CN114075515B - 一种产虫草素基因工程菌及其应用 - Google Patents
一种产虫草素基因工程菌及其应用 Download PDFInfo
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- CN114075515B CN114075515B CN202010808145.XA CN202010808145A CN114075515B CN 114075515 B CN114075515 B CN 114075515B CN 202010808145 A CN202010808145 A CN 202010808145A CN 114075515 B CN114075515 B CN 114075515B
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Abstract
本发明属于基因重组技术领域，涉及一种产虫草素基因工程菌及其应用。本发明通过转化携带pyr4序列、Ura3序列、Amp抗性基因序列和AMA1序列的质粒pQF1到宿主A.nidulans A1145及LO8030中，经验证得到正确的含有AMA1质粒的转化子，并研究了携带AMA1的质粒对这两种常用宿主的影响。UPLC结果显示在CD‑ST液体培养基中，AMA1转化子的发酵粗提物中虫草素(COR)产量增加了1.72～1.84倍，虫草素的最高产量为277mg/L。这项研究表明携带AMA1的pQF1质粒可以在这两个构巢曲霉宿主中促进虫草素的产生。同时，本研究为虫草素的产量提高提供了潜在的工程策略。
Description
技术领域
本发明属于基因重组技术领域，涉及一种产虫草素基因工程菌及其应用。
背景技术
虫草素(cordycepin,COR)是蛹虫草(Cordyceps militaris，或C.militaris)代谢产物中非常具有效益的一个核苷类化合物。研究发现COR具有抗菌、抗炎、抗病毒、抗肿瘤、抗氧化和免疫调节等多种药理活性(Soltani M,Malek R A,Elmarzugi N A,et al.:Cordycepin:A Biotherapeutic Molecule from Medicinal Mushroom,Biology ofMacrofungi,2018:319-349)。1997年，美国已将虫草素用于三期临床实验，用于治疗急性pre-B和pre-T淋巴细胞白血病患者[康冀川康,文庭池,钱一鑫,雷帮星.虫草菌素药理学研究进展[J].菌物学报,2011,30(2):191-197]。如今，来源于C.militaris的高纯度COR的售价已达到$50-$80/kg。
与存在局限(如工艺复杂、合成过程使用大量有机试剂等)的化学法相比，生物发酵法更有利于COR的生产。采用生物发酵法主要是利用构巢曲霉(Aspergillus nidulans，或A.nidulans)或C.militaris发酵制备COR。尽管COR初始产率在A.nidulans最高产量达到249.46μg/mL，略高于C.militaris(228.45μg/mL)，可以作为COR新的生物来源[张红霞,吴畏,陈伟,et al.无冠构巢曲霉与北冬虫夏草发酵液中虫草素及腺苷含量比较分析研究[J].上海农业学报,2006,(02):34-37]。然而目前关于COR的工业生产仍是使用C.militaris进行发酵[Zhang Q,Liu Y.The Strategies for Increasing CordycepinProduction of Cordyceps Militaris by Liquid Fermentation[J].Fungal Genomics&Biology,2016,6(1)]，而以A.nidulans进行发酵制备COR尚未形成规模化工业生产。
发明内容
本发明的目标之一在于提供一种产虫草素的基因工程菌，其以构巢曲霉(Aspergillus nidulans)为模式菌株构建而成，具有较高的COR产量。
本发明的目标之二在于提供上述产虫草素的基因工程菌在生产虫草素中的应用。
为此，本发明第一方面提供了一种产虫草素的基因工程菌，其为含有质粒pQF1的重组构巢曲霉菌。
根据本发明，所述质粒pQF1携带有pyr4序列、URA3序列、Amp抗性基因序列和AMA1序列的一种或几种。
本发明中，所述AMA1序列包括内源性AMA1序列，以及外源性AMA1序列。
在本发明的一些实施例中，所述外源性AMA1序列来源于构巢曲霉菌GO51株，所述构巢曲霉菌G051的保藏编号为ATCC 38163；优选地，所述AMA1序列如SEQ No.1所示。
在本发明的一些实施例中，所述pyr4序列来源于粗糙脉孢菌OR74A菌株；优选地，所述粗糙脉孢菌OR74A菌株的保藏编号为MYA-4619D-2；进一步优选地，所述pyr4序列如SEQNo.2所示。
在本发明的一些实施例中，所述URA3序列来源于酵母菌S288C菌株；优选地，所述酵母菌S288C菌株的保藏编号为ATCC 20450，进一步优选地，所述URA3序列如SEQ No.3所示。
在本发明的一些实施例中，Amp抗性基因序列来源于枯草芽孢杆菌2014-3557株质粒pGMrib，或者质粒PUC18，PBR322；优选地，所述Amp抗性基因序列如SEQ No.4所示。
本发明中，用于构建重组构巢曲霉菌的模式菌为原始构巢曲霉菌或改造后的构巢曲霉菌；优选地，用于构建重组构巢曲霉菌的模式菌包括构巢曲霉菌A1145株和/或构巢曲霉菌LO8030株。
本发明第二方面提供了如本发明第一方面所述的产虫草素基因工程菌的在生产虫草素中的应用。
根据本发明，所述应用包括将所述基因工程菌接入发酵培养基，进行发酵培养，然后对所获得的发酵培养液进行分离纯化，制得虫草素。
本发明中，所述发酵培养基包括CD-ST培养基。
在本发明的一些实施例中，所述发酵的温度为20-40℃，优选为25-28℃。
在本发明的另一些实施例中，所述发酵的时间为5-12天(d)，优选为10-12天。
本发明通过转化携带pyr4序列、Ura3序列、Amp抗性基因序列和AMA1序列的质粒pQF1到宿主A.nidulans A1145及LO8030中，经验证得到正确的含有AMA1质粒的转化子，并研究了携带AMA1的质粒对这两种常用宿主的影响。UPLC结果显示在CD-ST液体培养基中，AMA1转化子的发酵粗提物中虫草素(COR)产量增加了1.72～1.84倍，虫草素的最高产量为277mg/L。这项研究表明携带AMA1的pQF1质粒可以在这两个构巢曲霉宿主中促进虫草素的产生。同时，本研究为虫草素的产量提高提供了潜在的工程策略。
附图说明
为使本发明容易理解，下面结合附图来说明本发明。
图1示出COR生物合成路径。
图2为A.nidulans A1145(A)及LO8030(B)AMA1转化子PCR验证电泳图；其中，P：阳性对照，以质粒pQF1作为PCR模板；N：阴性对照，以野生型菌株A1145或LO8030 gDNA作为PCR模板。
图3示出野生型A.nidulans及AMA1转化子发酵萃取物UPLC图谱(A,B)。
图4为A.nidulans中COR合成相关基因图[35](A)及菌株RT-PCR验证琼脂糖凝胶电泳图(B)。
图5为COR标准曲线图
图6示出CD-ST液体培养基培养不同时间的UPLC谱图(210nm检测)。
图7为含有AMA1质粒的转化子COR产量柱状图。
具体实施方式
为使本发明容易理解，下面将结合附图详细说明本发明。但在详细描述本发明前，应当理解本发明不限于描述的具体实施方式。还应当理解，本文中使用的术语仅为了描述具体实施方式，而并不表示限制性的。
除非另有定义，本文中使用的所有术语与本发明所属领域的普通技术人员的通常理解具有相同的意义。虽然与本文中描述的方法和材料类似或等同的任何方法和材料也可以在本发明的实施或测试中使用，但是现在描述了优选的方法和材料。
Ⅰ、术语
本发明所述用语“内源性基因”是指宿主生物体自身基因组内的基因。
本发明所述用语“外源性基因”是通过基因工程技术等途径引入靶细胞(宿主生物体)中的基因序列。
本发明所述用语“基因工程菌”是指将目的基因导入宿主生物体(即宿主细胞或模式菌)内使其表达，如构巢曲霉菌等。基因工程的核心技术是DNA的重组技术，因此，本发明中也将基因工程菌称为为重组微生物。
本发明所述用语“重组”是指利用供体生物的遗传物质或人工合成的基因，经过体外或离体的限制酶切割后与适当的载体连接起来形成重组DNA分子，然后再将重组DNA分子导入到受体细胞或受体生物构建转基因生物，该种生物就可以按人类事先设计好的蓝图表现出另外一种生物的某种性状。
本发明中所述用语“模式菌”与“宿主菌”或“宿主”可以互换使用。
Ⅱ、实施方案
如前所述，尽管COR初始产率在A.nidulans最高产量达到249.46μg/mL，略高于C.militaris(228.45μg/mL)，可以作为COR新的生物来源[张红霞,吴畏,陈伟,et al.无冠构巢曲霉与北冬虫夏草发酵液中虫草素及腺苷含量比较分析研究[J].上海农业学报,2006,(02):34-37]。然而目前关于COR的工业生产仍是使用C.militaris进行发酵[ZhangQ,Liu Y.The Strategies for Increasing Cordycepin Production of CordycepsMilitaris by Liquid Fermentation[J].Fungal Genomics&Biology,2016,6(1)]，而以A.nidulans进行发酵制备COR尚未形成规模化工业生产。鉴于此，本发明人对于以A.nidulans进行发酵制备COR进行了大量研究。
本发明人研究发现，关于COR生物合成途径，Xia Y等的最新研究成果解析了完整的COR生物合成途径及关键基因，如图1所示。从图1可以看出，COR的合成有一个基因簇负责，从原料腺苷adenosine开始，Cns3磷酸激酶结构域催化3′-OH形成3′-脱氧腺苷(adenosine-3′-monophosphate,adenosine-3′-monophosphate(3′-AMP)，3′-AMP在Cns2水解酶作用下形成2-羰基-3'-脱氧腺苷2-carbonyl-3′-deoxyadenosine(2-carbonyl-3′-deoxyadenosine,2′-C-3′-dA)，最后经由氧化还原酶Cns1由2′-C-3′-dA得到COR。这三个基因与A.nidulans中的三个基因高度相似，此后实验也确定了其在构巢曲霉中负责COR的合成。此基因簇同时合成抗癌药物—喷司他丁(Pentostatin,PTN)。PTN与COR存在底物竞争关系，同时PTN可以抑制腺苷转氨酶(Adenosine deaminase,ADA)的活性，ADA正是与COR在体内的代谢脱氨作用相关的蛋白，在COR的合成量达到较高浓度，PTN的外排作用增加，解除ADA的抑制作用，将会使得COR脱氨失活。
构巢曲霉(Aspergillus nidulans)是常用的异源表达次级代谢产物的模式菌，具有较清晰的代谢背景和高效的基因编辑工具盒。AMA1序列(autonomously maintained inAspergillus，曲霉中自主复制序列)通常用于提高基因复制和原生质体异源表达过程中的转化效率。然而，对于宿主代谢过程究竟具有怎样具体的影响，目前还未见报道。
而本发明通过转化携带pyr4序列、Ura3序列、Amp抗性基因序列和AMA1序列的质粒pQF1到宿主A.nidulans A1145及LO8030中，经验证得到正确的含有AMA1质粒的转化子，并研究了携带AMA1的质粒对这两种常用宿主的影响。UPLC结果显示在CD-ST液体培养基中，AMA1转化子的发酵粗提物中虫草素(COR)产量增加了1.72～1.84倍，虫草素的最高产量为277mg/L。这项研究表明携带AMA1的pQF1质粒可以在这两个构巢曲霉宿主中促进虫草素的产生。同时，本研究为虫草素的产量提高提供了潜在的工程策略。
因此，本发明第一方面所涉及的产虫草素的基因工程菌，其为含有质粒pQF1的重组构巢曲霉菌；其中，所述质粒pQF1携带有pyr4序列、URA3序列、Amp抗性基因序列和AMA1序列的一种或几种。
本发明中，所述AMA1序列包括内源性AMA1序列，以及外源性AMA1序列；其中，所述外源性AMA1序列来源于保藏编号为ATCC 38163的构巢曲霉菌(Aspergillus nidulans)GO51株；优选地，来源于保藏编号为ATCC 38163的构巢曲霉菌GO51株的外源性AMA1序列如SEQ No.1所示，其GenBank登录号为X78051.1。
在本发明的一些实施例中，所述pyr4序列来源于粗糙脉孢菌OR74A菌株；优选地，所述粗糙脉孢菌(Neurospora crassa)OR74A菌株的保藏编号为MYA-4619D-2；优选地，来源于保藏编号为MYA-4619D-2的粗糙脉孢菌OR74A株的pyr4序列如SEQ No.2所示。
在本发明的一些实施例中，URA3序列来源于保藏编号为ATCC 20450的酵母菌(Saccharomyces cerevisiae)S288C株；优选地，来源于保藏编号为ATCC 20450的酵母菌S288C株的URA3序列如SEQ No.3所示。
在本发明的一些实施例中，Amp抗性基因序列来源于枯草芽孢杆菌(Bacillussubtilis strain)2014-3557株质粒pGMrib，或者质粒PUC18，PBR322；优选地，所述Amp抗性基因序列如SEQ No.4所示。
本发明中，用于构建重组构巢曲霉菌的模式菌为原始构巢曲霉菌或改造后的构巢曲霉菌，例如，可以采用本领域中常规的原始构巢曲霉菌模式菌或改造后的构巢曲霉菌模式菌；优选地，用于构建重组构巢曲霉菌的模式菌包括构巢曲霉菌A1145株(TN02A7)和/或构巢曲霉菌LO8030株，更优选为构巢曲霉菌A1145株。
在本发明的一些实施例中，所述构巢曲霉菌A1145株的保藏编号为FGSC(fungalgenetic stock center，美国真菌遗传学库存中心)A1145(TN02A7)，其基因型为：pyrG89；pyroA4；nkuA::argB；riboB2(Genetics 172:1557-1566)。
在本发明的另一些实施例中，所述构巢曲霉菌LO8030株为保藏编号为FGSC 4的构巢曲霉菌的突变株(Yi-Ming Chiang等，“Development of Genetic DereplicationStrains in Aspergillus nidulans Results in the Discovery of Aspercryptin”，Angew.Chem.Int.Ed.2015,54,1-5)，其通过从保藏编号为FGSC 4的构巢曲霉菌中删除25个负责合成毒素sterigmatocystin(ST)，及emericellamide,orsellinic acid,asperfuranone,monodictyphenone和terrequinone的次级代谢基因，删除构巢曲霉这些SM簇获得；由此获得的构巢曲霉菌LO8030株消除了不必要的毒素的产生，简化了化学背景从而降低对异源表达的SM通路基因可能产生的干扰，使其更适用于异源表达。
所述保藏编号为FGSC 4的构巢曲霉菌的基因组序列如SEQ No.7所示，其Genebank登录号为JQ435097.1。
根据本发明，本发明第一方面所涉及的产虫草素的基因工程菌的制备方法包括：
1.A.nidulans原生质体制备及转化
