b.amyloliquefaciens菌株，发现菌株b.amyloliquefaciensy-2能够产生7.1±0.5mg/l的mk-7。序列分析表明以下六个酶经历了错义突变：mena，menc，mend，mene，menh，heps。通过在枯草芽孢杆菌168中过表达菌株y-2的这六个酶，发现mena的过表达使得mk-7的含量增加1.6倍，优于其它酶；在解淀粉芽孢杆菌y-2中过表达自身的这六个酶，发现heps的过表达导致mk-7的产量最高，达到273±5.4克/干细胞重量(dcw)。到目前为止，还未发现有对mk-7的从头生物合成途径进行全面系统的研究。由于对枯草芽孢杆菌的生物化学、遗传学和分子生物学的研究有很多，且其生长速度快，并'通常被认为安全'(gras)的，所以枯草芽孢杆菌是最佳表征的模式微生物之一。而且枯草芽孢杆菌168的完整基因组序列已被测序完成，这有利于通过基因工程的手段构建工业生产菌株。因此，我们选择枯草芽孢杆菌168作为底盘细胞，对从甘油合成mk-7的整体合成路径进行全面系统的研究。在枯草芽孢杆菌168内，甘油通过促进扩散的方式进入细胞内，经甘油异化途径生成甘油醛-3-磷酸(g3p)，随后进入糖酵解过程分别合成赤藓糖-4-磷酸(e4p)、磷酸烯醇式丙酮酸(pep)、丙酮酸(pyr)。e4p和pep经莽草酸(shikimate)路径代谢合成分支酸，其是mk-7合成的前体物。分支酸经甲基萘醌-7(mk-7)路径的九步酶催化，最终合成目标产物mk-7。1，4-二羟基-2-萘甲酸庚异戊二烯基转移酶(mena)催化mk-7路径的第八步反应，即将庚异戊二烯基焦磷酸(heptaprenyl-pp)的七个异戊二烯基转移到1，4-二羟基-2-萘酸生成去甲基甲萘醌。其中heptaprenyl-pp是由g3p与pyr经由甲基赤藓糖醇-4-磷酸(mep)路径的十一步酶催化反应生成。技术实现要素：有鉴于此，本发明提供一种基因过表达及获得的菌株、应用。该枯草芽孢杆菌高产mk-7菌株，具有精确修饰目标基因、次级突变概率低、遗传育种周期短的优势。为了实现上述发明目的，本发明提供以下技术方案：本发明提供了过表达基因在提高微生物次级代谢产物的代谢通量中的应用；所述基因具有如下所示的核苷酸序列中的任意一项：i、具有如基因dxs、dxr和/或yacm-yacn的核苷酸序列；ii、具有如i所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；iii、与如i所示的核苷酸序列具有至少80％同源性的序列或翻译后所得蛋白与基因dxs、dxr和/或yacm-yacn表达的蛋白功能相同或相近的核苷酸序列；iv、如iv、ⅴ或ⅵ所示序列的互补序列。在本发明的一些具体实施方案中，本发明提供了过表达基因dxs、dxr和/或yacm-yacn在提高微生物次级代谢产物的代谢通量中的应用。在本发明的一些具体实施方案中，所述次级代谢产物为mep路径的产物。在本发明的一些具体实施方案中，所述微生物为原核生物；所述次级代谢产物为庚异戊二烯基焦磷酸或醌类物质。在本发明的一些具体实施方案中，所述原核生物为枯草芽孢杆菌；所述醌类物质为甲基萘醌-7。本发明还提供了菌株，其基因过表达；所述基因具有如下所示的核苷酸序列中的任意一项：v、具有如基因dxs、dxr和/或yacm-yacn的核苷酸序列；vi、具有如v所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；vii、与如v所示的核苷酸序列具有至少80％同源性的序列或翻译后所得蛋白与基因dxs、dxr和/或yacm-yacn表达的蛋白功能相同或相近的核苷酸序列；viii、如v、vi或vii所示序列的互补序列。在本发明的一些具体实施方案中，本发明还提供了一种菌株，其基因dxs、dxr和/或yacm-yacn过表达。在本发明的一些具体实施方案中，所述菌株为原核生物。在本发明的一些具体实施方案中，所述原核生物为枯草芽孢杆菌。本发明还提供了所述的菌株在提高微生物次级代谢产物的代谢通量中的应用。在本发明的一些具体实施方案中，所述次级代谢产物为mep路径的产物。在本发明的一些具体实施方案中所述微生物为原核生物；在本发明的一些具体实施方案中所述次级代谢产物为庚异戊二烯基焦磷酸或醌类物质。在本发明的一些具体实施方案中所述原核生物为枯草芽孢杆菌；所述醌类物质为甲基萘醌-7。本发明还提供了所述菌株的发酵方法，包括补料发酵的步骤。在本发明的一些具体实施方案中，所述补料发酵为在发酵过程中补加2～6％(v/v)的甘油和/或5～18.9％(w/v)的大豆蛋白胨。在本发明的一些具体实施方案中，所述补料发酵为在发酵48h和96h均补加1％(v/v)的甘油和/或2％(w/v)的大豆蛋白胨。本发明以革兰氏阳性模式菌株枯草芽孢杆菌168为出发菌株，对其进行研究，确定影响mk-7的关键基因，为将来遗传改造纳豆芽孢杆菌构建mk-7高产菌株提供理论依据。由于纳豆芽孢杆菌的基因组信息不完整及分子操作技术不成熟，故本发明选择与其生理生化特征相似的、革兰氏阳性模式菌株枯草芽孢杆菌168(bacillussubtilis168)进行基础研究。本发明以b.subtilis168为出发菌株，首先过表达mena和原位替换heps-meng-heot操纵子的启动子，来提高mk-7合成路径的代谢通量；然后过表达dxs、dxr和yacm-yacn基因，提高mep路径的代谢通量，增加前体物庚异戊二烯基焦磷酸(heptaprenyl-pp)的供应，最终促进mk-7的合成；最终确定mena编码的1，4-二羟基-2-萘酸庚异戊二烯基转移酶及该反应所需的前体物hepta-pp是mk-7合成的限制性因素，这为将来工业上通过代谢工程手段构建纳豆芽孢杆菌高产mk-7菌株提供理论依据。附图说明为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。图1示枯草芽孢杆菌168内mk-7的生物合成路径；物质简写：gly，甘油；g3p，甘油醛-3-磷酸；e4p，赤藓糖-4-磷酸；dahp，3-脱氧-阿拉伯糖-庚酮酸7-磷酸；cha，分支酸；icha，异分支酸；sephchc，2-琥珀酰基-5-烯醇丙酮酰-6-羟基-3-环己烯-1-羧酸酯；shchc，2-琥珀酰-6-羟基-2,4-环己二烯-1-羧酸叔丁酯；osb，2-琥珀酰苯甲酸；osb-coa，2-琥珀酰苯甲酰基-coa；dhna-coa，1,4-二羟基-2-萘甲酰基-coa；dhna，1,4-二羟基-2-萘甲酸；dmk，2-去甲基甲萘醌；mk-7，甲基萘醌-7；pep，磷酸烯醇式丙酮酸；pyr，丙酮酸；dxp，1-脱氧木酮糖-5-磷酸；mep，甲基赤藓糖醇-4-磷酸；cdp-me，甲基赤藓糖醇4-胞苷酰焦磷酸；cdp-mep，2-磷酸-4-(胞苷5'-焦磷酸)-2-甲基赤藓糖醇；mec，2-甲基赤藓糖醇2,4-环二磷酸；hmbpp，1-羟基-2-甲基-2-丁烯基4-二磷酸；dmapp,二甲基烯丙基焦磷酸；ipp，异戊烯基焦磷酸；gpp，香叶基焦磷酸；fpp，法尼基焦磷酸；hepta-pp，庚异戊二烯基焦磷酸；酶：menf，异分支酸合成酶；mend，2-琥珀酰-5-烯醇丙酮酰基-6-羟基-3-环己烯-1-羧酸酯合成酶；menh，2-琥珀酰-6-羟基-2,4-环己二烯-1-羧酸酯合成酶；menc，邻琥珀酰基苯甲酸合成酶；mene，邻琥珀酰基苯甲酸-coa连接酶；menb，1,4-二羟基-2-萘酰coa合成酶；meni，1,4-二羟基-2-萘甲酰-coa水解酶；mena，1,4-二羟基-2-萘甲酸庚异戊二烯基转移酶；meng，去甲基甲萘醌甲基转移酶；heps/hept，庚异戊二烯基焦磷酸合成酶组分i/ii；dxs，1-脱氧木酮糖-5-磷酸合成酶；dxr，1-脱氧木酮糖-5-磷酸还原异构酶；yacm，甲基赤藓糖醇4-磷酸胞苷酰转移酶；ispe，4-二磷酸胞苷-2-甲基赤藓糖醇激酶；yacn，甲基赤藓糖醇2,4-环二磷酸合成酶；ygfy，4-羟基-3-甲基丁-2-烯基二磷酸合成酶；yqfp，4-羟基-3-甲基丁-2-烯基二磷酸还原酶；ypga，异戊烯基焦磷酸δ-异构酶；yqid，香叶基/法尼基焦磷酸合成酶；图2示重组菌株mk3、mk4和mk34的基因过表达示意图(图2a)、菌体生长情况(图2b)和mk-7产量图(图2c)；图3示示重组菌株mk3-mep1、mk3-mep2和mk3-mep3的基因过表达示意图(图3a)、菌体生长情况(图3b)和mk-7产量图(图3c)；图4示重组菌株mk3-mep12和mk3-mep123的基因过表达示意图(图4a)、菌体生长情况(图4b)和mk-7产量图(图4c)。具体实施方式本发明公开了一种基因过表达及获得的菌株、应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本
发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。枯草芽孢杆菌(bacillussubtilis)是重要的工业菌株，基因组信息完整、分子操作技术及发酵技术成熟，且生理生化特征与纳豆芽孢杆菌相似。枯草芽孢杆菌可以利用甘油作为碳源，依次经过莽草酸路径、mep路径及mk-7路径合成mk-7。通过过表达mk-7路径的基因mena及heps-meng-hept，以及mep路径的dxs，dxr及yacm-yacn，考察其对mk-7合成的影响，确定影响mk-7合成的限速步骤，为将来构建纳豆芽孢杆菌mk-7的高产菌株提高基础研究和理论依据。1、本发明首次通过代谢工程手段尝试构建枯草芽孢杆菌高产mk-7菌株。2、对枯草芽孢杆菌内mk-7路径的基因mena及heps-meng-hept进行过表达，确定其是影响mk-7合成的关键基因，尤其是mena。3、对枯草芽孢杆菌内mep路径的基因dxs、dxr及yacm-yacn进行过表达，确定其是影响mk-7合成的关键基因。4、本发明表明mk-7路径的第八步反应是影响mk-7合成的限速步骤。mena编码的1，4-二羟基-2-萘酸庚异戊二烯基转移酶(全长311aa)催化第八步反应，将庚异戊二烯基焦磷酸(hepta-pp)的七个异戊二烯基转移到1，4-二羟基-2-萘酸上生成去甲基甲萘醌。增加前体物hepta-pp的供应，且同时过表达该酶，使得在250ml摇瓶中发酵120h后，mk-7产量达到12.044mg/l，是出发菌(3.137mg/l)的3.84倍。以上研究为将来工业上通过代谢工程和合成生物学手段构建纳豆芽孢杆菌高产mk-7菌株提供理论依据。与传统诱变相比，通过模块化设计构建枯草芽孢杆菌高产mk-7菌株，具有精确修饰目标基因、次级突变概率低、遗传育种周期短的优势。根据本发明的实验方法及结论，可将该模块化设计同样应用于纳豆芽孢杆菌和解淀粉芽孢杆菌，但由于二者的分子信息不完整，首先需要测序确定其基因序列，再进行后续实验设计及基因改造；分子技术缺乏即只能用整合质粒。在本发明的基础上结合分子生物学的技术获得的相应技术方案均在本发明的保护范围之内。材料：菌株、质粒和培养基：本发明涉及的所有质粒和菌株信息详见表1。含有启动子的质粒puc57-1.8k-p1/p2/p3均由genscriptbiotech公司合成；引物均由genewiz公司合成。lb培养基(蛋白胨10g/l、酵母提取物5g/l、氯化钠10g/l)用于b.subtilis及e.coli的一般培养，固体培养基添加15g/l琼脂粉，需要时加入氨苄青霉素100μg/ml，或新霉素16μg/ml，或氯霉素8μg/ml。发酵培养基：甘油30ml/l，大豆蛋白胨60g/l，酵母提取物5g/l，k2hpo43g/l，mgso4·7h2o0.5g/l，ph7.3。表1实验中涉及的菌株和质粒试剂及仪器：限制酶购自fermentas公司；fasttaq酶、hifidna聚合酶、dntp均购自北京全式金生物技术有限公司；质粒小提试剂盒、普通dna产物纯化试剂盒、琼脂糖凝胶dna回收试剂盒、rnapreppure培养细胞/细菌总rna提取试剂盒、fastquantcdna第一链合成试剂盒、superreal荧光定量预混试剂盒均购自天根公司；标准品mk-7购自chromadex公司；其余生化试剂为进口或国产分析纯试剂。所用仪器：漩涡混合器、真空离心浓缩仪、高效液相色谱仪(waters)、lightcycler480(roche)。引物用于pcr的引物见表2。表2pcr引物序列方法：dna操作：所有dna片段都来自pcr扩增；用于转化的dna片段均采用重叠pcr方法拼接形成。b.subtilis感受态细胞的制备及转化采用spizizen方法。本实验采用了两种无标记修饰方法：第一种方法是利用反选择盒(para-neo)和选择盒(cat-arar)来完成。反向选择盒(para-neo)就是在阿拉伯糖操纵子的启动子控制下的新霉素抗性基因的结构基因，其表达受arar蛋白的阻遏，在arar基因缺陷背景下，新霉素抗性基因表达；存在arar蛋白时，新霉素抗性基因被抑制。首先将反向选择盒整合在枯草芽孢杆菌染色体上的arar基因位点，同时缺陷arar基因，使菌株带有新霉素抗性；然后利用上游同源臂u和下游同源臂g将带有选择盒cr的片段整合在基因组上，使重组子的新霉素抗性消失，用氯霉素筛选重组子；最后当引入的arar基因由于染色体内同源重组(片段d)而被弹出时，细胞的新霉素抗性恢复，用于反向筛选因染色体内同源重组而形成的转化子。第二种方法是利用连接cat-upp选择盒且其两端含有多克隆酶切位点用于插入目的片段的pss质粒。首先将上游同源臂u和下游同源臂d利用酶切链接到质粒pss上，其中u的3’端和d的5’端均含有相同的dr区；然后将构建好的质粒转入受体菌中，cat-upp选择盒通过双交换整合到基因组上，利用氯霉素培养基来筛选转化子；然后基因组上dr区发生单交换，弹出cat-upp选择盒，利用10μm的5-氟尿嘧啶(5-fu)基本培养基筛选目的转化子(含upp基因的枯草芽孢杆菌不能在10μm的5-fu基本培养基上生长，upp缺陷的枯草芽孢杆菌可以在10μm的5-fu基本培养基上生长)。所有基因在染色体上的过表达位点与mk-7合成路径没有关系且经枯草芽孢杆菌最小基因组研究表明，其的敲除不影响细胞正常生长特性。本发明提供的基因过表达及获得的菌株、应用中所用菌株、原料及试剂均可由市场购得。下面结合实施例，进一步阐述本发明：实施例1过表达mena选择b.subtilis染色体的yxla位点过表达mena基因。首先以b.subtilis168的染色体为模板，分别用引物yxla-mena-u1/yxla-mena-u2q，yxla-mena-1q/yxla-mena-2，yxla-mena-d1q/yxla-mena-d2，yxla-mena-g1q/yxla-mena-g2扩增片段u(如seqidno.59所示，1115bp)、a(如seqidno.61所示，1057bp)、d(如seqidno.62所示，1053bp)、g(如seqidno.64所示，806bp)；以质粒puc57-1.8k-p1为模板，用引物yxla-mena-p1/yxla-mena-p2扩增含有启动子plaps的片段p(如seqidno.60所示，442bp)；以实验室保存的bs168num的染色体为模板，用引物yxla-mena-cr1q/cr2扩增片段cr(如seqidno.63所示，2069bp)。然后通过重叠pcr方法，利用引物yxla-mena-u1/yxla-mena-2，将片段u、p和a拼接成片段upa(2614bp)；再利用引物yxla-mena-u1/yxla-mena-d2将片段upa和片段d拼接成片段upad(3667bp)；最后利用引物yxla-mena-u1/yxla-mena-g2将片段upad、片段cr和片段g拼接成片段upadcrg(6542bp)。将upadcrg片段转化受体菌bs168nu的感受态细胞，经过两步筛选，最终获得plaps-mena基因在yxla位点整合的重组菌株mk3。利用引物yxla-mena-p1/yxla-mena-2进行测序，无额外点突变。upadcrg片段(如seqidno.65所示)。实施例2原位替换heps-meng-hept操纵子的启动子用组成型启动子plaps原位替换bs168nu染色体上heps-meng-hept操纵子的启动子。首先以b.subtilis168的染色体为模板，用引物取sgt-u1m/sgt-u2qm扩增片段u(1319)，通过bglii和xhoi酶切连接到载体pss，转化e.colitranst1感受态，得到重组质粒pss-sgt-u。然后以质粒puc57-1.8k-p1为模板，用引物plaps1/plaps2扩增含有启动子plaps的片段p(443bp)；以b.subtilis168的染色体为模板，用引物取sgt-d1q/sgt-d2扩增片段d(1359bp)，二者通过重叠pcr的方法，利用sgt-plaps1qm/sgt-d2m拼接成片段pd(952bp)，最后通过bamhi和kpni酶切连接到质粒pss-sgt-u上，转化e.colitranst1感受态，得到重组质粒pss-sgt-upd。将构建好的质粒转化受体菌bs168nu感受态细胞，然后通过两步筛选后最终获得重组菌株mk4。用引物cx-sgt-1/cx-sgt-2进行测序，发现heps-meng-hept操纵子的启动子按原设计被替换。pss-sgt-upd质粒序列(如seqidno.66所示)。其中，如seqidno.67所示序列为上游同源臂u；如seqidno.68所示序列为第一双交换片段dr区；如seqidno.69所示序列为cat；如seqidno.70所示序列为upp；如seqidno.71所示序列为第二双交换片段dr区；如seqidno.72所示序列为启动子plaps；如seqidno.73所示序列为下游同源臂d。实施例3摇瓶发酵培养及菌体生长情况的测定表3发酵培养基及发酵条件参数范围甘油20～80ml/l大豆蛋白胨60～180g/l酵母提取物0～20g/lk2hpo41～5g/lmgso4·7h2o0.1～0.8g/lph6.5～7.5接种量1％～6％温度35～45℃转速100～250r/min发酵时间72-144h摇瓶发酵：挑取新活化的平板单菌落接入装有5mllb培养基的试管中，200r/min、37℃振荡培养14h；按1％接种量转接装有30ml发酵培养基的250ml锥形瓶中(三个平行)，37℃、黑暗条件下、200r/min振荡培养144h。生物量的测定：首先间隔6h，中间间隔12h，后面间隔24h取适量发酵液，13000r/min离心1min沉淀细胞，洗涤后重悬、稀释适当倍数，测定菌悬液的od600值。实施例4提高mk-7路径的代谢通量，促进mk-7的合成在枯草芽孢杆菌内，mk-7路径共有九步反应，mena编码1，4-二羟基-2-萘甲酸庚异戊二烯基转移酶(全长311aa)，该酶催化第八步反应，将七个异戊二烯基转移到1，4-二羟基-2-萘酸生成去甲基甲萘醌。催化第九步反应的酶的编码基因meng与heps/hept构成一个heps-meng-hept操纵子。meng编码去甲基甲萘醌甲基转移酶(全长233aa)，该酶将s-腺苷-l-甲硫氨酸(sam)的甲基转移到去甲基甲萘醌上生成甲基萘醌-7即mk-7。heps编码庚异戊二烯基焦磷酸合成酶组分i(全长251aa)，hept编码庚异戊二烯基焦磷酸合成酶组分ii(全长348aa)。因此为了促进枯草芽孢杆菌168能够合成mk-7，本发明采用组成型启动子plaps在染色体的yxla位点过表达自身的mena基因，并原位替换heps-meng-hept操纵子的启动子，分别得到重组菌株mk3和mk4。通过摇瓶发酵，得到重组菌株的菌体生长情况和它们在发酵96h和120h的mk-7产量。由表4可知，上述基因过表达未对菌体生长造成明显影响，生长趋势基本不变。实时定量反转录pcr分析表明(表5)，利用plaps启动子过表达mena后，重组菌mk3中mena的表达水平是出发菌中mena的表达水平的6.22倍；利用plaps启动子原位替换heps-meng-hept操纵子的启动子后，重组菌mk4中该操纵子的表达水平是出发菌中该操纵子的表达水平的3.70倍，说明该启动子是有效果的。由表6可知，发酵96h后，出发菌bs168nu的mk-7产量为2.331mg/l，重组菌mk3的mk-7产量为4.499mg/l，较出发菌提高93％，重组菌mk4的mk-7产量为2.878mg/l，较出发菌提高23.5％；发酵120h后，出发菌的mk-7产量为3.137mg/l，重组菌mk3的mk-7产量为6.571mg/l，较出发菌提高109.5％，重组菌mk4的mk-7产量为4.335mg/l，较出发菌提高38.2％。结果表明，在枯草芽孢杆菌168内，mena和heps-meng-hept是影响mk-7合成的关键基因，尤其是mena。因此，为了进一步提高mk-7的合成量，本发明替换菌株mk3基因组上heps-meng-hept操纵子的启动子，得到重组菌株mk34，由表4可知，其生长与对照菌mk3相比，在发酵24～72h时od600降低，但生长趋势基本不变。由表5可知，发酵96h后，重组菌mk34的mk-7产量为4.427mg/l，较对照菌mk3降低1.6％；发酵120h后，重组菌mk34的mk-7产量为5.991mg/l，较对照菌mk3降低8.8％。由此可见，过表达mena同时原位替换heps-meng-hept操纵子的启动子，并不利于mk-7的大量合成，因此本发明选择菌株mk3进行后续研究。如图2所示。表4菌体生长情况发酵时间(h)od600(bs168nu)od600(mk3)od600(mk4)od600(mk34)63.370±0.0303.309±0.0343.528±0.1273.820*±0.079128.101±0.2098.602*±0.0838.294±0.1958.316±0.0302413.926±0.11314.378*±0.25314.846#±0.3746.435#±0.0274819.349±0.09817.451*±0.26916.872#±0.5749.683#±0.0487217.699±0.10317.114±0.67515.268*±0.84714.015#±0.2849619.210±0.46519.599±0.24822.448#±0.22819.568±0.19912024.479±0.44720.153#±0.13223.191±0.13420.722#±0.65314412.627±0.28615.203#±0.66717.529#±0.94716.947#±0.372注：*示与出发菌bs168nu相比具有显著差异(p＜0.05)；#示与出发菌bs168nu相比具有极显著差异(p＜0.01)。表5mena和heps-meng-hept的相对转录水平菌株mena的相对转录水平heps-meng-hept的相对转录水平bs168nu1.001.00mk36.22#±0.13－－mk4－－3.70#±0.01注：*示与出发菌bs168nu相比具有显著差异(p＜0.05)；#示与出发菌bs168nu相比具有极显著差异(p＜0.01)。表6mk-7产量发酵时间(h)c(bs168nu)(mg/l)c(mk3)(mg/l)c(mk4)(mg/l)c(mk34)(mg/l)962.331±0.0234.499#±0.1592.878±0.1754.427#±0.4361203.137±0.1936.571#±0.0624.335*±0.0255.991#±0.136注：*示与出发菌bs168nu相比具有显著差异(p＜0.05)；#示与出发菌bs168nu相比具有极显著差异(p＜0.01)。实施例5过表达dxs选择b.subtilis染色体的yjob位点过表达dxs基因。首先以b.subtilis168的染色体为模板，分别用引物yjob-dxs-u1/yjob-dxs-u2q，yjob-dxs-1q/yjob-dxs-2，yjob-dxs-d1q/yjob-dxs-d2，yjob-dxs-g1q/yjob-dxs-g2扩增片段u(1236bp，如seqidno.74所示)、s(包含整个dxs基因，1947bp，如seqidno.76所示)、d(808bp，如seqidno.77所示)、g(697bp，，如seqidno.79所示)；以质粒puc57-1.8k-p2为模板，用引物p431/p432扩增含有启动子p43的片段p(232bp，如seqidno.75所示)；以实验室保存的bs168num的染色体为模板，用引物yjob-dxs-cr1q/cr2扩增片段cr(2069bp，，如seqidno.78所示)。然后通过重叠pcr方法，利用引物yjob-dxs-u1/yjob-dxs-2，将片段u、p和s拼接成片段ups(3415bp)；再利用引物yjob-dxs-u1/yjob-dxs-d2将片段ups和片段d拼接成片段upsd(4223bp)；最后利用引物yjob-dxs-u1/yjob-dxs-g2将片段upsd、片段cr和片段g拼接成片段upsdcrg(6989bp)。将upsdcrg片段转化受体菌mk3的感受态细胞，经过两步筛选，最终获得p43-dxs基因在yjob位点整合的重组菌株mk3-mep1。利用引物cx-dxs1/cx-dxs2/cx-dxs3进行测序，无额外点突变。实施例6过表达dxr选择b.subtilis染色体的ydeo位点过表达dxr基因。首先以b.subtilis168的染色体为模板，分别用引物ydeo-dxr-u1/ydeo-dxr-u2q，ydeo-dxr-1q/ydeo-dxr-2，ydeo-dxr-d1q/ydeo-dxr-d2，ydeo-dxr-g1q/ydeo-dxr-g2扩增片段u(1129bp，如seqidno.80所示)、r(1182bp，如seqidno.81所示)、d(1005bp，如seqidno.82所示)、g(514bp，如seqidno.83所示)；以质粒puc57-1.8k-p2为模板，用引物p431/p432扩增含有启动子p43的片段p(232bp，如seqidno.75所示)；以实验室保存的bs168num的染色体为模板，用引物ydeo-dxr-cr1q/cr2扩增片段cr(2069bp，如seqidno.78所示)。然后通过重叠pcr方法，利用引物ydeo-dxr-u1/ydeo-dxr-2，将片段u、p和r拼接成片段upr(2543bp)；再利用引物ydeo-dxr-u1/ydeo-dxr-d2将片段upr和片段d拼接成片段uprd(3548bp)；最后利用引物ydeo-dxr-u1/ydeo-dxr-g2将片段uprd、片段cr和片段g拼接成片段uprdcrg(6131bp)。将uprdcrg片段转化受体菌mk3的感受态细胞，经过两步筛选，最终获得p43-dxr基因在ydeo位点整合的重组菌株mk3-mep1。利用引物cx-dxr1/cx-dxr2进行测序，无额外点突变。实施例7过表达yacm-yacn选择b.subtilis染色体的yqal位点过表达yacm-yacn基因。首先以b.subtilis168的染色体为模板，分别用引物yqal-yacmn-u1/yqal-yacmn-u2q，yqal-yacmn-1q/yqal-yacmn-2，yqal-yacmn-d1q/yqal-yacmn-d2，yqal-yacmn-g1q/yqal-yacmn-g2扩增片段u(1121bp，如seqidno.84所示)、mn(1519bp，如seqidno.85所示)、d(834bp，如seqidno.86所示)、g(812bp，如seqidno.87所示)；以质粒puc57-1.8k-p2为模板，用引物p431/p432扩增含有启动子p43的片段p(232bp，如seqidno.75所示)；以实验室保存的bs168num的染色体为模板，用引物yqal-yacmn-cr1q/cr2扩增片段cr(2069bp，如seqidno.78所示)。然后通过重叠pcr方法，利用引物yqal-yacmn-u1/yqal-yacmn-2，将片段u、p和mn拼接成片段upmn(2872bp)；再利用引物yqal-yacmn-u1/yqal-yacmn-d2将片段upmn和片段d拼接成片段upmnd(3706bp)；最后利用引物yqal-yacmn-u1/yqal-yacmn-g2将片段upmnd、片段cr和片段g拼接成片段upmndcrg(6587bp)。将upmndcrg片段转化受体菌mk3的感受态细胞，经过两步筛选，最终获得p43-yacm-yacn基因在yqal位点整合的重组菌株mk3-mep2。利用引物cx-yacmn1/cx-yacmn2进行测序，无额外点突变。实施例8实时定量反转录pcr分析取菌体生长稳定期初期(48h)的发酵液1ml，12000r/min离心1min收集菌体，利用rnapreppure培养细胞/细菌总rna提取试剂盒提取rna，然后根据fastquantcdna第一链合成试剂盒说明加入反应体系进行反转录得到cdna，最后根据superreal荧光定量预混试剂盒说明加入反应体系进行荧光定量pcr，得到ct值，根据公式根据2-△△ct法计算待测基因的相对表达量。