(1)含营养缺陷补充的GMM固体培养基，37℃培养2-3天，用0.1％Tween 80收集孢子，显微镜观察计数。
(2)30mL含营养素的LMM液体培养基，孢子浓度109/mL接种，28℃，200rpm摇床培养6-10h使孢子萌发。
(3)8000rpm，4℃，离心15min，收集萌发好的孢子和菌丝，50mL ddH2O洗涤2次。
(4)配制酶解液，称量50mg Lysing Enzymes from T.harzianum和20mg Yatalase溶解于10mL Osmotic Medium。
(5)28℃，80rpm，摇床酶解5-12h，4h后每30min镜检1次,观察原生质体形成情况。
(6)酶解液转移至50mL无菌透明圆底离心管内，加入10mL Trapping Buffer，溶液上下分层，5000rpm，离心15min。
(7)收集上下交界面处的原生质体于15mL无菌EP管中，(移出剩余的TrappingBuffer，将菌体沉淀重悬于残余的Osmotic Medium中，加入10mL Trapping Buffer，如前离心，将两次收集到的原生质体合并)。
(8)用1倍体积的STC Buffer与原生质体混合，6000rpm，4℃离心5-8min。用STCBuffer重复洗涤1次。
(9)STC Buffer重悬原生质体沉淀，冰上保存备用。
(10)倒板：每个平板15mL的含筛选营养素的SMM培养基，晾干备用。
(11)每个EP管加入100μL原生质体，1-2μg pQF1质粒DNA，混匀后冰浴50min。
(12)每管加入1.25mL60％PEG溶液，缓慢翻转离心管混匀，室温静置20min。加5mLSTC Buffer，混匀，每平板平均加1-1.5mL菌体混合液。
(13)盖板，取Top SMM，加入营养素，每板倒入5mL，混合均匀，放置晾干。
(14)25℃培养箱培养5-7天，挑取转化子，提基因组验证。
2.转化子(gDNA)的提取
采用苯酚氯仿抽提法提取基因组。原理：在细胞裂解后，小的质粒DNA等将会缠绕在细胞碎片上，有机溶剂处理是蛋白变性溶于有机相，而基因组DNA将会被萃取到水相，再经乙醇沉淀出DNA，乙醇洗涤后，RNA酶降解掉RNA，得到基因组DNA，琼脂糖凝胶电泳初步检测基因组提取是否成功。
具体步骤如下：
(1)含适宜营养补充剂的LMM液体培养基或PDB培养菌株2-3天。
(2)菌体去除水分后放入1.5mL EP管。
(3)每管放入2-3颗钢珠，加入400μL LEST Buffer，组织研磨仪破碎90s。
(4)破碎均匀后再加入300μL LEST Buffer混合均匀，室温放置5min。
(5)加入500μL PCI溶液(苯酚：氯仿：异戊醇＝25:24:1)，混合颠倒10-15次，室温放置5min。
(6)4℃，13000rpm，离心10min。
(7)上清液转移至新的EP管，加入1mL纯乙醇，13000rpm，4℃，离心10min。
(8)弃上清，加入500μL 70％乙醇，稍震荡，4℃，13000rpm，离心2min，重复洗涤两次。
(9)弃上清，室温干燥。
(10)加20μL TE Buffer，0.5μL RNase A，37℃水浴30min，-20℃长期保存。
本发明第二方面提供了如本发明第一方面所述的产虫草素基因工程菌的在生产虫草素中的应用。
根据本发明，所述应用包括将所述基因工程菌接入发酵培养基，在20-40℃，优选为25-28℃下进行发酵培养5-12天，优选为10-12天，最优先为10天，然后对所获得的发酵培养液进行分离纯化，制得虫草素。
本发明中，所述发酵培养基包括CD-ST培养基。
在本发明的一些实施例中，所述CD-ST培养基组成为：淀粉20g/L，胰蛋白胨20g/L，20×氮盐溶液50mL，1000×微量元素储液1mL，额外添加营养缺陷补充。
本发明中产物的分离与鉴定包括：
1.对发酵培养液进行分离纯化
(1)对发酵培养液进行萃取分离
在发酵培养液中加入等体积的萃取剂，60℃下超声1h，将得到的萃取剂在旋蒸仪中蒸干，得到粗浸膏。
例如，在一些例子中，对发酵培养液进行分离纯化包括：利用等体积乙酸乙酯或MEA(甲醇：乙酸乙酯：三氟乙酸＝100：900：1,v/v/v)超声萃取发酵液1h，将上层有机相萃取物用旋转蒸发仪蒸干，得到粗浸膏，加入色谱级甲醇复溶。
又如，在另一些例子中，对发酵培养液进行分离纯化包括：将发酵液与等体积的蒸馏水混合，用HCl调节pH到3，于60℃条件下超声1h，离心得到上清液，将上清液用旋蒸仪蒸干，得到粗浸膏，加入色谱级甲醇复溶。
再如，再又一些例子中，对发酵培养液进行分离纯化包括：将发酵液与等体积的50％乙醇混合，于60℃条件下超声1h，离心得到上清液，将上清液用旋蒸仪蒸干，得到粗浸膏，加入色谱级甲醇复溶。
(2)对发酵培养液进行超临界萃取分离
将发酵培养液旋蒸成粗浸膏，干燥粉碎后放入超临界萃取提取容器，静态萃取50min，之后在40MPa，40℃，甲醇流速0.05mL/min，CO2流速2.0L/mL的条件下，动态萃取2h，得到提取物。
本发明中优选采用MEA、乙酸乙酯萃取、乙醇或水对发酵培养液进行萃取分离，进一步优选采用乙醇或水对发酵培养液进行萃取分离。
2.对虫草素粗浸膏进行分离纯化
(1)ODS硅胶柱层析
1)装柱：将适量的硅胶粉加入纯甲醇使硅胶颗粒膨胀均匀，填入色谱柱中，使硅胶柱填料均匀紧实。
2)拌样：用适量的甲醇溶解粗浸膏样品，然后与质量为样品质量的1～3倍的硅胶粉混合搅拌，将溶剂彻底挥发。
3)上样：将拌好样的硅胶粉填入硅胶柱。
4)梯度洗脱：选择5％-100％的甲醇/水，每次200mL洗脱液进行梯度洗脱，期间加压提高洗脱速率。
5)旋蒸：将不同的组分分别利用旋转蒸发仪35℃蒸干。
6)检测：甲醇溶解各洗脱梯度的样品，13000rpm室温离心20min，取上清装入液相瓶中，UPLC检测不同梯度的样品。
(2)半制备液相色谱分离
半制备液相色谱使用仪器安捷伦1260，色谱柱使用UniSil10-120 C18反相色谱柱(10μm，10×250mm)，流动相25％甲醇/75％水，流速2.5mL/min，恒梯度洗脱，进样量30μL。
(3)离子交换柱分离
首先，将少量蒸馏水装到空柱中以驱除气泡，然后将含水树脂连续填充到柱中。安装后，使用pH＝4的盐酸平衡液，以3倍柱体积流过柱，并在流出物的酸碱度恒定时停止。将样品沿管壁缓慢加入到柱中，加入相应的盐酸平衡液固定树脂吸附的虫草素，用蒸馏水通过柱去除杂质。洗脱液(0.15mol/L氨水)由恒流泵以40滴/分钟的流速加入色谱柱，由全自动收集器收集。
3.对虫草素进行鉴定
(1)核磁共振(NMR)
样品用600μL氘代甲醇(CD3OD)溶解，装入清洁干燥的核磁管中，利用核磁共振波谱仪(例如，AV600，布鲁克公司)检测1H-NMR、13C-NMR(400MHz)确定结构。
(2)UPLC及LC-MS检测
使用Waters高效液相色谱仪，流速0.4mL/min，流动相为A相(水，添加0.5‰三氟乙酸)，B相(乙腈)10％B-100％B梯度洗脱7min，100％B冲洗1min，10％B平衡2min，PDA检测器检测。分析柱：ACQUITY UPLC BEH C18 1.7μm×2.1×100mm柱。液质使用thermo scitificUltimate3000液相-HRMS质谱联用，PDA液相检测器，得到正负离子ESI谱图。
Ⅲ、实施例
以下通过具体实施例对于本发明进行具体说明。下文所述实验方法，如无特殊说明，均为实验室常规方法。下文所述实验材料，如无特别说明，均可由商业渠道获得。
发酵过程：将所述基因工程菌接入发酵培养基，在25-28℃下进行发酵培养5-12天，然后采用前述方法对所获得的发酵培养液进行分离纯化，制得虫草素。
实施例1：
在添加了适宜营养补剂的LMM培养基(Pyridoxine HCl+Riboflavin)中培养A.nidulans A1145和LO8030转化子，提取gDNA，在质粒pyr4的一端和AMA1序列的一端设计引物，P1-F：GTCGACATGTTGGCTGTTGTG；P1-R：GTGATGGCAGACATGGTAGG，进行PCR验证，琼脂糖凝胶电泳结果如图2所示，其中A.nidulans A1145转化体系阴性对照用A.nidulans A1145的gDNA作PCR模板，阳性对照用质粒pQF1作为PCR模板，结果显示A.nidulans A1145转化子T1，T2，T3成功转化了质粒pQF1(图2A)。A.nidulans LO8030转化体系阴性对照用A.nidulans LO8030的gDNA作PCR模板，阳性对照用质粒pQF1作为PCR模板，结果显示转化子T4，T5，T6，T7成功转化了质粒pQF1(图2B)。
实验成功获得了A.nidulans A1145和LO8030转化子，为验证空质粒上AMA1序列对宿主次级代谢的影响提供了实验材料。利用转化子A.nidulans A1145 T1和LO8030 T7与A.nidulans A1145和LO8030菌株在相同条件下进行发酵培养，以探究含pyr4、Ura3、Amp和AMA1的空质粒对宿主的影响。
实施例2：
通常菌株A.nidulans A1145使用的培养基有GMM，大米，及CD-ST，经过实验发现在CD-ST液体培养基中发酵，野生型菌株次级代谢谱图背景清晰，自身代谢产物较少，对新产物的检测有利，同时出现了化合物产量的变化，因此选用CD-ST培养基进行后续的发酵。经过初步发酵，对于代谢谱图，发现在1.6min或1.4min转化子中多出一个液相峰。
如图3所示，对比A1145，LO8030与转化子A1145 T1，LO8030 T7的UPLC图谱，发现1.5min化合物(COR)的产量有明显提高，而其他保留时间并未出现明显新的液相峰。
经过发酵后，进行产物分离，获得纯品。发酵液粗提物经过ODS反相硅胶柱甲醇/水梯度洗脱，共得到35个组分，旋转蒸发仪(常规)蒸干后，UPLC检测发现目标产物位于10％-2的馏分中。取此极性段(约60mg)进行下一步的分离。
恒梯度洗脱，液相色谱结构显示共有9个峰，利用半制备液相色谱，接收不同馏分，分别进行旋蒸，峰4和峰9质量分别为30mg和10mg。峰9经过UPLC检测是单一峰，核磁测试1H-NMR(400MHz,CD3OD-d4)信号(δppm)：8.41(s,1H),8.20(s,1H),5.95(d,J＝1.6Hz,1H),4.71(m,J＝2.4,4.0Hz,1H),4.52(m,J＝2.4,5.2,7.2Hz,1H),3.92(dt,J＝2.4,5.2,7.2Hz,1H),3.67(dt,J＝1.6,6.0,8.4Hz,1H),2.37(m,J＝1.2,9.2Hz,1H),2.07(m,J＝1.6,3.2Hz,1H)及13C-NMR(400MHz,CD3OD-d4)149.92,153.62,157.42,141.14,120.68,93.61,82.55,76.59,64.21,34.53，经过与文献1H-NMR(Seul Gi Lee S-H H,Gi-Ho Sung&Hyung-KyoonChoi.Simple and Rapid Determination of Cordycepin in Cordyceps militarisFruiting Bodies by Quantitative Nuclear Magnetic Resonance Spectroscopy[J].Analytical Letters,2014,47:1031–1042.)、13C-NMR数据[Huichun X,Liang Y,Lao D,etal.Preparative Separation of High-Purity Cordycepin from Cordyceps Militaris(L.)Link by High-Speed Countercurrent Chromatography[J].Journal of LiquidChromatography&Related Technologies,2011,34(7):491-499]比对完全符合，此化合物鉴定为虫草素(COR)。
实施例3：
如图4A所示，在A.nidulans中，与COR生物合成相关的基因AN3333,AN3331,AN3330是分别于蛹虫草中三个基因cns1，cns2,cns3高度同源的，Xia Y[Xia Y,Luo F,Shang Y,etal.Fungal Cordycepin Biosynthesis Is Coupled with the Production of theSafeguard Molecule Pentostatin[J].Cell Chem Biol,2017,24(12):1479-1489]等报道cns1,cns2是必需基因，敲除将不再产生COR，cns3是可被替换的。图4B中RT-PCR结果显示，cns1，cns2基因表达，而cns3基因不表达，即必须的两个基因是起作用的，而cns3基因催化的生成3′-AMP的反应由于可以被mRNA降解产物2′,3′-cAMP由磷脂酶催化生成，所以此结果符合先前的报道。而且，cns3基因与保护分子PTN合成相关，而PTN与COR存在底物竞争作用，在cns1-3同时异源表达时COR的产量反而低于cns1,cns2同时表达[Xia Y,Luo F,Shang Y,et al.Fungal Cordycepin Biosynthesis Is Coupled with the Production of theSafeguard Molecule Pentostatin[J].Cell Chem Biol,2017,24(12):1479-1489]。而在转化子与原始菌中COR产量的差异原因很可能就在于AMA1对于另一种途径中，中间产物2’,3’-cAMP向3′-AMP转化过程存在促进作用。COR抑菌作用的起因是其可以抑制mRNA的poly A结构的生成[Johns D G,Adamson R H.Enhancement of the biological activity ofcordycepin(3'-deoxyadenosine)by the adenosine deaminase inhibitor2'-deoxycoformycin[J].Biochemical Pharmacology,1976,25(12):1441-1444]，同时另一方面，AMA1可以促进基因的复制[Aleksenko A,Clutterbuck A J.The plasmid replicatorAMA1 in Aspergillus nidulans is an inverted duplication of a low-copy-numberdispersed genomic repeat[J].Molecular Microbiology,1996,19(3):565-574]，所以在此过程中可能促进mRNA的降解产物2’,3’-cAMP的产生，而最终导致COR产量的提高。
实施例4：
HPLC检测在活性天然产物的定量研究中具有广泛的应用，本章采用外标法对发酵液中COR产量进行测定，首先，由标准品拟合得到图5中所示的标准曲线公式为：Y＝0.004X，RHPLC2＝0.9999；其中，X表示COR液相峰积分面积，Y表示样品中COR样品的浓度。在浓度1-100000mg/L的范围内，COR浓度与积分面积具有良好的线性关系。
因为样品制备过程中，以50mL培养基出发，经过萃取，旋蒸浓缩后得到1.4mL粗提物样品，所以发酵液中COR产量的计算方法为Z(发酵液中COR产量)＝1.4Y/50，单位mg/L。后续发酵液中的COR浓度评估采用此处所述计算方法。
实施例5：
时间设为10天是较为合适的。Fan D[Fan D-D,Wang W,Zhong J-J.Enhancementof cordycepin production in submerged cultures of Cordyceps militaris byaddition of ferrous sulfate[J].Biochemical Engineering Journal,2012,60:30-35]等报道，C.milises培养过程中，添加离子的实验组与对照组分别在第5-10天和15天菌丝的生物量达到顶值，而本章实验在图6UPLC谱图结果显示，随着发酵时间的延长，COR的峰呈现先增长后减少的趋势，第10天时，COR的液相谱图峰面积达到最高，即COR的产量最高，所以发酵第10天，COR的产量达到峰值，与之相比，生产速率较快。
实施例6：
经过多次重复CD-ST液体摇瓶发酵，UPLC测试萃取粗提物，发现始终存在这样的规律：AMA1转化子A.nidulans A1145 T1和LO8030 T7与构巢曲霉A.nidulans A1145和LO8030相比，COR的产量有所提高，利用高效液相色谱外标法，计算出发酵液中的COR浓度。
计算得到发酵液中COR的浓度，得到如图7所示柱状图，发现AMA1转化株A1145 T1，LO8030 T7发酵液中COR的产量分别是A.nidulans A1145和LO8030的1.72倍和1.84倍，最高产量达到277mg/L，与A.nidulans中已知的产量数据相比稍高[张红霞,吴畏,陈伟,et al.无冠构巢曲霉与北冬虫夏草发酵液中虫草素及腺苷含量比较分析研究[J].上海农业学报,2006,(02):34-37]。因为菌株LO8030删除了25个负责合成毒素sterigmatocystin(ST)，及emericellamide,orsellinic acid,asperfuranone,monodictyphenone和terrequinone的次级代谢基因，删除构巢曲霉这些SM簇，能够消除不必要的毒素的产生，简化化学背景从而降低对异源表达的SM通路基因可能产生的干扰，使其更适用于异源表达的过程。在UPLC结果上LO8030整体峰数量较少，COR的产量较A1145降低了12倍，提示删除25个基因后可能会影响其他代谢产物的合成。实验结果说明在CD-ST摇瓶培养条件下，AMA1序列促进了宿主中COR产量的提升，结合RT-PCR结果，COR合成基因簇的基因cns1和cns2处于表达状态，所以以本实验的结果来看，COR的合成第一步反应底物是mRNA降解产物2’,3’-cAMP在磷脂水解酶作用下生成3’-AMP，AMA1序列导致了COR合成产量的提高。
Aspergillus nidulans是一个在异源表达过程应用较多的模式菌株，AMA1序列因为可以提高转化效率和质粒复制的能力，在质粒构建过程中具有良好的应用，然而对于宿主代谢过程究竟是怎样具体的影响，目前还未见报道。本章通过转化携带pyr4，Ura3，Amp，AMA1序列的质粒pQF1到宿主A.nidulans A1145及LO8030中，得到转化子菌株，此菌株也可作为参照消除后期异源表达产物的内源性影响，经过液态发酵，使用液相色谱分析次级代谢产物情况，比对初始菌株代谢谱图，主要结论如下：
(1)成功构建构巢曲霉A.nidulans A1145和LO8030转化体系，得到空质粒转化子A1145 T1，LO8030 T7。使用高效液相色谱外标法检测，得出在CD-ST培养基摇瓶培养下质粒pQF1的转入使得两株构巢曲霉A.nidulans A1145及LO8030代谢产物COR产量分别提高了1.72和1.84倍，在A.nidulans A1145 T1中最高产量达到277mg/L。
(2)RT-PCR结果显示，A.nidulans中COR合成相关的三个基因，AN3333，AN3331处于表达状态，而AN3330不表达或低表达，结合COR生物合成路径，说明COR的合成主要是通过mRNA的降解过程起始的。
(3)初步探究了COR合成的时间依赖性，得出10天的培养时间有利于COR的产生，培养时间与蛹虫草相似。
应当注意的是，以上所述的实施例仅用于解释本发明，并不构成对本发明的任何限制。通过参照典型实施例对本发明进行了描述，但应当理解为本发明中，所述的词语为描述性和解释性词汇，而不是限定性词汇。可以按规定在本发明权利要求的范围内对本发明做出修改，以及在不背离本发明的范围和精神内对本发明进行修订。尽管其中描述的本发明涉及特定的方法、材料和实施例，但是并不意味着本发明限于其中公开的特定例，相反，本发明可扩展至其他所有具有相同功能的方法和应用。
序列表
<110> 北京化工大学
<120> 一种产虫草素基因工程菌及其应用
<130> RB2002441-FF
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5252
<212> DNA
<213> （外源性AMA1序列）
<400> 1
aagcttattt tttgtatact gttttgtgat agcacgaagt ttttccacgg tatcttgtta 60
aaaatatata tttgtggcgg gcttacctac atcaaattaa taagagacta attataaact 120
aaacacacaa gcaagctact ttagggtaaa agtttataaa tgcttttgac gtataaacgt 180
tgcttgtatt tattattaca attaaaggtg gatagaaaac ctagagacta gttagaaact 240
aatctcaggt ttgcgttaaa ctaaatcaga gcccgagagg ttaacagaac ctagaagggg 300
actagatatc cgggtaggga aacaaaaaaa aaaaacaaga cagccacata ttagggagac 360
tagttagaag ctagttccag gactaggaaa ataaaagaca atgataccac agtctagttg 420
acaactagat agattctaga ttgaggccaa agtctctgag atccaggtta gttgcaacta 480
atactagtta gtatctagtc tcctataact ctgaagctag aataacttac tactattatc 540
ctcaccactg ttcagctgcg caaacggagt gattgcaagg tgttcagaga ctagttattg 600
actagtcagt gactagcaat aactaacaag gtattaacct accatgtctg ccatcaccct 660
gcacttcctc gggctcagca gccttttcct cctcattttc atgctcattt tccttgttta 720
agactgtgac tagtcaaaga ctagtccaga accacaaagg agaaatgtct taccactttc 780
ttcattgctt gtctcttttg cattatccat gtctgcaact agttagagtc tagttagtga 840
ctagtccgac gaggacttgc ttgtctccgg attgttggag gaactctcca gggcctcaag 900
atccacaaca gagccttcta gaagactggt caataactag ttggtctttg tctgagtctg 960
acttacgagg ttgcatactc gctccctttg cctcgtcaat cgatgagaaa aagcgccaaa 1020
actcgcaata tggctttgaa ccacacggtg ctgagactag ttagaatcta gtcccaaact 1080
agcttggata gcttaccttt gccctttgcg ttgcgacagg tcttgcaggg tatggttcct 1140
ttctcaccag ctgatttagc tgccttgcta ccctcacggc ggatctgcat aaagagtggc 1200
tagaggttat aaattagcac tgatcctagg tacggggctg aatgtaactt gcctttcctt 1260
tctcatcgcg cggcaagaca ggcttgctca aattcctacc agtcacaggg gtatgcacgg 1320
cgtacggacc acttgaacta gtcacagatt agttagcaac tagtctgcat tgaatggctg 1380
tacttacggg ccctcgccat tgtcctgatc atttccagct tcaccctcgt tgctgcaaag 1440
tagttagtga ctagtcaagg actagttgaa atgggagaag aaactcacga attctcgaca 1500
cccttagtat tgtggtcctt ggacttggtg ctgctatata ttagctaata cactagttag 1560
actcacagaa acttacgcag ctcgcttgcg cttcttggta ggagtcgggg ttgggagaac 1620
agtgccttca aacaagcctt cataccatgc tacttgacta gtcagggact agtcaccaag 1680
taatctagat aggacttgcc tttggcctcc atcagttcct tcatagtggg aggtccattg 1740
tgcaatgtaa actccatgcc gtgggagttc ttgtccttca agtgcttgac caatatgttt 1800
ctgttggcag agggaacctg tcaactagtt aataactagt cagaaactag tatagcagta 1860
gactcactgt acgcttgagg catcccttca ctcggcagta gacttcatat ggatggatat 1920
caggcacgcc attgtcgtcc tgtggactag tcagtaacta ggcttaaagc tagtcgggtc 1980
ggcttactat cttgaaatcc ggcagcgtaa gctccccgtc cttaactgcc tcgagatagt 2040
gacagtactc tggggacttt cggagatcgt tatcgcgaat gctcggcata ctaatcgttg 2100
actagtcttg gactagtccc gagcaaaaag gattggagga ggaggaggaa ggtgagagtg 2160
agacaaagag cgaaataaga gcttcaaagg ctatctctaa gcagtatgaa ggttaagtat 2220
ctagttcttg actagattta aaagagattt cgactagtta tgtacctgga gtttggatat 2280
aggaatgtgt tgtggtaacg aaatgtaagg gggaggaaag aaaaagtcgg tcaagaggta 2340
actctaagtc ggccattcct ttttgggagg cgctaaccat aaacggcatg gtcgacttag 2400
agttagctca gggaatttag ggagttatct gcgaccactg gactctcagg ctttccattg 2460
gggtactaac atagccatca aatgccggcc cgtgtttaat ctcctcgaga tggctgggct 2520
cacatgcgtt ctactcaaaa actcttatac ctaggccctc tcccaatcgt gtatgtttgg 2580
accttcctct ctcacacttt ggataacatc aagcatacct gcagattttg acctagttaa 2640
caaggcacat catgctatca cttccaacac tcttgctgag cgcgttctgc cttgcctttc 2700
tggttctacg aagtatccag tcccgcctgc aacgccgcag aaacgccaaa aggtggggtt 2760
gtcaaccgcc agctagagct ccatcgggat tctttggcat tccgccgttc ctcggtggtc 2820
gcagataact ccctaaattc cctgagctaa ctctaagtcg accatgccgt ttatggttag 2880
cgcctcccaa aaaggaatgg ccgacttaga gttacctctt gaccgacttt ttctttcctc 2940
ccccttacat ttcgttacca caacacattc ctatatccaa actccaggta cataactagt 3000
cgaaatctct tttaaatcta gtcaagaact agatacttaa ccttcatact gcttagagat 3060
agcctttgaa gctcttattt cgctctttgt ctcactctca ccttcctcct cctcctccaa 3120
tcctttttgc tcgggactag tccaagacta gtcaacgatt agtatgccga gcattcgcga 3180
taacgatctc cgaaagtccc cagagtactg tcactatctc gaggcagtta aggacgggga 3240
gcttacgctg ccggatttca agatagtaag ccgacccgac tagctttaag cctagttact 3300
gactagtcca caggacgaca atggcgtgcc tgatatccat ccatatgaag tctactgccg 3360
agtgaaggga tgcctcaagc gtacagtgag tctactgcta tactagtttc tgactagtta 3420
ttaactagtt gacaggttcc ctctgccaac agaaacatat tggtcaagca cttgaaggac 3480
aagaactccc acggcatgga gtttacattg cacaatggac ctcccactat gaaggaactg 3540
atggaggcca aaggcaagtc ctatctagat tacttggtga ctagtccctg actagtcaag 3600
tagcatggta tgaaggcttg tttgaaggca ctgttctccc aaccccgact cctaccaaga 3660
agcgcaagcg agctgcgtaa gtttctgtga gtctaactag tgtattagct aatatatagc 3720
agcaccaagt ccaaggacca caatactaag ggtgtcgaga attcgtgagt ttcttctccc 3780
atttcaacta gtccttgact agtcactaac tactttgcag caacgagggt gaagctggaa 3840