(其中，△△ct＝[(ct(target,test)-ct(ref,test)-(ct(target,calibrator)-ct(ref,calibrator))]；target：目标基因；ref：内参基因，该实验选择ccpa为内参基因；test：待测样本；calibrator：参照样本)。实施例9mk-7的提取及hplc检测取96h和120h发酵液750ml，向其加入1ml的异丙醇和2ml的正己烷，漩涡震荡2min，3000r/min离心10min，取上清750μl进行真空离心浓缩，最后加入500μl甲醇进行溶解。经0.25μm滤膜过滤，取滤液进行hplc检测。所有过程应尽量避光。色谱条件：色谱柱，c18色谱柱(150mm×4.6mm，5μm)；检测器，waters2998pda；流动相，纯甲醇；柱温，50℃；检测波长为270nm；流速1.0ml/min。定量方法：使用相同的色谱条件测定标准品mk-7的标准溶液，绘制浓度-峰面积标准曲线，对mk-7定量。实施例10提高mep路径的代谢通量，促进mk-7的合成甲基赤藓糖醇-4-磷酸(mep)路径通常存在于真细菌、藻类、蓝细菌和植物叶绿体中，主要提供异戊二烯基焦磷酸(ipp)及其异构体二甲基烯丙基焦磷酸(dmapp)用于生物合成类异戊二烯。在枯草芽孢杆菌168内，mep路径共包括十步反应，甘油醛-3-磷酸(g3p)和丙酮酸(pyr)依次经dxs、dxr、yacm、ispe、yacn、ygfy、yqfp、yqid的顺序催化生成法尼西焦磷酸(fpp)，后者经heps/hept催化生成庚异戊二烯基焦磷酸(heptaprenyl-pp)，其是mk-7合成的重要前体物。dxs编码1-脱氧-d-木酮糖-5-磷酸(dxp)合成酶(全长633aa)，催化g3p和pyr合成生成dxp，这是mep路径的第一步反应；dxr编码dxp还原异构酶(全长383aa)，在辅因子nadph的辅助下，催化dxp还原成甲基赤藓糖醇4-磷酸(mep)，这是mep路径的第二步反应；yacm编码mep胞苷酰转移酶(全长232aa)，催化mep和胞苷酰三磷酸(ctp)生成甲基赤藓糖醇4-胞苷酰焦磷酸(cdp-mep)，这是mep路径的第三步反应；yacn编码甲基赤藓糖醇-2,4-环二磷酸(hmbpp)合成酶(全长158aa)，催化2-磷酸-4-(胞苷5'-焦磷酸)-2-甲基赤藓糖醇(mec)裂解生成hmbpp和胞苷酰一磷酸(cmp)，这是mep路径的第五步反应。在枯草芽孢杆菌基因组上，yacm与yacn与未知蛋白的编码基因yack、yacl构成一个操纵子yack-yacl-yacm-yacn。因此为了促进mk-7的合成，本发明利用启动子p43分别在染色体的yjob、ydeo和yqal位点选择过表达枯草芽孢杆菌mep路径的基因dxs、dxr和yacm/yacn，分别得到重组菌株mk3-mep1、mk3-mep2、mk3-mep3。通过摇瓶发酵，得到重组菌株的菌体生长情况和它们在发酵96h和120h的mk-7的产量。由表7可知，上述三个基因过表达对菌体生长影响不大。实时定量反转录pcr分析表明(表8)，利用p43启动子分别过表达上述三个基因后，重组菌mk3-mep1中dxs的表达水平是对照菌mk3中dxs的表达水平的2.35倍；重组菌mk3-mep2中dxr的表达水平是对照菌mk3中dxr的表达水平的3.50倍；重组菌mk3-mep3中yacm-yacn的表达水平是对照菌mk3中yacm-yacn的表达水平的3.70倍，以上结果说明p43启动子是有效果的，上述三个基因的mrna水平提高。由表9可知，发酵96h后，重组菌mk3-mep1的mk-7产量为7.847mg/l，较对照菌mk3提高78.4％；重组菌mk3-mep2的mk-7产量为6.139mg/l，较对照菌mk3提高39.6％；重组菌mk3-mep2的mk-7产量为5.053mg/l，较对照菌mk3提高14.9％。发酵120h后，重组菌mk3-mep1的mk-7产量为9.713mg/l，较对照菌mk3提高47.8％；重组菌mk3-mep2的mk-7产量为9.144mg/l，较对照菌mk3提高39.2％；重组菌mk3-mep2的mk-7产量为8.812mg/l，较对照菌mk3提高34.1％。结果表明，在枯草芽孢杆菌168内，mep路径的dxs，dxr和yacm-yacn是影响mk-7合成的关键基因。如图3所示。表7菌体生长情况发酵时间(h)od600(mk3)od600(mk3-mep1)od600(mk3-mep2)od600(mk3-mep3)63.309±0.0343.402±0.0083.585*±0.0853.773#±0.121128.602±0.0838.250±0.1498.514±0.1658.838±0.0692414.378±0.25313.728±0.19210.469#±0.92814.131±0.1344817.451±0.26914.326#±0.25415.352*±0.68714.020#±0.0357217.114±0.67514.194#±0.25118.186±0.45017.955±0.4059619.599±0.24817.725*±0.06218.235±0.91618.647±0.13612020.153±0.13219.861±0.13720.004±0.91411.917#±0.54814415.203±0.66714.238±0.87312.225#±0.9647.369#±0.457注：*示与mk3相比具有显著差异(p＜0.05)；#示与mk3相比具有极显著差异(p＜0.01)。表8dxs，dxr和yacm-yacn的相对转录水平菌株dxs的相对转录水平dxr的相对转录水平yacm-yacn的相对转录水平mk31.001.001.00mk3-mep12.35#±0.01－－－－mk3-mep2－－3.50#±0.26－－mk3-mep3－－－－3.70#±0.36注：*示与mk3相比具有显著差异(p＜0.05)；#示与mk3相比具有极显著差异(p＜0.01)。表9mk-7产量注：*示与mk3相比具有显著差异(p＜0.05)；#示与mk3相比具有极显著差异(p＜0.01)。因此，为了进一步提高mk-7的合成量，本发明在菌株mk3-mep1的基础上，依次过表达dxr和yacm-yacn，分别得到重组菌株mk3-mep12和mk3-mep123。由表10可知，同时过表达dxs、dxr及yacm-yacn后，菌株生长明显下降。由表11可知，发酵96h后，重组菌mk3-mep12的mk-7产量为8.399mg/l，较对照菌mk3-mep1提高7％，重组菌mk3-mep123的mk-7产量为9.456mg/l，较对照菌mk3-mep1提高20.5％，；发酵120h后，重组菌mk3-mep12的mk-7产量为10.562mg/l，较对照菌mk3-mep1提高8.7％，重组菌mk3-mep123的mk-7产量为12.044mg/l，较对照菌mk3-mep1提高24.0％。由此可见，同时过表达mep路径的基因dxs、dxr及yacm-yacn，可显著提高前体物庚异戊二烯基焦磷酸(hepta-pp)的供应，从而促进mk-7的大量合成。如图4所示。表10菌体生长情况发酵时间(h)od600(mk3-mep1)od600(mk3-mep12)od600(mk3-mep123)63.402±0.0082.379*±0.2501.790#±0.173128.250±0.1498.122±0.4298.203±0.0132413.728±0.19211.435*±0.1109.627#±0.0784814.326±0.25415.773*±0.28611.161#±0.1587214.194±0.25115.462*±0.77710.793#±0.6169617.725±0.06218.472±0.51411.646#±0.48112019.861±0.13719.898±0.06016.541±0.73714414.238±0.87315.151±0.51812.326±0.189注：*示与mk3-mep1相比具有显著差异(p＜0.05)；#示与mk3-mep1相比具有极显著差异(p＜0.01)。表11mk-7产量发酵时间(h)c(mk3-mep1)(mg/l)c(mk3-mep12)(mg/l)c(mk3-mep123)(mg/l)967.847±0.2858.399±0.3259.456*±0.0241209.713±0.17910.562±0.39912.044#±0.008注：*示与mk3-mep1相比具有显著差异(p＜0.05)；#示与mk3-mep1相比具有极显著差异(p＜0.01)。以上所述仅是本发明的优选实施方式，应当指出，对于本
技术领域：
的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。序列表<110>天津大学;赤峰制药股份有限公司<120>基因过表达及获得的菌株、应用<130>mp1809069<160>87<170>siposequencelisting1.0<210>1<211>36<212>dna<213>人工序列(artificialsequence)<400>1agtgaactagtagatctgatgcaagcgaattgtcta36<210>2<211>51<212>dna<213>人工序列(artificialsequence)<400>2atcctctcgagggttaaatgctcctgactctaaagaattggctgtatgctc51<210>3<211>20<212>dna<213>人工序列(artificialsequence)<400>3cgacacggaaatgttgaata20<210>4<211>18<212>dna<213>人工序列(artificialsequence)<400>4ttgtcctctctcccctct18<210>5<211>38<212>dna<213>人工序列(artificialsequence)<400>5agaggggagagaggacaaatgcaagacatctacggaac38<210>6<211>19<212>dna<213>人工序列(artificialsequence)<400>6agggaaatctgtggctgat19<210>7<211>51<212>dna<213>人工序列(artificialsequence)<400>7ctagtggatccgaggatacagccaattctttagagtcaggagcatttaacc51<210>8<211>32<212>dna<213>人工序列(artificialsequence)<400>8catctggtaccgtgaatttagaggcacaagtg32<210>9<211>19<212>dna<213>人工序列(artificialsequence)<400>9gttgaggacggagtgaatg19<210>10<211>21<212>dna<213>人工序列(artificialsequence)<400>10gaagccaatggtgacataatc21<210>11<211>18<212>dna<213>人工序列(artificialsequence)<400>11cagcactcacaaccatca18<210>12<211>42<212>dna<213>人工序列(artificialsequence)<400>12gagtattcatcatttgcgtgtcaaggttccccatataatagc42<210>13<211>21<212>dna<213>人工序列(artificialsequence)<400>13gacacgcaaatgatgaatact21<210>14<211>21<212>dna<213>人工序列(artificialsequence)<400>14ttttcctctctcccctctaat21<210>15<211>42<212>dna<213>人工序列(artificialsequence)<400>15attagaggggagagaggaaaaatgaacgaaacaaataagggt42<210>16<211>20<212>dna<213>人工序列(artificialsequence)<400>16gaactttatccgcagtccat20<210>17<211>40<212>dna<213>人工序列(artificialsequence)<400>17atggactgcggataaagttctgcgttctgtggtttactat40<210>18<211>18<212>dna<213>人工序列(artificialsequence)<400>18atatcgccgcctgtatca18<210>19<211>38<212>dna<213>人工序列(artificialsequence)<400>19tgatacaggcggcgatattcttcaactaaagcacccat38<210>20<211>20<212>dna<213>人工序列(artificialsequence)<400>20ttattcattcagttttcgtg20<210>21<211>41<212>dna<213>人工序列(artificialsequence)<400>21gcacgaaaactgaatgaataagatgatggagcaatataggc41<210>22<211>18<212>dna<213>人工序列(artificialsequence)<400>22agacagaacgaacgcaat18<210>23<211>19<212>dna<213>人工序列(artificialsequence)<400>23tcttcctgatgctggtgaa19<210>24<211>40<212>dna<213>人工序列(artificialsequence)<400>24caagagcgtgatgtttgtccaatccgtgtgataaggtaac40<210>25<211>19<212>dna<213>人工序列(artificialsequence)<400>25gacaaacatcacgctcttg19<210>26<211>21<212>dna<213>人工序列(artificialsequence)<