atgatcagga caatggcgag ggcccgtaag tacagccatt caatgcagac tagttgctaa 3900
ctaatctgtg actagttcaa gtggtccgta cgccgtgcat acccctgtga ctggtaggaa 3960
tttgagcaag cctgtcttgc cgcgcgatga gaaaggaaag gcaagttaca ttcagccccg 4020
tacctaggat cagtgctaat ttataacctc tagccactct ttatgcagat ccgccgtgag 4080
ggtagcaagg cagctaaatc agctggtgag aaaggaacca taccctgcaa gacctgtcgc 4140
aacgcaaagg gcaaaggtaa gctatccaag ctagtttggg actagattct aactagtctc 4200
agcaccgtgt ggttcaaagc catattgcga gttttggcgc tttttctcat cgattgacga 4260
ggcaaaggga gcgagtatgc aacctcgtaa gtcagactca gacaaagacc aactagttat 4320
tgaccagtct tctagaaggc tctgttgtgg atcttgaggc cctggagagt tcctccaaca 4380
atccggagac aagcaagtcc tcgtcggact agtcactaac tagactctaa ctagttgcag 4440
acatggataa tgcaaaagag acaagcaatg aagaaagtgg taagacattt ctcctttgtg 4500
gttctggact agtctttgac tagtcacagt cttaaacaag gaaaatgagc atgaaaatga 4560
ggaggaaaag gctgctgagc ccgaggaagt gcagggtgat ggcagacatg gtaggttaat 4620
accttgttag ttattgctag tcactgacta gtcaataact agtctctgaa caccttgcaa 4680
tcactccgtt tgcgcagctg aacagtggtg aggataatag tagtaagtta ttctagcttc 4740
agagttatag gagactagat actaactagt attagttgca actaacctgg atctcagaga 4800
ctttggcctc aatctagaat ctatctagtt gtcaactaga ctgtggtatc attgtctttt 4860
attttcctag tcctggaact agcttctaac tagtctccct aatatgtggc tgtcttgttt 4920
tttttttttg tttccctacc cggatatcta gtccccttct aggttctgtt aacctctcgg 4980
gctctgattt agtttaacgc aaacctgaga ttagtttcta actagtctct aggttttcta 5040
tccaccttta attgtaataa taaatacaag caacgtttat acgtcaaaag catttataaa 5100
cttttaccct aaagtagctt gcttgtgtgt ttagtttata attagtctct tattaatttg 5160
atgtaggtaa gcccgccaca aatatatatt tttaacaaga taccgtggaa aaacttcgtg 5220
ctatcacaaa acagtataca aaaaataagc tt 5252
<210> 2
<211> 2144
<212> DNA
<213> （pyr4序列）
<400> 2
aacttttttg gggtccaccc aacgtcctcg gcgacagggc actgcatcga gagttcggcg 60
cttcccaatc aatcccgtcg tctttttctc tttttcgtgc cctgggccag acactgacac 120
atcccttcac ttcttcctcc cgctcgtacc tacatatgta gatcgatgca gctgcttcct 180
caacctctcg attcttcctc tcaactttcc caatccaaca aacaccacca ccggccactc 240
cgatcttggt ctctccttgc cgagtcacta cctacttcca cttggaccac acgagtcaag 300
cttagacttc aacaacccca ccatcccacc tcgtgccggc aattcttttt agttgttacc 360
tcgagtgtat tcgacaatat cttgcttgga ttgccttccg ctgtacatcc aacaaacgca 420
aatcacaaca gccaacatgt cgacaagtca ggaaacgcag ccacactggt ccctcaagca 480
gtcgtttgct gagcgggtag agagctcgac gcatcccctc accagctacc tcttccgcct 540
gatggaggtc aagcagtcca acctctgcct cagcgccgat gtcgagcacg cgcgggatct 600
cctcgccctt gccgacaagg tgggcccctc gattgtcgtc ctcaagaccc actacgacct 660
gatcacaggg tgggactacc acccgcacac gggcaccggc gccaagctgg ccgcccttgc 720
ccggaagcac ggcttcctca tcttcgagga ccgcaagttc gtcgacattg gcagcaccgt 780
ccagaagcag tacacggccg gcaccgcgcg cattgtcgaa tgggcccaca tcaccaacgc 840
cgacatccac gccggagagg ccatggtgag cgccatggcc caggccgcgc aaaagtggag 900
ggagcgcatc ccctacgagg tcaagacgtc ggtttcggtg ggcaccccgg tcgcggacca 960
gttcgccgac gaggaagccg aggaccaggt tgaggagctg cgcaaggtcg tcacccgcga 1020
gaccagcacc accacaaagg acacggatgg gaggaagagt agcatcgtct ccatcacgac 1080
cgtcacgcag acatatgagc cggccgactc gccacgtctg gtcaagacca tctcggagga 1140
cgatgagatg gtgttccccg gcatcgagga ggcgcctctg gaccgcggcc tgctgatctt 1200
ggcccagatg tcgtccaagg gctgcctcat ggacggcaag tacacatggg agtgtgtcaa 1260
ggcggcccgc aagaacaagg gctttgtcat gggctacgtt gcgcagcaga acctgaacgg 1320
cattaccaag gaagctttgg ccccaagcta cgaagacggc gaaagcacga cagaggaaga 1380
agcgcaagca gacaacttca tccacatgac acccggctgc aagttgccgc caccaggaga 1440
ggaagcgcct cagggcgacg gactgggtca gcagtacaac acgccggata accttgtcaa 1500
catcaagggc accgatatcg cgattgttgg gcgtggcatc atcaccgcgg cggatcctcc 1560
ggccgaggct gagcgctaca ggaggaaagc ctggaaggcg taccaggatc gccgggagcg 1620
tctggcatag aagactgggg aataagaaat ggcggaggat atttcgtttt catatgactg 1680
ggatgagcgt ctacataatt cttggtggca tcatgtaatc acactcggtt attcttctgg 1740
ccgggactgt tatatcaggt cgcaggccat ggatatattg tggaagaaaa cttaccatag 1800
tcatttgtaa ggcacacggg caaggaggga ggttgttttt ttcattttgg gccaagggtt 1860
tctctaagga tttgggcggc ttcgaacggc gtttgggcga taatagatgc ttggcacttt 1920
tggcctgctg ttttttgctg tgtataccac tgcataatgt tactttctgt tcactatgtc 1980
ttgaactcta actttatttg cctgtgtttt ctcttttcgt tgcattcgat aagtagccag 2040
ttcccgaaag cgacgaatga tgaagatctt tttacacaga aaagacagac cggggtcgcg 2100
gagttaactg gcttatcccc gggctcgctt tcccctcgac gacg 2144
<210> 3
<211> 1713
<212> DNA
<213> （URA3序列）
<400> 3
cattatacga agttatgcac cacgcttttc aattcaattc atcatttttt ttttattctt 60
ttttttgatt tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag 120
aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa 180
catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacctgc aggaaacgaa 240
gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg 300
ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg 360
ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac 420
taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa 480
aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg 540
gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca 600
ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag 660
aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct 720
ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt 780
ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt 840
tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata 900
gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat 960
ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag 1020
catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta 1080
tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc ataacttcgt 1140
atagcataca ttatacgaag ttatcccggg taccgagctc gaattcgtaa cttacacgcg 1200
cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat 1260
gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa 1320
aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta 1380
ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa 1440
atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat 1500
acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct 1560
tttacatctt cggaaaacaa aaactatttt ttctttaatt tcttttttta ctttctattt 1620
ttaatttata tatttatatt aaaaaattta aattataatt atttttatag cacgtgatga 1680
attcgtaatc atggtcatag ctgtttcctg tgt 1713
<210> 4
<211> 858
<212> DNA
<213> （Amp抗性基因）
<400> 4
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 60
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 120
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 180
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 240
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 300
tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 360
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 420
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 480
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 540
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 600
ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 660
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 720
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 780
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 840
gaaatgttga atactcat 858
<210> 5
<211> 21
<212> DNA
<213> （引物P1-F）
<400> 5
gtcgacatgt tggctgttgt g 21
<210> 6
<211> 20
<212> DNA
<213> （引物P1-R）
<400> 6
gtgatggcag acatggtagg 20
<210> 7
<211> 33227
<212> DNA
<213> （保藏编号为FGSC 4的构巢曲霉菌的基因组序列）
<400> 7
aataggaacc ccttataaag attaactatt attaattttt atataatata aaattcagta 60
tattatatta aaataaaaat ctttttagag gttttatatg aaatatgtat tcaataaagg 120
tatatagtaa tatcctcaat ccctatatca aaataaatta gttaataata agtataaata 180
taaatatatt tatatttact tattaaattt tagataataa ataaatatac aatatctaaa 240
ataaataatt atattttaag gcatgttaat tcaatggtag aatattgtag tacggccaca 300
agaatataag ttcaaatctt atacatgtct tagagatata gtatagataa atcaaaaaaa 360
aatgtaaaaa agttattatg aatttttcaa tatttttatt tctaatagga atattaggtt 420
ttgttttaaa tagaaaaaat ataatattaa tgttaatttc aattgaaatt atgttattat 480
caataacatt tttaatacta ataagttcac taagttttga cgatatttta gggcaaacat 540
ttgcaatata tatcataact atagctggag ctgaatctgc aataggttta ggaatattag 600
tagcatatta tagattaaga ggaagtatat caatacaata taaataatgt atttaacatt 660
aataatttta cctttattag gatcaatagt ttcaggtttt tttggtagaa aagtaggagt 720
aacaggtgca catttaataa catgtgtttc agttgttact acaacaatat tagctatatt 780
agctttctta gaagtaggtt ttaataatat accagtaaca ataaatatag caagatgatt 840
agatgtagaa tcattatatg tattatgaaa ttttagattt gattctttaa cagtatcaat 900
gttattacca gtattaatag tatcaagttt agtacatata tattctataa gttatatgag 960
ccatgaccca cacaaccaaa gattttttag ttatttaagt ttattcactt ttatgatgat 1020
tatacttgta acaggaaata attatttaat aatgtttgta ggttgagaag gtgtaggtgt 1080
ttgttcatat ttattagtaa atttctgatt tacaagaata gcagctaatc aaagttcttt 1140
atcagcatta ttaactaata gagttggtga tactttatta actgttggta tgtttgctat 1200
actatgatct tttggtaata tagattatag tacagtattt gctttagctc cttattataa 1260
tgaaaatatt ataacattaa ttggtatttg tttattaata ggtgctacag ctaaaagttc 1320
acaggttggt cttcatattt gattaccaca ggcgatggag ggacctactc ctgtatctgc 1380
tttaattcac gctgctacta tggttacagc aggtgtatat ttattaatga gatcatctcc 1440
tttaatagag tatagttcaa ctgttttagt actttgttta tgattaggtg ctattactac 1500
agtatttagt tctttaattg gtttattcca acaagatatt aaaaaagtta ttgcttattc 1560
aactatgagt caattaggaa tgatggtaat agctgttggt ttatctagtt ataatttagc 1620
tttattccat ttagttaatc atgcttttta taaagcatta ttattcttag gtgctggttc 1680
agttattcac gctgtagctg ataatcaaga tttcagaaaa tatggtggtt taagagaatt 1740
cttaccttta acttattcag ttatgttaat agcttcttta agtttagttg ctgttccttt 1800
tatgactgga ttctattcta aagattttat tcttgaatct gcttatggac aatattattt 1860
atcaagtact attgtttatt tcgttgctac tattggtgct atgtttacta cactttactc 1920
tgctaaagtt ttatatctaa ctttcttaac taatcctaat ggtcctttag ttaattataa 1980
acatgctcat gaaggtgact tatttatgac tactccttta attattttag ctatcttctc 2040
tattttcttt ggatatttaa ctaaagatat ttttataggt ttaggtactg gttttttctc 2100
tgataatagt ttatttattc atcctagtca tgaaattatg ttagatactg aatttgctgt 2160
acctacattc tttaaattat taccttttat atttactatc tcattaagtg ttatttctgt 2220
tttattatca gaatttttac ctaaattact tattaatttt aaattctcta gattaggtta 2280
taatattttt agtttcttta atcaaagatt ttatatagaa ttattctata ataaatatat 2340
tgtagaaggt gttcttaaat taggtggtca aacttctaag agtttagata aaggttctgt 2400
agaattatta ggaccttatg gtttagaaaa agcattatta agtttaagta atagtattgg 2460
taaattaagc actagtgttg ttactactta tgctttatat attttaatag gattaatctt 2520
ttatatttct ctattatatt tctcttataa tgataataat ctatttattt tagttatttt 2580
tacattattt gttctattaa ataacaatat tattaataaa ttttaataat taaagttata 2640
ttttattata attattaaaa ctttttaata gcactgaatt actttatgta atgttattaa 2700
aataacaaat atatatatat atatataaat ttgatacgtt ttcaataaag gactccatcc 2760
taacgtactt aatgtaaaat caccttttta gatatatctg gagcttataa ccatttatct 2820
actcgtgaac ttgaagagga atgtagacgt tttcgtagtt tacgttaaaa actaatatat 2880
atatatgtta taatacaata atatcattat tgattataag tatacaaatc gtacaaaatt 2940
tttgatataa ttattattct atcttattta gatgatagta aaatacaata taatattgta 3000
ttttaggagc ttgagcagat ggttttgcgt tttgattgca aattgaaata tagggattcg 3060
atttccccga gctcctcaaa atgtttttta tattaattaa atataataaa aaatattatt 3120
acataatttg ctatatatta ataaataatt tttatattcg ttttaattta tctatttaaa 3180
tttatattaa cgtgcgacat gcttttatct tcgatattta gtttattgtt atcaaatgct 3240
ttgtctttta gacgtgatac tgcaattcta tatagtagaa ttggtataat agtattatta 3300
tattgtatct atttatctta taataattta tttataacat atttagataa tgggtttggg 3360
ctatttggag gattattttt catatcttct ataacacaaa catttcatat attaattttt 3420
ataattagta ttttaatatt aaatatgaca ggattttatc ctagaaagct tgtaactagt 3480
aaatatgcta gtttacataa attattattt actaaattac aatttagtaa aaatataaca 3540
gtatctaata ttattttaaa aaaaggagaa caatatttaa tattagagta tacattgatg 3600
ttatttttta ttataacagg aagtgtatta ttaatatcaa gtagtgactt agtttctatt 3660
tttttatcta tagaattaca aagttatgga ttatatttat tatgtgctat atacagaaat 3720
tctgaatcat caacttcagc tagtttaact tacttcttat taggtggttt atcttcttgt 3780
tttattttat taggtatagg tttaatatat tctaatttag gtgttacaaa tttagatagt 3840
ttatatacta taattaattt agcaggtata gataatgagt tagaaataat tacatatata 3900
ccttattgtt tattattaat ttctgtagga ttcttattta aaatatcagc tgcaccattc 3960
catttttgat ctcctgatgt ttatgatagt atacctacta ttgttacaac ttttgttgct 4020
attatagcaa aaatatctat attaacatta ttattacatt tagtacataa tactaatagt 4080
attaatatta cttctcaata tccttgaact ataagtttct taattagttc tttattatct 4140
ttaataatag gaactgtttt aggtttaact caatttagaa ttaaaagatt atttgcttat 4200
agtactatct ctcatttagg ttttatgtta ttagctttag ctattaatac tatagaatct 4260
attcaatctt tcatatttta tttaatacaa tacagtttat ctaatttaaa tgcttttgtt 4320
ttattagtag ctataggtta tagtttatat gcttatacag ataataatat taatcataat 4380
aatttaatag ataaaaataa ttctccaatt caattaatta gtcaacttaa aggttatttc 4440
tttattaata gtattttagc tttaagttta tctattactt tattatcatt tgcaggtatt 4500
cctcctttaa taggattctt tgctaaactt atggtattct ctgctgcttt acaagaagga 4560
tttattttct taactattat agctgtatta actagtgtaa ttagtgctgt ttattattta 4620
ttaattgtaa aaacaatgtt ctttgatcaa cattcttata catactttag taaattaaaa 4680
gctcttaaat taccagctct tgttttacaa aaagataaag ttgtagataa aatctatttt 4740
tctaattttt cattatctaa ttctttatct ataactatct ctacattaac tttatttatc 4800
cttttattta tggcttcacc taatgaatta attcatttta ttaatatctt aggtattttg 4860
tagaattttt acattattta tttttcttgt taactgatat ttatatatat attatatata 4920
tatattatat gttagttttt tttataatgt tatatttaat tgtgtctaga tatgttataa 4980
atagaatatt tatatacatt atattattta ttaatacttt atgtgaagat agctataatt 5040
cttataatat taaacgtaaa tttgatgaag attcaggtga atcttctaat agaaaacgta 5100
aattaaatcc taaaaattct gctagttcag aatctgaacc tgaaccagaa tcaagttcac 5160
aatctggatc tatatcagaa tcagatagat tagaatctgg atcagaatca gaagcctcta 5220
atagtatttc tagtgttcca tctttagatc attcagatga aacattagat gatgaaccta 5280
tgtattctga atctgattct ccttcacata gagaatcatt tgaagatata ttgggataca 5340
taagggtaat ttaatcttat tatttatttt tagattcctg aattttaata tatatatata 5400
tatatatatt cataagtgtg aggatattat atccttggtt gggaggaggg tattttagcc 5460
tttatagctc aatggtagag cggaatactg ttaatatttt gatagatgtt caattcatct 5520
taagggctta tattataagt tataactata atatagatat atataaatta gatccttaac 5580
tagcataaat taaaaaatag caagagagct attagaaaaa aaaaattata aatataaaac 5640
atgtcacaac taatactatt cttttttgta aatcaagtaa tatttgcatt tatagtgtta 5700
actatattaa tctatgcatt taataaatat atattaccta gattattata tatatataca 5760
acacataaaa aagttataaa aaataaagta aaagttttaa ataatgatat caataaaact 5820
tgtgaatttt tagaaataac attagttgaa aaaataatta ttagaattaa tactgtaaaa 5880
attataacta tttttttaat aagaccatta ataaaaaaat tattagatat attacaagaa 5940
atattacaac aactaggtta tagcatagga gctgtaccag aaaacataat atattatatt 6000
tctatcctag ttggatttat tatgttatta ggtatatttg aatttgagtt gttaaactga 6060
ataaaatctt taaaacccaa tttaggaatt atggaatgtt taataaatta tataaagaca 6120
aataaaatac aagcaacaat aaaatcaata ggtcctggtg ttttaacaaa tattaaatgc 6180
gtggacaaag aaggaagaat attaggtcca gatggttcat tattaggaga agaaaataaa 6240
ggtaatatat atgaatatta tatacaagat gaagttatag ttagatcaga tggctttaaa 6300
cgtggatttt atgataaaga ttgaggtata gcttctgaag aaatgcctaa aggtagtaat 6360
tacactaata agtgtaaggt tcataataca aaggttgaaa ttgttaacaa taaaattaat 6420
tatctgagag attgagctgc tcatttaaaa aataattaca catttgaatt tattgtatgc 6480
cctaattata gaattaataa tgagcaaata aatattgcaa tcaaccatat aaatgaacat 6540
atgaaggttt atgatcaatt aaatgagtta ataactagat ataataatta ttataatcat 6600
attaaaataa aaaaaccata tctaccatta gtgttaccac cacttaatgt agaagctgac 6660
agggatttaa tacgtagcct tgaaaattta gaacaacatt taaaaataca aaaagataat 6720
tctaatttaa gttcataagt tatagtatat tttttacatg agtagatctt cttaaaaact 6780
aacctatttg tgaaattcta ataattatat attcttctga tattaataca tatctatata 6840
ataacacaat aaatgtgtca aatagatttt gtattaaatc caataaacca atttgaaata 6900
atagatttat aattaacatt agtttatcgc ttattagtta caaaataatt ataaaataat 6960
atcaaacagt tgatcaataa gataaaaagt tataaatgta atagtatata gtattgtaat 7020
aaaacaacta aatctaataa ataatatttc cttttatata agtattataa ctgataagtt 7080
tgtaattaat taagtaaaaa tgcctataaa attcatacaa tataaatgat ataaaaaatt 7140
tctgaaaagg aaagtccgac actaattaag tatatatgct tattaaataa gctttattaa 7200
aaaataagga atttaacaga aactaaaaat tattaaaaac gttaatataa cgtttatata 7260
taattgcgaa cattactcct atatttagta ttaatttaga attataaaaa tatttttaac 7320
agaaaccggc ttaattatat ctttatatta gtatgataat tttttcaaaa agtgtaaaaa 7380
aaaatataat cttgcactaa taaaaaaaaa aacaataaaa aaagcgttaa aaacaacaat 7440
aataaattta atataaattt ttaaatttaa ttttgggtaa agttagggtg taatatccga 7500
gggtttaagg tgggtaaatt attagttata agtaactata cctattataa gtaatattat 7560
atagttaatt aactatataa aacagtaatt aataaataaa ataattactt taatctttag 7620
atttttaaat ctgataaata aaaaaaaaaa aataaataaa taaattaagt gaagaaaaaa 7680
aaaaataaaa aaataaaaaa tgagaatttt aaaaagtcat cctttactaa aaatagtaaa 7740
ttcgtatata atagattcac ctcaaccagc taatttaagt tatttatgaa atttcggatc 7800
attattagct ttatgtttag gtatacaaat agtaacaggt gttacattag ctatgcatta 7860
tacacctagt gtatcagaag catttaattc tgtagagcat attatgagag atgtaaataa 7920
tggatgatta gtacgttact tacactctaa tacagcttca gctttcttct ttttagtata 7980
cttacacata ggaagaggtt tatattatgg atcttacaaa acacctagaa ctttaacatg 8040
agctattgga acagtaatac taatagttat gatggccaca gccttcttag gttatgtttt 8100
accttatggt caaatgagtt tatgaggtgc tacagttatt actaacctaa tgagtgctat 8160
accttgaata ggtcaagata ttgttgagtt tatttgagga ggtttataca cagatgaacc 8220
acaatgcggt gacgtattgt taaaaatcct gcttaatgct ggaaaatccc caatcttagg 8280
atttgcatac gacttattct ttataatagt attattaata ggcgtgaaaa ttgcaatgac 8340
acggggaaaa tcagcagggg tgagaagttt acatacttca gaagcctctc agagactaca 8400
tgcaggagat cttacatatg cctacttagt aggattgttt gaaggtgatg gttatttttc 8460
cattacaaaa aaaggtaaat atttaaccta tgaattaggt attgaattat caattaaaga 8520
tgtacaattg atttacaaaa taaaaaaaat tttaggaatt ggtattgtaa gttttaggaa 8580
aagaaatgaa atagaaatgg tagccttaag aataagagac aaaaaccatt taaaaagttt 8640
cattttacct atatttgaga aatatcccat gttttctaat aagcaatatg actatttaag 8700
atttagaaat gcattacttt caggtattat ttctttagaa gatttacctg attatactag 8760
aagtgatgaa cctctaaatt ctatagagtc tattattaat acatcttatt tttctgcttg 8820
attagtagga tttatagaag ctgaaggttg ttttagcgtt tataaattaa ataaagatga 8880
tgattattta atagctagtt ttgatattgc tcaaagagat ggggatattt taatatcagc 8940
catacgtaaa tatttatctt ttactactaa agtttattta gataaaacta actgttcaaa 9000
gttaaaagtt acaagtgtaa gatcagtaga aaatattatt aaatttttac aaaatgcacc 9060
tgtaaaatta ctaggtaata aaaaattaca atatttatta tgattaaaac agttacgtaa 9120
aatatctaga tattcagaaa aaataaaaat accttcaaat tattaaaaga gatcatgata 9180
tagtccgatc aataaagaaa tttattgcgt atagtaagag gatttaatat ttatattaaa 9240
tctgtaacta tcaacataaa tgctctgtaa ataatgcaac tttaaacaga ttctttgcat 9300
tacatttctt attacctttt gtattagctg ctttagcatt aatgcattta atagctatgc 9360
atgatacagt aggatcaggt aatcctttag gtatttctgc taattacgat agattacctt 9420
ttgctcctta ttttatattt aaagatttaa taactatatt tatattcttt attgtattat 9480
caatatttgt tttctttatg cctaatgctt taggtgatag tgaaaattat gttatggcta 9540
atcctatgca aactccacct gctatagttc cagaatgata tcttttacct ttctatgcta 9600
ttttaagatc tatacctaat aaattattag gtgttatagc tatgtttgct gctatattag 9660
cattaatggt tatgcctata actgatttat ctaaattaag aggagtacaa tttagacctt 9720
taagtaaagt agtattctat atttttgtag ctaacttctt aatattaatg caaataggtg 9780
caaaacacgt tgaaactcca tttattgaat ttggacaaat ttctactatt atttattttg 9840
catatttctt tgtaatagtt cctgttgtta gtttaattga aaatacttta gtagaattag 9900
gaactaaaaa aaacttttaa ttcttagtcc tcttaggaaa aaaaaacaaa tttattaaaa 9960
cagtcgaaat ttaatttatg aaaatgatat tagacaaaaa attttaaaaa gaattagata 10020
gctacatttg attataatca atttattaat attttggttt tcatctatac tttgtagtta 10080
atcataagta tgatgtaata aatagtaata tctttttaaa gtagacttga cctttaaaat 10140
ttttaatata attattatta tcttgttaga gtataattaa atacaatata atattgtata 10200
ttaggagttt gagcaaatgg ttttgcgttt tgattgcaaa ttgaaatata gggattcgat 10260
ttccccggac tcctcaaaat attttatgaa atgtagaata gtaagttgta tcttatgaac 10320
ttaaaaaata tatagcaaaa agctatatat tttttataat aggtcatatt atataaactt 10380
ttaagataaa aaatacaaat tttatttata ctaaacttaa taaataaact gctagtatca 10440
aaatatagca aaaagctata tatctttata tattagtatg ttataatata tgttataaaa 10500
aaaattatat ttataatata tatcaaaaca ataaataaat aatttcgaat aaagcattaa 10560
tataaagata tttgtatttt tacaaagata ttataaaaaa atgttatatt taatttcaat 10620
aattgaaggt cttttagtaa ttgttcctgc tttattatca gttgcttttg taacagtagc 10680
agaaagaaaa actatggcaa gtatgcaaag aagattaggt cctaatgctg taggttatta 10740
tggtttatta caagcatttg cggatgcttt aaaattatta ttaaaggaat atatagcgcc 10800
aactcaagct aatatagtat tattctttct tggtcctata ataactttaa ttttctcttt 10860
attaggttat cttgttattc catttggttc aggtttatat atatctgatt ataatttagg 10920
tatattatat atgttagctg ttagttcttt agctacatat ggtatattat tagctggttg 10980
atcagctaat tctaaatatg catttttagg ttcattaaga agtacagctc aattaattag 11040
ttatgaatta attttaagtt ctgtaatttt attagtaata ttattaacag gaagtttaaa 11100
tattattact ataatagaat cacaaagagt agtaaattta ttattccctt tattcccttt 11160
attcttagta ttctttatag gttctatagc agaaactaat agagctcctt ttgatttagc 11220
agaggcagaa tcagaacttg taagtggttt catgacagaa cattcagcta gtatatttgt 11280
attctttttc ttagctgaat atgctagtat tgttttaatt tgtattttaa atagtatctt 11340
atttttaggt ggttatttaa gtattatacc tttagatttt attgtaaata tcttaaattt 11400
atttgttaat actaataata ctattttatt tgatatattt gtacatattt cttcttctcc 11460
tattaattta gcattaaaaa cagctttctt aatctttgta ttcatttgag ttagagcttc 11520
tttccctaga atacgttttg atcaattaat gtctgtttgt tgaactgttt tattacctat 11580
aattattgct tatgttgtat taataccttg tattgtatat ggtctaggta taataccaac 11640
aaacatttca ttattatagt aaaaaaataa aattatatta taaatagtat aatttttaaa 11700
ttatatgcgt ttttaataaa ggattagggt actatcctat atatatttta ggagtattaa 11760
gttaattaat tattaatatt agttatactt aatataattg gcatacaata agtaaaaaaa 11820
aataaatgat atattacatt atctaatgta atatatatat tttataaata gtaaataaat 11880
tatttaaatt atatatatat aatatatgtc tttattatta ttaataacac ctttaatagg 11940
aataacttta gtaacattac aaggtaatta tggtttatcc ataattaata atgttaaaat 12000
aaaatcaatt gcgttagtaa caacaataat aaatttgatt atatcactgg taatgtttat 12060
cttatttgat tttagtagta aacaatacca atttatagaa gaacattatg aaattaatca 12120
ttttgatatc tatttaggag tagatggttt atcaatatat tttgtgttat taacaacaat 12180
aataatgcca atagctatat tatctaattg aaattcaata gaatctaaaa atgtattatc 12240
atttatagta ataatgctat tgttagaaac acttttatta gcagtgttct tagtattaga 12300
tatactattg ttttacatct tttttgagag tatattacca ccattatttt tgttaatagg 12360
attatttggt tcaagtaata aagtaagagc tagtttttat ttatttttat atacattatt 12420
aggatcatta tttatgttat tatcaataat agctattact tctattatgg gtacatcaga 12480
ttttgatgca ttaacaaaag caaactttaa ttatataact caaatatttt tattttatgg 12540
tatatttata gcttttgctg taaaaacacc agtaatcttt ttaaatactt gattattaaa 12600
agctcacgtt gaatcacctt tatcaggaag tattatttta gctggtatag ttttaaaatt 12660
aagtttatac ggtatattta gattaatttt acctttatta cctaaagctt ctataaatta 12720
tacttatata atttatgtta taggtgtaat aactatatta tatgctagtt ttagtacatt 12780
aagaactata gatattaaag aacttattgc ttattcatct gtatctcatg cagctgtata 12840
tttaataggt gcatttagta atactataca aggtattgaa ggatcaattg ctttaggttt 12900
agctcacggt tttgtttctt caggtttatt tatttgtgct ggtggtatct tatacgatag 12960
atcatctact agattaataa cttattatag aggtatggct caaattatgc ctattttctc 13020
tgtgttattc ttcatattag cattaggtaa tagtggaact cctttaactt taaatttcat 13080
aggtgagttt atgtcattat atggagtatt tgaaagaatg cctatcttag gtgttttagc 13140
tagtacttct atagttttct ctgctgctta tactatattt atgtataata gaatagtatt 13200
tggtggttca tattctatct attttagaga aaatataggt gatgtaacta gaagagaatt 13260
tataatgtta ttagttttcg ttatattaac tgtattattt ggtatatacc ctgctcctat 13320
tttagatggt ttacattatt cagtttctta tttaatatat aatattaatt aaaaaattta 13380
atatataata ttaatataaa aatataatat ataaatatat atctaaatag ataagtattc 13440
ttattagctc aatggtagag caaaatactt ctaatatttt gatctaagtt cgagtcttag 13500
ataagaaggt aattatatat aaactattag cctttatagc tcaacggtag agcggaatac 13560
tgttaatatt ttgatagatg ttcaattcat cttaagggct tatattataa gttaaaacta 13620
ttatagtata gttatatata aattactacc ttaaccagca taaattaaaa aatagcaaga 13680
gagctattag aaaaatttat aagtttaata taaaatatgc cacaactagt accattcttt 13740
tttgtaaatc aagtagtatt tgcatttata gtattaactg tattaatcta tgcatttagt 13800
aaatatatat tacctagatt attacgtaca tatatatcac gtatatatat aaataaatta 13860
taattataat tataattata aacaaaataa aacttgtata tagaaatata taagaaaaat 13920
ttatcaattt aaatttattt aaataataat gacatgtatc aatttaattt tatattaagt 13980
ccattagatc aatttgaaat aagagattta ttctctttaa atgctaatgt attagggaat 14040
atacatttat caataacaaa tattggttta tatttatcaa taggtttatt attaacatta 14100
ggttatcact tattagcagc aaataataaa ataataccta acaattgatc aataagccaa 14160
gaagctatat atgcaacagt acatagtatt gtaataaatc aattaaatcc tacaaaaggg 14220
caattatatt tcccttttat atatgcatta tttatattta ttttagtaaa taatttaata 14280
ggaatggttc catatagttt tgcatcaaca tctcatttta ttttaacatt ctcaatgagt 14340
ttcactatag tattaggagc aacattttta ggattacaaa gacatggatt aaaattcttt 14400
tcattatttg taccttcagg ttgtccttta ggattattac ctttattagt tttaatcgaa 14460
tttatttctt atttatctag aaatgtatct ttaggattaa gactagcagc aaatatatta 14520
tcaggtcaca tgcttttatc tattttaagt ggatttacat ataatataat gactagtgga 14580
atattattct tctttttagg tttaatacct ttagcattta ttatagcttt ctcaggatta 14640
gagttagcta tcgcatttat tcaagctcaa gtttttgtag ttttaacttg ttcttacatt 14700
aaagatggat tagatttaca ctaattaaat attaatttct aaatttgttt attatatcca 14760
tgaaatataa atgatatgta atgaaaattt tatgataagt aaagtttatc attaattaag 14820
tataaatgct taaaaaatcc atgtaagata aatgatataa aaattcctga taaagtccga 14880
tactaattaa atatatatgc ttattaaata agctttatta aaaaataagg aatttaacag 14940
aaactaaaaa ttattaaaac gttataataa cgttttttat ataattgcga acattactcc 15000
tatatttagt attaatttat aattataaat gtttttaaca taaacgggct taattatatc 15060
tttatactaa tatgataagt aaaaaatttt tttttttcaa aaagtgtaaa aaaaaatata 15120
atcttgcact atatattata aaaaaacaac ataaaatata agataaaaaa gtatttatct 15180
atacaataaa ggtatagagt aatatcccca tttccctata ctatgaaaaa ttttgagttt 15240
gatgatggct ctgattgaac gctgtccaag tacttgacac atgctaatcg aacgactaat 15300
tagttattaa actaagtagt agtggtgtac aggtgagtaa aagataattt ggctacctta 15360
aagtaagggg aaaaatccct tataaaaaga aaggaaaaag aaggtccgct ttaagatgtt 15420
aattattatc acggtgagta gtagtaaagg taatgacttt actagctaaa tccgtagtcg 15480
tgactgagag gtcgatcgac cacattgggt ctgaaaaaac cccaatgcgt tttagtacag 15540
cagtgaggaa tattggtcaa tggccgaaag gctgaaccag taacttggaa gaatgaaagt 15600
gtattataat aatacaataa cgattatatc gtataaaatt ctaaatagaa taatgataat 15660
gacaattttc tatttataag tcttgaccaa actacgtgcc agcagtcgcg gtaatacgta 15720
gaagactagt gttaatcatc tttattaggt ttaaagggta cctagacggt aaattaaact 15780
ctaaatgagt acttatttac tagagtttta tgtaagaagg aagaatttct ggagtagtga 15840
tataatacgt atataccaga gagactggta acggcgaagg catccttcta tgtaaaaact 15900
gacgttgagg gacgaaggct tgggtagcaa gaaggattag ataccctagt agtccaagca 15960
gacaatgatg aatgtcatag actagaaaaa gtcgtttaga ctataaattt agtctataaa 16020
tgaaagtgta agcattccac ctcaagagta atatggcaac atataaactg aaatcattag 16080
accgtttctg agaccagtag tgaagtatgt tatttaattc gatgatccgc gaaaaacctt 16140
accacagttt gaatatttta caagcgctgc acggctgttt ttagttaatg tcgtgagatc 16200
tggttaagtc ctttaattaa cgaaaaccct cactttattt gcatttataa agttgatcgc 16260
ctttatattg gttagataat agggattaag acaagtcatc atggccttaa tactgtgggc 16320