400>26ttgtaaattcctcccttacct21<210>27<211>44<212>dna<213>人工序列(artificialsequence)<400>27aggtaagggaggaatttacaaatggatctcttatcaatacagga44<210>28<211>22<212>dna<213>人工序列(artificialsequence)<400>28gccatcttgccactaataatac22<210>29<211>40<212>dna<213>人工序列(artificialsequence)<400>29gtattattagtggcaagatggctcaatggcacgaagaagg40<210>30<211>18<212>dna<213>人工序列(artificialsequence)<400>30gattctgcggacggtgta18<210>31<211>38<212>dna<213>人工序列(artificialsequence)<400>31tacaccgtccgcagaatctcttcaactaaagcacccat38<210>32<211>39<212>dna<213>人工序列(artificialsequence)<400>32gcacgaaaactgaatgaataaggttgattggagcctgag39<210>33<211>18<212>dna<213>人工序列(artificialsequence)<400>33cgtccagcgtattgagaa18<210>34<211>19<212>dna<213>人工序列(artificialsequence)<400>34accttatcacacggattgg19<210>35<211>19<212>dna<213>人工序列(artificialsequence)<400>35gctaagtgctgtatgaacc19<210>36<211>20<212>dna<213>人工序列(artificialsequence)<400>36ccgactttgtttctttccat20<210>37<211>22<212>dna<213>人工序列(artificialsequence)<400>37gtgtgctgatataaagagagtg22<210>38<211>39<212>dna<213>人工序列(artificialsequence)<400>38caagagcgtgatgtttgtcaagcgataccattccgataa39<210>39<211>46<212>dna<213>人工序列(artificialsequence)<400>39aggtaagggaggaatttacacatgaaaaacatctgtcttttaggag46<210>40<211>22<212>dna<213>人工序列(artificialsequence)<400>40acgctataactgtattcacgaa22<210>41<211>40<212>dna<213>人工序列(artificialsequence)<400>41ttcgtgaatacagttatagcgtcggtgaaggtctgaatca40<210>42<211>19<212>dna<213>人工序列(artificialsequence)<400>42aggaaggctgataggtcat19<210>43<211>39<212>dna<213>人工序列(artificialsequence)<400>43atgacctatcagccttccttcttcaactaaagcacccat39<210>44<211>41<212>dna<213>人工序列(artificialsequence)<400>44gcacgaaaactgaatgaataacggctaacatagaaggagat41<210>45<211>20<212>dna<213>人工序列(artificialsequence)<400>45gctacatcatacacggtaac20<210>46<211>19<212>dna<213>人工序列(artificialsequence)<400>46ttagcatcgttggttcaca19<210>47<211>20<212>dna<213>人工序列(artificialsequence)<400>47tggaagaacattagaacctg20<210>48<211>20<212>dna<213>人工序列(artificialsequence)<400>48caacaaggagcaacaatgaa20<210>49<211>39<212>dna<213>人工序列(artificialsequence)<400>49caagagcgtgatgtttgtctgaagtatggtggcgatgag39<210>50<211>42<212>dna<213>人工序列(artificialsequence)<400>50aggtaagggaggaatttacaaatgagttatgatgtcgtgatt42<210>51<211>19<212>dna<213>人工序列(artificialsequence)<400>51ctctgactgacggtatgga19<210>52<211>38<212>dna<213>人工序列(artificialsequence)<400>52tccataccgtcagtcagagaccaatgcctaagtcagtt38<210>53<211>20<212>dna<213>人工序列(artificialsequence)<400>53cacaataacagatgccgaaa20<210>54<211>40<212>dna<213>人工序列(artificialsequence)<400>54tttcggcatctgttattgtgtcttcaactaaagcacccat40<210>55<211>41<212>dna<213>人工序列(artificialsequence)<400>55gcacgaaaactgaatgaataagatatggtgacggaagatga41<210>56<211>20<212>dna<213>人工序列(artificialsequence)<400>56acggattgttggtgtaagat20<210>57<211>19<212>dna<213>人工序列(artificialsequence)<400>57tcactagaccgaatggaga19<210>58<211>18<212>dna<213>人工序列(artificialsequence)<400>58acttaccgccttgattgc18<210>59<211>1115<212>dna<213>人工序列(artificialsequence)<400>59cagcactcacaaccatcagcgcgcagaatacaaataaaatgatctcttgaagaagttttt60ttctgtattcagctttcatttgtgacagctggtgaagcagttctttttgattcggtatag120agggattggctgtttgatcaatttttttccattctgactttaaatgatcagaaagctttt180ctttattcatcatcccactcctttctgatttgctggagccctttatgaactctggacttt240acagtgccttctttgatttgaagcatcaccccgatttcagcgtaggtatagccgtagtaa300tgccttaataaaaccggtgtgcgcagtttagggtccagctttgaaaacagatcgagcgtt360tcgctccactctgcccctttcgccgacacgtcccacttgatcttgcggattgtttcctcc420gtaactgtctgattccgtttccattctctctttctctttttctggtgatcgagatagagc480cgcgatgcaatggagatcagccatgtggagaattttgaacggccctgaaagcttcttaaa540tggatgtaccctttcagaaaggtttcctgaacaagttcctcgctgagatcggggtgaagg600gaaagctttaacaagtatttatataagaaagaatagtgatagtggaaaagggctgtaaac660gcctcgtcgtttccctctttcgcctgctggatcagccgctgttcttcttgtgtatccaat720gaccgtgatccccccttttttgtttttctgccttatgagacgacaccggattcaaaagcg780ttcatcttttttacaattcttttcagccattgacagatcaggcttcccggccgtttggct840gcatatccttatttgtaaacccccttatagtctgttaagctatgactttatgtcgatttc900aaaaaaacatccaatatggaggacgatgcattgaaagtagaaaggcgaacgattgaatat960attccgaatgaagaaagacatggtaaagcgaaagatttatttcccgtatggttcggggct1020aatatgcatatcaccacgttagtgaccggcacgattcccgttgcaatgggacttaatctg1080ttttggagtgttgccgctattatatggggaacctt1115<210>60<211>442<212>dna<213>人工序列(artificialsequence)<400>60gacacgcaaatgatgaatactcatactcttcctttttcaatattattgaagcatttatca60gggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatagg120ggttccgcgcacatttccccgaaaagtgccacctgacgtcccaatgatgaattcgcggcc180gcttctagagtcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatt240tactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaa300atagcgagagatgatacaagaacgtcctgatcttattataatataagcaaaaaactcata360aaaaggaaaagcattgacctgaaaacttatcggtaaagtatgatataatacaaaaagacc420gattagaggggagagaggaaaa442<210>61<211>1057<212>dna<213>人工序列(artificialsequence)<400>61atgaacgaaacaaataagggtgagggtcagacagcgccgcaaaaagaaagcatggggcag60atcctttggcagttaacccgtcctcatacgttaaccgcatcgtttgtgcctgtgctgctc120ggaaccgttttggcgatgttttatgtgaaggttgatctgctgctgtttttggctatgctg180ttttcttgcctatggatccagatcgcgacgaacttatttaatgaatactatgattttaaa240cgcggattagatacagcagaatcagtcggaatcggaggggccattgtacgccacggaatg300aagcctaaaacgattttgcaattagctcttgcctcatacgggattgccattttgctcggt360gtctatatttgtgccagcagcagctggtggcttgcgctgatcggccttgtcggcatggcg420atcggctacctgtatacaggcgggccgctgccgattgcgtacacgccgttcggtgaatta480ttctcaggcatttgcatgggttcggtgtttgtgctgatttcgtttttcattcagacagat540aagatcaatatgcagagcattttgatttccatcccgattgcgattcttgtcggcgcgatt600aacctgtcgaacaacattcgtgatattgaagaggacaaaaaaggcggacgcaaaacattg660gcgattttgatggggcataagggagctgttactctgttagctgcgtcgtttgccgtcgct720tatatctgggttgtcggcttggttattaccggtgccgcaagcccatggctgtttgtcgtc780tttttgagcgtgcctaagccggttcaggcagtgaagggcttcgtccagaacgaaatgccg840atgaatatgattgtcgcaatgaaatcaacagcccaaacaaatacatttttcggattcctg900ctctcgatcggattattgatcagctatttccgataataaaaaagaccgctcgtttcatgc960ggtctttttttgttacaatcgaccgcattttgtaaaaaaattcatagaaccttgcagcag1020acagggacgtctagtacatggactgcggataaagttc1057<210>62<211>1053<212>dna<213>人工序列(artificialsequence)<400>62tgcgttctgtggtttactatgtattaatgaaaccgcgtctgaaaaaacgggcggggtatc60aagaaaagctgtcttctctctgaagacagcttttttatcattcaaatcgtttccacacgc120ggggcaggatgtcagatagctcatgagcgagcaacgtatgagcggagtgctcgtccgtcc180agagctcggcgcacgcaccatgcaaatagaccgcgtttaatacggcgtgctttggatctt240catggcagcagagcatccccaatatcatgcccgtcagcgtgtcaccggttccgcccttgg300cgagcgccccgtttccggttggattgagccagcagtctccatcaggaaacgcgatgacgg360tttggtttccttttaacacaatgacagtctgcagctgagccgcccattcctttgcatatt420ccgctctttttttctgcaattcgttcacgggcacccctgtcattcggaaaaactcgcccg480ggtgcggcgtgaggatgacggggccctcccgtttcggatatgtgcgcttcgccagcgctc540cggcatccaaaataacaggacaatcagccgtaagcacatgatcgactgcctgctgtacgc600tttccgtttgcggaaggcccggtccgatggcaatggcccggtacgtctcttcaagctgag660catccgccgcctttttccagccgtcacgccaataggtcgcctcgggcaaaacaggaacaa720ttagcgggattacgttttctgatgtaccgatgacaagcttgccgagaccgctccgcatgg780ccccaagccctgcaagcagcgctgctcccggcatgtcgtcacttccagccagcagcaaag840ccgtaccgtacgtgcctttatggctctccgcatctctttcaggaagcgtggcccttacat900gctcttccgtccaaaatggaacattcatctctatccctcctctgctttcttttcctattt960ttcctgtcccttttctttttcaaacaaaaaagacagccttttagggctgtcttttatttt1020acaggaacatcaaggtgatacaggcggcgatat1053<210>63<211>2069<212>dna<213>人工序列(artificialsequence)<400>63tcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatattttt60aaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaag120aaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaa180tgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatt240tgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaa300acataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtga360taaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattggg420ataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtattt480ggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagaga540aatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctc600tttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagta660attaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatt720taccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgttta780tgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataa840tgccgactgtactttttacagtcggttttctaatgtcactaacctgccccgttagttgaa900ggcattttctgtcaatgttttcttacaaagaacgctgtgatatactgaaatttgtccgta960tacattttggaggaatggatatgttaccaaaatacgcgcaagtaaaagaagaaatcagtt1020cttggattaatcaaggcaaaatactgcccgatcaaaaaatccctaccgaaaacgaattaa1080tgcagcaattcggcgtcagccggcataccatccgcaaagcgatcggagacctcgtatcac1140aaggtctgctgtacagcgtgcaaggcggaggcacctttgtcgcttcacgctctgctaagt1200cagcgctgcattccaataaaacgatcggtgttttgacaacttacatatcagactatattt1260tcccgagcatcatcagaggaatcgagtcctatttaagcgagcaggggtattctatgcttt1320tgacaagcacaaacaacaacccggacaatgaaagaagaggcttagaaaacctgctgtccc1380agcatattgacggactcatcgtagaaccgacaaaaagcgcccttcaaaccccaaacatcg1440gctattatctgaacttggagaaaaacggcattccttttgcgatgattaacgcgtcatatg1500ccgagcttgccgcgccaagttttaccttggatgatgtgaaaggcgggatgatggcggcgg1560agcatttgctttctctcggccacacgcatatgatgggtatttttaaagctgatgacacac1620aaggcgtgaaacggatgaacggatttatacaggcgcaccgggagcgtgagttgtttcctt1680ctccggatatgatcgtgacatttacaacggaagaaaaagaatcaaaacttctggagaaag1740taaaagccacactggagaaaaacagcaagcacatgccgacagccattctttgttataacg1800atgaaattgcgctgaaggtgattgatatgctgagggagatggatcttaaagtgccggagg1860atatgtctattgtcgggtacgatgattcacatttcgcccaaatctcagaagtgaaactaa1920cctctgtcaaacatccgaaatcagtgcttggaaaagcagccgccaaatatgtcattgact1980gcttagagcataaaaagccgaagcaagaggatgtcatatttgagcctgagttgatcattc2040gccagtccgcacgaaaactgaatgaataa2069<210>64<211>806<212>dna<213>人工序列(artificialsequence)<400>64gatgatggagcaatataggcttaagtgccgtatgttttttactcaccattttcggccatg60atttgattcacaaaatgcaaaaaatcctttcttggacctcttttgctgtattttttgctg120caacgattctcatttttcaattgccgattccggcaggcagctggatacccggagcgattg180atctgccaattttcctagtggcagtcagcgcagttgcgacgtggcagcttgcctacgcgc240cgtatgttgccgattattcaagatatctgccagttaaaacaccggcttcaaaaacatttt300ggtacagctatgcaggtacatctgtcagctccatctggatgatgctgctcggcgcattgc360tgacaacctcacttcctgactttacggcaaactcaggcagccagattgtccaattgttcg420gtcccttctcgtttattatgctgatcattgtgctatttgggcaaatggcaatcaatgttt480ttaacctgtatggggctttcatgtccacgacaacaacgctggagccgtttctgaaattaa540aagtcacgcccaaagtcagaatcatcatgatcctgggtgtcacgcttgtcggcacagtct600taagcctactgggacagagcaattttatggaactgtttttgaattttatctttttcatca660gttatttcttaattccgtggacggcaatcaatttagtcgattactacttcgtccgccacg720gaaagtatcaagtcaaagccatgttcgacgtaaacgggccttatggaaaggtcaattgga780tcaccacaattgcgttcgttctgtct806<210>65<211>6542<212>dna<213>人工序列(artificialsequence)<400>65cagcactcacaaccatcagcgcgcagaatacaaataaaatgatctcttgaagaagttttt60ttctgtattcagctttcatttgtgacagctggtgaagcagttctttttgattcggtatag120agggattggctgtttgatcaatttttttccattctgactttaaatgatcagaaagctttt180ctttattcatcatcccactcctttctgatttgctggagccctttatgaactctggacttt240acagtgccttctttgatttgaagcatcaccccgatttcagcgtaggtatagccgtagtaa300tgccttaataaaaccggtgtgcgcagtttagggtccagctttgaaaacagatcgagcgtt360tcgctccactctgcccctttcgccgacacgtcccacttgatcttgcggattgtttcctcc420gtaactgtctgattccgtttccattctctctttctctttttctggtgatcgagatagagc480cgcgatgcaatggagatcagccatgtggagaattttgaacggccctgaaagcttcttaaa540tggatgtaccctttcagaaaggtttcctgaacaagttcctcgctgagatcggggtgaagg600gaaagctttaacaagtatttatataagaaagaatagtgatagtggaaaagggctgtaaac660gcctcgtcgtttccctctttcgcctgctggatcagccgctgttcttcttgtgtatccaat720gaccgtgatccccccttttttgtttttctgccttatgagacgacaccggattcaaaagcg780ttcatcttttttacaattcttttcagccattgacagatcaggcttcccggccgtttggct840gcatatccttatttgtaaacccccttatagtctgttaagctatgactttatgtcgatttc900aaaaaaacatccaatatggaggacgatgcattgaaagtagaaaggcgaacgattgaatat960attccgaatgaagaaagacatggtaaagcgaaagatttatttcccgtatggttcggggct1020aatatgcatatcaccacgttagtgaccggcacgattcccgttgcaatgggacttaatctg1080ttttggagtgttgccgctattatatggggaaccttgacacgcaaatgatgaatactcata1140ctcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatac1200atatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaa1260gtgccacctgacgtcccaatgatgaattcgcggccgcttctagagtcaggagcatttaac1320ctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttctttt1380ctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatacaagaacgt1440cctgatcttattataatataagcaaaaaactcataaaaaggaaaagcattgacctgaaaa1500cttatcggtaaagtatgatataatacaaaaagaccgattagaggggagagaggaaaaatg1560aacgaaacaaataagggtgagggtcagacagcgccgcaaaaagaaagcatggggcagatc1620ctttggcagttaacccgtcctcatacgttaaccgcatcgtttgtgcctgtgctgctcgga1680accgttttggcgatgttttatgtgaaggttgatctgctgctgtttttggctatgctgttt1740tcttgcctatggatccagatcgcgacgaacttatttaatgaatactatgattttaaacgc1800ggattagatacagcagaatcagtcggaatcggaggggccattgtacgccacggaatgaag1860cctaaaacgattttgcaattagctcttgcctcatacgggattgccattttgctcggtgtc1920tatatttgtgccagcagcagctggtggcttgcgctgatcggccttgtcggcatggcgatc1980ggctacctgtatacaggcgggccgctgccgattgcgtacacgccgttcggtgaattattc2040tcaggcatttgcatgggttcggtgtttgtgctgatttcgtttttcattcagacagataag2100atcaatatgcagagcattttgatttccatcccgattgcgattcttgtcggcgcgattaac2160ctgtcgaacaacattcgtgatattgaagaggacaaaaaaggcggacgcaaaacattggcg2220attttgatggggcataagggagctgttactctgttagctgcgtcgtttgccgtcgcttat2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lsequence)<400>73atgcaagacatctacggaactttagccaatctgaacacgaaattaaaacaaaagctgtct60catccttatttagcgaagcatatttctgcgccgaaaattgatgaggataagcttcttctt120tttcatgctttatttgaagaagccgacataaaaaacaacgacagagaaaattatattgta180acagcgatgcttgtacaaagcgcccttgatacccatgatgaagtgacgacagctagagtc240ataaaacgagacgaaaacaaaaaccgccaattgactgttctcgcgggcgattatttcagc300gggctgtactactctttactatctgaaatgaaggatatctacatgattcggacgcttgct360acagccattaaagaaatcaacgaacataaaattcgtctgtatgaccgttctttcaaggac420gaaaacgattttttcgaaagtgtcggcatcgttgaatcagctttattccatcgtgtggcg480gaacacttcaacctcccgcgctggaaaaagctgtcgagtgatttttttgtatttaagcgg540cttatgaacggaaatgatgcatttctggatgtgatcggcagttttatacagctgggaaaa600acaaaagaagagatattagaagattgttttaaaaaagcgaaaaacagcattgagtcactt660ctgcctctaaattcac676<210>74<211>1236<212>dna<213>人工序列(artificialsequence)<400>74tcttcctgatgctggtgaaaggcggggaacaaatgcaatatgccaatggctagcgtagca60ctcggaatactgccgatggctaagtattttaccaatttaaagttaatcgttttctgccgc120caatgggaggcaacgccaaacagctttgtgatcgagttatatacaagatccgtaccgacc180gcgatggaaggattaattccaagcacaattaaaaggggagttaataaggccgctccaccc240actcccgtcaatccgactaaggctccgacaaacaaacccatcaatatgatgcttacactc300atgataaaggcctcctgaaaacccgactatttttatcagattatatatgattaggacgcg360ttaatcaattaaattttttgaatttttgcacgttagttgcttgaaattttggtatgctca420atatctctattcatttgccctataatgagtgtctggacgaaatcgagaagtactaaaggc480gcaaaaaaaagatacctcttaacaggtatcttttttcttgtttacgctacatgcgctgct540gatagaaaggaagctcagtgtggcccatttcccgcacatacacgaacaggttgcctttat600ggtgaatttcatgctccattgcaagctgaagcagcgcgcggcctgtgacttttctgccga660aagcggatgtcagatcaatttctctgtccagctgctcttcagtcagctgttcaaggatag720cgaccgttttttctgtatatgtttttgccagaacattgagatctgtttctgtttcttcct780gcttgttttgaaatggtgaggcattgccttccttaatgacatttgcgaacaagtggaaag840acgtaagaatgtgtttgaccagctcttctgctgacattgatgtttctgccggtttgtagc900tgtaatggtctttgctgattttttcggcaagctcattggtcacgtttcgatgggataaaa960aatggctgacaatttgattggattggcacattatgatctcctccatatgtaaaaataaat1020ggtgcaatgaaatcctatatcaacggttatgaattcacaagtaatatgctgtgggatgaa1080acaaaatgctatgtcaatcgtatatataacgttcacaaaacatgaaatggagtataaaca1140tgactaacatacctttcatttatcagtacgaagaaaaagagaatgaaagagctgcggcgg1200gctacggcactttcggttaccttatcacacggattg1236<210>75<211>232<212>dna<213>人工序列(artificialsequence)<400>75gacaaacatcacgctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtt60tttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctg120aaaattcttacatttattttacatatttagaaatgggcttgaaaaaaagcgcgcgattat180gtaaaatataaagtgatagcggtaccattataggtaagggaggaatttacaa232<210>76<211>1947<212>dna<213>人工序列(artificialsequence)<400>76atggatctcttatcaatacaggacccgtcgtttttaaaaaacatgtccattgatgaatta60gagaaattaagtgatgaaatccgtcagtttttaattacaagtttatccgcttccggcggc120cacatcggcccaaacttaggtgtcgtagagcttactgttgccctgcataaggaatttaac180agcccgaaagacaaatttttatgggatgtaggccatcagtcgtatgtccataagctgctg240acaggacgcggaaaagaatttgcgacgcttcgccagtacaaagggctttgcggatttcca300aagcggagtgaaagcgagcacgatgtttgggaaaccgggcacagctcgacttctctttca360ggcgcgatgggaatggcagctgcccgtgatattaaaggaacggatgaatatattattccg420atcattggtgacggcgcgctgaccggcggtatggcgctcgaagcccttaaccacatcggc480gacgagaaaaaagacatgattgtcatccttaatgataatgaaatgagtattgcgccaaac540gtcggtgccattcactctatgctcggacggctccgcactgcggggaaataccagtgggtc600aaagatgagcttgaatacttatttaaaaagattccggcagttgggggcaagcttgccgcc660acggcggaacgggtcaaagacagcctgaaatacatgctcgtctccggaatgtttttcgag720gagctcggttttacgtatttgggcccagtggacggacattcttatcatgagctgattgag780aatcttcaatacgccaaaaaaacgaaaggccctgttcttctgcacgtcattacgaaaaaa840gggaaggggtacaaaccggctgagaccgatacgattgggacatggcatggtaccggacca900tataaaattaataccggtgactttgtaaagccgaaagccgcagctccttcgtggagcggt960cttgtcagcggaactgtgcagcgaatggcgcgcgaggacggacgcattgtagccattacg1020ccggctatgcctgtcggttcaaagcttgaaggcttcgcaaaggaattccctgaccggatg1080ttcgacgtaggaatcgcagaacagcatgccgcaacaatggctgcagctatggcaatgcag1140ggtatgaagccgtttttggcgatttactcaaccttcctgcaaagggcatatgaccaagtt1200gttcatgacatctgccgccaaaacgctaatgtgtttattggaattgaccgtgctggactc1260gttggcgctgatggagagacacatcaaggcgtgtttgatattgcgtttatgcgccacatt1320ccaaacatggtcttaatgatgccgaaagacgaaaatgaaggccagcacatggttcataca1380gcacttagctatgacgaaggcccgatagcaatgcgttttccgcgcggaaacggactcggc1440gtaaaaatggatgaacagttgaaaacgattccgatcggtacgtgggaggtgctgcgtcca1500gggaacgatgctgtcatcttaacattcggcacaacaatcgaaatggcgattgaagcagcc1560gaagagctgcagaaagaaggcctttccgtgcgcgttgtgaatgcgcgttttattaagccg1620attgatgaaaagatgatgaagagtatcctaaaagaaggcttgccaattttaacaattgaa1680gaagcggtcttagaaggcggtttcggaagctcgattttagaattcgctcatgatcaaggt1740gaatatcatactccgattgacagaatgggtatacctgatcggtttattgaacacggaagt1800gtaacagcgcttcttgaggaaattggactgacaaaacagcaggtggcaaatcgtattaga1860ttactgatgccaccaaagacacacaaaggaattggatcatgacgtcaaagaaagaacgat1920tagatgtattattagtggcaagatggc1947<210>77<211>808<212>dna<213>人工序列(artificialsequence)<400>77tcaatggcacgaagaagggaatcaccatattgaaaccatggtgaaagacatgacaggaga60gcaaagaaaaagccagcggggaagctggatggaaagaaacaaagtcggttttcactaaaa120gaaagcacgggtgtttgaaaaacccgtgcttttttgttgcggttagccgaaattcgacaa180ttgcggttattttgcgttcttctttttcttgtaaatatgataaaatatgacatatctcgg240gtaattcaaaaggggggattaattgaggatgaagcagacgattccgtcctcttatgtcgg300gcttaaaattaatgaatggtatactcatatccggcagttccacgtcgctgaagccgaacg360ggtcaagctcgaagtagaaagagaaattgaggatatggaagaagaccaagatttgctgct420gtattattctttaatggagttcaggcaccgtgtcatgctggattacattaagccttttgg480agaggacacgtcgcagctagagttttcagaattgttagaagacatcgaagggaatcagta540caagctgacagggcttctcgaatattactttaatttttttcgaggaatgtatgaatttaa600gcagaagatgtttgtcagtgccatgatgtattataaacgggcagaaaagaatcttgccct660cgtctcggatgatattgagaaagcagagtttgcttttaaaatggctgagattttttacaa720tttaaaacaaacctatgtttcgatgagctacgccgttcaggcattagaaacataccaaat780gtatgaaacgtacaccgtccgcagaatc808<210>78<211>2069<212>dna<213>人工序列(artificialsequence)<400>78tcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatattttt60aaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaag120aaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaa180tgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatt240tgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaa300acataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtga360taaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattggg420ataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtattt480ggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagaga540aatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctc600tttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagta660attaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatt720taccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgttta780tgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataa840tgccgactgtactttttacagtcggttttctaatgtcactaacctgccccgttagttgaa900ggcattttctgtcaatgttttcttacaaagaacgctgtgatatactgaaatttgtccgta960tacattttggaggaatggatatgttaccaaaatacgcgcaagtaaaagaagaaatcagtt1020cttggattaatcaaggcaaaatactgcccgatcaaaaaatccctaccgaaaacgaattaa1080tgcagcaattcggcgtcagccggcataccatccgcaaagcgatcggagacctcgtatcac1140aaggtctgctgtacagcgtgcaaggcggaggcacctttgtcgcttcacgctctgctaagt1200cagcgctgcattccaataaaacgatcggtgttttgacaacttacatatcagactatattt1260tcccgagcatcatcagaggaatcgagtcctatttaagcgagcaggggtattctatgcttt1320tgacaagcacaaacaacaacccggacaatgaaagaagaggcttagaaaacctgctgtccc1380agcatattgacggactcatcgtagaaccgacaaaaagcgcccttcaaaccccaaacatcg1440gctattatctgaacttggagaaaaacggcattccttttgcgatgattaacgcgtcatatg1500ccgagcttgccgcgccaagttttaccttggatgatgtgaaaggcgggatgatggcggcgg1560agcatttgctttctctcggccacacgcatatgatgggtatttttaaagctgatgacacac1620aaggcgtgaaacggatgaacggatttatacaggcgcaccgggagcgtgagttgtttcctt1680ctccggatatgatcgtgacatttacaacggaagaaaaagaatcaaaacttctggagaaag1740taaaagccacactggagaaaaacagcaagcacatgccgacagccattctttgttataacg1800atgaaattgcgctgaaggtgattgatatgctgagggagatggatcttaaagtgccggagg1860atatgtctattgtcgggtacgatgattcacatttcgcccaaatctcagaagtgaaactaa1920cctctgtcaaacatccgaaatcagtgcttggaaaagcagccgccaaatatgtcattgact1980gcttagagcataaaaagccgaagcaagaggatgtcatatttgagcctgagttgatcattc2040gccagtccgcacgaaaactgaatgaataa2069<210>79<211>697<212>dna<213>人工序列(artificialsequence)<400>79ggttgattggagcctgagcatgtggcatatatatttgaaaagcttgaaaagaaaaccttt60gcttacgatgaagatgagaaagaaccggattataccgtccataagtctataagaaacagt120gtatacgcgtatcctgaaaagggagttgcttttgccagaattccatattttcaagatggg180agcattatgagttttgattgtctgtttgcggttaacgatgaaaagatgcgtgcatttctg240gagggagtcagaccgcgtctttgggaaaaaagcaagcggaaagtgaccgtgtttacggat300ggagatggagggacttcaagagagcaggaagccattgtcagagaggttcagcggagtcaa360gtcatcatgaatccgctattgaaaaaagagatatacagatcaattgatcagttttttcat420agtgataaatcgttttatcaaacatatgacatcccttacaagcgcggcattctgttatat480ggacctcctggaaacggaaagacgacgttagtgaagtcgatcgcaggcagtatcgatgca540cctgttgcttattggcaaattactgaatttacgtcgagcgagacaatagaagaagtcttt600caggcagcgagacgcctcgctcctgcagttctggtcatcgaggatatagattcgatgccg660gaagatgtgcggtccttttttctcaatacgctggacg697<210>80<211>1909<212>dna<213>人工序列(artificialsequence)<400>80gtgtgctgatataaagagagtgtatccagccaatcctcaagtcttggctggagcggatta60ggcatgtttaaaatgtctgcttggaccccatctgcgagcagacgttttttcagccaaggg120aaccaatgatttgttgaggaggctctgtagccatgaatgatatagacttgttttgtcatt180atttcctcctaagaaatattttcattcagtataaaattcctctcattgggaaacaagtac240gcacttttttgtaagtagctttacatatgaggccctccttgtgccattccccacttacac300atctcatctatgaggggatacaaagatcgccctctgtcagataacgaatactctactttc360agtggggtgtgagaaacctcagaccgatgaataaggccgtcagtttgcaattccttaagc420tgtgcgcttaacattttatgggtgatgaagggcatagcccttttcagctcgccgtatctc480atcgttttttcacaccctaactcatagagaattctcattttccatttgccccctataaga540gataaggtgtattcaaatggggcttgtttactgcggcacaggctattcattgtaatcaaa600cctccttttttaagtatcatatcagaataaattaggattttcggtgttgaagtttgaggg660cgattcacgatctgaagtgaaaggggccgcttgctcaacaggcggcacggtacatgaatg720ttctaagtcaaagagggttaccttactgttagttactttatttaaaaaaacgcatgtttt780gtgtgctgatataaagagagtgtatccagccaatcctcaagtcttggctggagcggatta840ggcatgtttaaaatgtctgcttggaccccatctgcgagcagacgttttttcagccaaggg900aaccaatgatttgttgaggaggctctgtagccatgaatgatatagacttgttttgtcatt960atttcctcctaagaaatattttcattcagtataaaattcctctcattgggaaacaagtac1020gcacttttttgtaagtagctttacatatgaggccctccttgtgccattccccacttacac1080atctcatctatgaggggatacaaagatcgccctctgtcagataacgaatactctactttc1140agtggggtgtgagaaacctcagaccgatgaataaggccgtcagtttgcaattccttaagc1200tgtgcgcttaacattttatgggtgatgaagggcatagcccttttcagctcgccgtatctc1260atcgttttttcacaccctaactcatagagaattctcattttccatttgccccctataaga1320gataaggtgtattcaaatggggcttgtttactgcggcacaggctattcattgtaatcaaa1380cctccttttttaagtatcatatcagaataaattaggattttcggtgttgaagtttgaggg1440cgattcacgatctgaagtgaaaggggccgcttgctcaacaggcggcacggtacatgaatg1500ttctaagtcaaagagggttaccttactgttagttactttatttaaaaaaacgcatgtttt1560tagctgaaaatatctattatggtatatttaaatataaagaaatatttgagaaatgaggaa1620tggtatgaatacacccaaaaaacataagaaatttaaagcgaaaatgattctccaaatcat1680catggttatcattggaggaatcatagcggcatatggactagaaacagtgctcattccaaa1740cagtgtatctgatggaggggtgacgggtcttagcatcgttggttcacagctattcaactt1800gccgctggggattttgattgctgtgatcaacattccatttgtctggttaggatataagca1860aattgggaaaagctttgcattattatcaattatcggaatggtatcgctt1909<210>81<211>1182<212>dna<213>人工序列(artificialsequence)<400>81atgaaaaacatctgtcttttaggagcaacaggatcaatcggcgaacagactcttgatgta60ctgcgtgcacatcaagaccaatttcagctggtatctatgtcgtttggcagaaatattgat120aaggccgttccaatgattgaggtctttcagccgaagtttgtctctgtcggtgatctggat180acatatcataaattaaaacaaatgtctttttcttttgaatgccaaatcgggctgggagaa240gaaggactgatagaagcagcagtgatggaggaggttgacattgttgtcaacgctttgctt300ggaagcgtcggtcttatcccgacattaaaggcaattgaacagaaaaaaacaattgcgctt360gcaaataaggaaactcttgtcactgcagggcatatagtaaaagaacatgctaagaaatat420gatgttccgctgctgcctgttgacagtgagcattcagccatttttcaagctcttcaaggc480gaacaggctaaaaacattgaacgcctcatcattacggcatccggcggaagttttcgggac540aagacgcgggaggagctcgaatcagtaacggttgaagatgcattaaaacatccaaactgg600tcaatgggtgcaaaaattacaattgattcggctacaatgatgaataagggattagaggtg660atcgaggcacactggcttttcgatataccttatgaacaaattgatgtggttttacataag720gagagcatcatccattcaatggttgagttccatgataaaagtgtgatcgcacagctcgga780actccggatatgagagtccccattcaatatgcgctcacttatcctgaccgattgccatta840ccagatgcgaaaaggcttgaattatgggaaatcggcagccttcattttgagaaagctgat900tttgacaggttccgatgcttacaatttgcttttgaatcaggtaaaataggaggaacaatg960ccgacagtgctaaatgcggcaaacgaagtagctgtcgctgcctttttagctggcaagata1020ccgtttttggctattgaagactgtatcgaaaaggcactaacccgccatcaattactgaaa1080aaaccgagctggcggacattcaagaagtggacaaagatacccggggatacgtcaattcaa1140tactcacataaggtggtatgttcgtgaatacagttatagcgt1182<210>82<211>1005<212>dna<213>人工序列(artificialsequence)<400>82cggtgaaggtctgaatcacagaaataccaagggcgtcaaaacccttggtattttttctgg60ttattagattttatcttcagctttacgtgctgcttcgagcttatcaatcctgtctttata120tttttctccccagtccttcatgtgcaggataacaggttctaatgttcttccaaactcagt180taaagagtattcgacttttggagggacctgtttataaacctcgcggtgaatcacgccatc240ccgttccaattctcttaattgtagcgtgagcatacgctgggtaatttttgggtatagttt300tcgaaactcattaaacctctttttaccgtcaatgagatgataaagaagaatgcctttcca360cttacctccgatgatatctaatgttgtctccaccggacatccttcaaagtcatcagaaga420ataatcaatgccgagtttacgatttctcaagctgttcactcctaatagtatactttttga480tactatatacattcaatgtgcgtactttttatattttaatatacatcatataatgatgtc540acatctaaggaatgaaaggaatggatcatatgaaaacactcgtactcgttgtacatccga600atatagaatcctctcgtatcaataaaaagtggaaagaagccgttttaagtgaaccagatg660taactgtccatgatctttatgaaaaatatcgcgatcaaccaattgatgtggaatttgaac720aacagcagctcctggcccatgaccgtatcgtttttcagtttccattatactggtacagca780gcccaccgcttttaaaacagtggtttgatgaagtgtttacgtttggctgggctcatggtc840ccggcggaaataaattgaaggggaaagagtgggtaactgccatgtccatcggttcacctg900aacactcttatcaagccggcggatataacttgttttcgataagcgagctgacaaaaccgt960tccaagcatctgcccatttagtaggcatgacctatcagccttcct1005<210>83<211>514<212>dna<213>人工序列(artificialsequence)<400>83cggctaacatagaaggagataccctgttaattaccgttgtcggcgggattatccttggat60tcggaatggggctggcgctgcgtaatggcggcgcattagatggaattgacatgcttgctg120tgctgctatcccgaaaactgccattcggaacgagcgatctcattctattcttaaacctat180ttgtgtttatttttgtttccacagtatttggcttacaaggggcattgctttcagtcattg240cttactacattgcatccaaagtgattcatgttgtggaggaaggcttaagcggctctaaaa300cctttcaaattattacaacccaacctgaattaatggtagagacaatacgtgatcaattag360gccgaagcgccacatataaagaagcttacgggggtttttcccacgagaaatttaaagaaa420tcacatgtgtcatcaaccgtttagaagaaaccaaactaaaggaaattattaatgatattg480ataaaactgcctttgttaccgtgtatgatgtagc514<210>84<211>1121<212>dna<213>人工序列(artificialsequence)<400>84caacaaggagcaacaatgaaagggctactttcctcaccatctgttattaaacgatttgag60gaagtgttagggaagagggctacacagtttactgcttctatcttaagcctttataacagc120gagcagatgctacagaaaacagatcctatgagcgtcatatcctcagcaatggtggcagct180acactcgatttacctatagataaaaacttagggtatgcctggattgttccttacggaggt240aaggctcaatttcagcttggatacaaaggatatatccagctagccttacgaacaggccaa300tataaatccatcaattgcataccgattcatgaaggcgaattgcagaagtggaatccgttg360actgaggagatcgagattgattttgaaaaacgagaatcagacgcggtaattggttatgca420gcttattttgagttgataaatggcttccgaaaaacagtgtactggacaaaggcacaagta480gagaagcacaaaaagaaattcagtaagtctgattttggatggaaaaatgactgggatgcg540atggctcttaagactgtattaaaagcagttttgagcaagtgggggattctctctgttgaa600atgcaaaaagcagttattgaggaagatgaaacaagggaacggattgacattaccaatgag660gcagacagttcagaaattatcgattccgagccttcaaacaaagatgaaacggaaaaacca720agcgcccaagaaaccgatccttttgacggcaagcctgtagatataaaagacgatgaactt780ccgtttgattgagccggcatctctgacataactgcactctgtgaaaggaagtaggtgatt840gacttggacataaaagcaatggggtatgtggtcataccccgactaccattcaaagagttt900agggatgaaaaaatttatgatcacttgttcaaaagagctgaatacaggccaaatcaagag960ctagaacttgggcagaccattatcaaagttgtggaactagcaaaagattttaactggtca1020gctgcacagatcaaatactcactagaccgaatggagaaacagggatatatcaaattgcac1080cgtcttccacaaaaaagagggctcatcgccaccatacttca1121<210>85<211>1519<212>dna<213>人工序列(artificialsequence)<400>85atgagttatgatgtcgtgattcctgcagccggacagggaaagcggatgaaggcagggaga60aataaactgttcattgagctgaagggagacccggtgatcatacacacgttaagagtgttt120gacagccaccggcagtgcgataaaatcattttggtgattaacgagcaggagcgggagcac180tttcagcaattgctgtccgattacccgtttcaaacttcaattgagcttgttgcaggcgga240gatgagcgacagcacagtgtgtataaggggctgaaagccgtaaagcaggaaaagattgtc300cttgtacatgacggtgcccgtccatttataaaacatgaacaaattgacgaactgatcgca360gaggcggaacagacaggagcggccatccttgctgttccggtaaaagatacgattaaacgc420gttcaagatttacaagtcagtgagacgattgaacgttcaagcttgtgggctgtccaaacg480ccacaagcttttcgtctttctttattgatgaaggctcacgctgaggccgagcgcaaggga540tttttagggacggatgacgccagcctcgttgaacagatggagggcggttcggtccgtgtt600gtagaaggcagctatacaaatattaagctgacgacgccagacgatttgacgtcagctgaa660gctatcatggaatcagaaagtgggaataaacatgtttagaattggacaaggatttgatgt720gcatcaattagtggagggccgtcctctcattattggcggaattgaaatcccgtacgaaaa780agggctgcttggccattctgatgcagacgtattgttgcataccgtcgctgacgcctgcct840gggagctgtgggtgaaggagacataggcaagcattttcctgacacagatcccgagttcaa900ggacgcagattctttcaaattacttcagcatgtctggggaatcgtgaaacagaaggggta960tgtccttgggaacattgattgcaccatcatagcgcaaaagccgaagatgctgccgtacat1020agaagatatgagaaaaagaattgctgaaggccttgaggcagatgtttctcaagtaaatgt1080aaaagcaacaacgacagaaaagcttggatttacaggccgggcggaaggaatagcggctca1140ggcgacagtactgatacaaaaaggctaacttgttttgatgccgggtctatttggtggtag1200aatagattcatacatttttgcctgaggccatacatgacatgaaaggaagtatttgaaaat1260gggaaacgaagtacgcgtccgttatgcaccgagtccaaccggacatttgcatattggaaa1320tgccagaacggcgctttttaattatttatttgcccgcaatcaaggcggtaagtttatcat1380tcgagttgaggatactgataaaaagcgcaatattgagggcggagaacaaagccagctgaa1440ttatctgaagtggctaggtattgactgggatgagagtgtggatgtcggaggagagtacgg1500tccataccgtcagtcagag1519<210>86<211>834<212>dna<213>人工序列(artificialsequence)<400>86accaatgcctaagtcagtttcaaactcaaaaaagaagaaagagaaagctgaaaagggcct60tctgcggccgactacaaagcccgacgttgataattatgtaaagggcgtgaaagatgcttt120gaatcatctgatatacaaagatgatagtcaggttgtggatttgaaagtcagtaagtttta180tagcgaagagccaagggtggaagtcatgataagagaggtttctgcctaaaaataaaaaac240accgaagcgcttagcctcagtgttcttgatatgaactggtacttctatcataacataggg300ggcgctttgagtgtacaatccaagagaaataaacatcaaaaaagacttcactattcagca360gaaaattgacccaggaaaagttcagatcattgttttagatgggaatcagggaactgcaca420tgttttagatgctccggagcacggcaaaacagtgattcaaactgtaaagggcagctttgc480aagggttgatcatgagatagggtttaaggtgaaatgagaagaagatataatactttataa540aaaggtaaacaagaaggaggatatctcctccttcggcttggatgtgcaggttattcctgt600tcaggtaaattgtcagctaatgtttctaataattgttgaggctcttctaaattcttttgg660ggttcaggaaaggcttcaactgtttcagaaggtgcatctaaagatgggtggaattgggta720ccttcatggagtaattctgcaaagattttaaccgattgttttatatgtcctattgattct780gttgaataatttgctccttcagtaacattttttatttcggcatctgttattgtg834<210>87<211>812<212>dna<213>人工序列(artificialsequence)<400>87gatatggtgacggaagatgattttctgttaggtaaggtttgtacgccggataaagctagc60gaatggaaagatacttattcaaaacagtgtgaatgtgtaagacgaaagaaaattgccaga120ctcatggcagctagtggcattacagaagagtttgaaaagcttctctttggtaacttcata180acggacggtaagccagacatgatcaaggacgcttatgagtgtgcagtggaatactataaa240gattttcaaaagatcaaaggagaaaggcaaaacagtatcgcattacttggacagccaggt300agcggtaaaactcatttgctcacggcgattatgaataatctgatcaagaaaaaatcagta360cactgcatgtatttcccttatgtagagggcatgggtgatttgaaagctaactttgataac420ttagaagcaaaactcgatgccatgagaaaggtcgaagttctattcattgatgatttattt480aaaccaataaacggtcaaccaagggcaacagactggcaggttgaacaaatccagtcagtc540ttgaactaccggtatttaaatcacaaacctttgctgatttcttcggagttaacaatcgat600gagattttggatatagacgaggctcttggttcacgaattcaccagatgtgccgtgattac660atagtgattattagaggcgatcgaatgcaattaaatcataggttaggtgattgggaatga720aaaagaaaacgaatataaaagcaactggtggactttatatatttggcccttcaaatcgaa780atgaaggtaaagatcttacaccaacaatccgt812当前第1页12