tatagacgtg ccacatatgc ctttacaaag ggatgcgatt ttgtgaaatt gagctaatcc 16380
cccaaaaaag gatataatat ggattgtagt ctgtaactcg actacatgaa taaggaatta 16440
ctagtaatcg tgaatcacca tcgtcacggt gaattaaaaa tcagctgggt actaaccact 16500
cgtcaggcgc tgaaagaagt atgtgcaaga agtttgattt acttatattt ataatatata 16560
taatcagtta tatatattta taagttaaat tttcgcatgc atgactttga ttggtgttaa 16620
gtcgaaatat ggttcgtgta atggaaattg cacgggatga attaaccact tagcaataaa 16680
taaatgcata tatataccaa attttatata cttttgtata taaaagatgg aacaaatata 16740
tatatagcat tatctttaaa taaaggtaaa ttgaaaaaat tttttcaatc tggttcaaat 16800
ccaggaaaag ataaaatttt aaggaagggt ccgtatgttg gtttacgggt tgagctgtaa 16860
actcaatggc tatgagccgt cgaaggttcg attccttttc ttcctaataa ctagaagtct 16920
ataaagattc aataccataa aaatttatct gctttattct agcattaaat aattaaaggt 16980
atgatgactt aaatttctaa taacactata aataaactat ttagatatta tatataataa 17040
tgaaagatta attaatttaa aattttaatg aataatatat tttataacga ttatatctct 17100
aatggattat tagaggtttt attaataaat gattatataa ctaatggatt caaagtagaa 17160
tttttagata ttttttatat aatatcaata acatttggag tatttacaat tataagtaga 17220
aaccctgttg tatctgtatt atttttaata ggtttatttg taaatatagc aggaatatta 17280
atattagcag gaataaatta tttaggatta tcatatattt tagtatatgt aggtgctgtt 17340
tcaattttat tcctttttat attaatgtta ataaacatta gaatttctga actattaagt 17400
gaaacaaata atgatatacc attagcagta ttaacagttt tattatttta ttacataata 17460
ggacaagtat taccttgtaa tttaacagat aaaacaataa tttcatcatt atcaaacaga 17520
tttacaggta tttataatat agatataagt aatcaatcaa gtatagtagg tataaatcaa 17580
gaaataggat acgtaagtag taaaggttga gataatacat tagtagagtt tacacaaata 17640
tcaggaatag gaaatataat gtatactaat tattcaatat gattaataat aagttcagta 17700
atattattat taggaatggt aggtgccata gtaataacaa taaaacaaaa gtaattattg 17760
aaaacttgta aaataaaaaa taaaaagtga tatatcaatc taaaagaaat tttcaaaacc 17820
atccgttcca tttagtatca ccatcacctt gaccattatt tactagtatt tcattattta 17880
tacttacaac agcaacagta ttatttatgc atggtttcga aggattccaa tatttagtac 17940
cagtagcagt aattaatgta atgtatgtaa tgggtttatg atttagagat gtaatatcag 18000
aaggaacata tttaggaaat catactaatg ctgtacaaaa aggattaaac ttaggagtag 18060
gtttatttat tatctctgaa gtattctttt tcttagcaat attctgagca ttcttccata 18120
gtgctatctc acctagtgta gaattaggtg cacaatgacc accattaggt atacaaggaa 18180
taaatccttt tgaattacct ttattaaaca caataatttt attatcatca ggtgttacta 18240
ttacatatgc acatcattca ttaatacaag gaaatagaaa aggtgcatta tatggtacag 18300
ttgtaacaat actattagca atagtattca cattcttcca aggagtagaa tatacagtat 18360
catcattcac aatatctgat agtgtttatg gatcatgttt ctattttgga acaggtttcc 18420
acggtttaca cgttataatt ggtacagcat ttttagcagt aggtttatga cgtttagctg 18480
cttaccactt aactgatcat catcatctag gatacgaatc aggaatctta tactgacatt 18540
ttgttgatgt tgtatgatta tttttatata tatcagtata ttactgaggt tattaaatta 18600
aaatttatca catagtataa attatatata ttatatagtt ttagatatat aaatatctaa 18660
gagactttag tttaatggta aaacatatga cttttaatca ttctactata ggttcgagtc 18720
ctataagtct tatatagata atgactataa gttaataggt agactgttcg tcttccaaac 18780
gaagtgtgtc gattcgaatt cgactagtca taaaataata taattattta aagtaatatg 18840
aatatatact aagatatata atataaacgg ctataagtta atggtagact acttagctac 18900
cacctaagat gtgtcgattc gaattcgact agccgtaaag ggttagtagt ttaataggta 18960
aagcgctttt ttgtcaagga agtttatgcc ggttcaagtc cggcctaacc cgagattact 19020
cgtttaatta attgaaatgt aaacgtatta attctaaaaa tcaactaatt aagggaaagt 19080
ttccatgggt agggtaagat atttgctaaa tattgtgcgt ttgcacatag atgttcgaat 19140
catctctttc ccgaaagagt atagtttaat ggtaaaactg taagcttcaa ccttatattt 19200
cttagttcaa atctaagtac tcttgaatat aaagagatat atgtatatat aaaatgatat 19260
atatatatta ataataaatc cacaaaaaaa aaaagagtaa agtaataaat aaaataagca 19320
aataggaagg gaatatattt attaaaatat ttttttataa aaactgcgaa ttgtaggttc 19380
tttaacttaa ccggtaaagt gtgttcttga taaggatatg ttcagtgttc gagtcactga 19440
agaatcaaaa tactaaaata attaaaaaga gagttggctg agtggtaagg cgactagctt 19500
gagtctagtt aagttaaaac tttcatatgt tcgaatcata tactctctga ataaataaaa 19560
atataaacag gttagagcca ggtggttagg cgtcttgttt gggtcaagaa attgttatgt 19620
tcgaatcata ataacctgaa tataaaaagt ataaattgaa tatctattca ataaaggtga 19680
tatatatcat atataacttg agaaatcaag gctagtatat aaagaaacta ttatggataa 19740
ttagctatat attaaagaaa taaatgacta gataaatcta ggattctaag aaaaatcaag 19800
taataaacga agtgaattga aatatcttag taactttagg aaaagaaatc aaacgagatt 19860
aatatttagt gtaaactaaa tattaaaagc ctaataacaa aacttgttaa gtaaaatgga 19920
ttcaaatctg tttgaataaa ggggaacctt cctctaaggc taaatataat atataagcga 19980
tagtgaaaag taccgtgagg taaaatttga aatagtagtt ttataagcag ctcaaataaa 20040
atttaaataa aaaataagag cgtacctttt gcataatggg tcagcaagtt aattttagat 20100
gcaagtaaag ataaccaatt taaattataa aaagttatga ttaaaaaggt tattttaact 20160
tagataaacc aattatgaaa aaatgagtta gtatctaaaa ttagacccga aggctagtga 20220
tcttaccatg gtcagggtta taaaagcccg aacgggttat cgttgtatag atatccgaag 20280
aattgtggta agttagtgaa agacaaaact gactagtata gctggttttc cgcgaaacct 20340
atatgagtag gtaatttaat taacatctta gcaggtacag aactgtgatc tcggataata 20400
tcaattgatt ttatcaacat cggggggttg tagtgacttt actggagagt atttgcactc 20460
ggaatggcaa agatgaatgt tgaataatcg gacatagtgc gataaggttg tatgtcaaaa 20520
gggaaacagc ccagaacaag agttaaggtt ccaaaattat tgttgagtga aattaaggat 20580
gtttattatt aatacaatca ggaaataggc ttagaagcgg ccattttttg aagacctcgt 20640
aacagagcac tgattaaatt ttagtaataa acaccaaaaa tttaacggat ctaaacaata 20700
taccgaaacc ttgtacacat attataataa ttatatatat taattaatat atattgtata 20760
tgtggggtag cggaacattg gataaatatt atttattatt acttctaaga tataataagt 20820
ataaatatta ttttaataat aataatttat caatataatc caagagagaa tgctgacatg 20880
agtaacgaaa aaggaataac ctcgccttaa gcttatggta ttgtattaaa ttttcggtgt 20940
ctaaaaatat taatcaaaag gtaattttga tgatatttat atatgtacta aatattaatt 21000
agagtatata atactcttag ttaataaaag taaaaaataa aacctttaaa aagtaataaa 21060
ggttctggaa aattttttac tatattatat atatatttta tttagagcct tagggactag 21120
ttatataatt tcttgttaaa gttttcaaga ttgtataaag tataatttta atataaaatt 21180
atacaataat atatatatat aataagttat atatataccg tacctagata ctatcacaag 21240
taagcaagta gagaatacaa aggcgtttga gtgaacaatc attaaggaac tcggcaaatt 21300
gactaccgta acttcgggat aaggagggcc attattataa tatttaacgt atatatgtct 21360
gtaaaaagat atatatatga tataattaaa tatataataa gaggaaacat aaaataatgt 21420
tgtacgactg tttaataaaa acacagcact ctgcaaagat taaatatcaa agtattgagt 21480
gtgatgtctg cccaatgtcg gcagggtaac gaatcttcct tggtatataa ctaaggtttt 21540
gaaggacacc ccgataaatg gcggccttat ctatgagggt cctaaggtag cgaaatacct 21600
tggccgttaa atgcggtcct gcatgaatga cataacgata caacagctgt ctcgatgatt 21660
gtctcagtga aattggaata gcagtgcaga tactgtttac ctctagatag acgagaagac 21720
cctatgcagc tttactgtta ccagttgcaa aagttaaatt ttaggatgtt ttcgagagta 21780
taaggtagtt gaaagataac aatgaaacac ctttattcgt ttccttatat atgcttttga 21840
agattggaag gcagtttatg cggggcacag atcccacaaa aagtacctgg gtatatccaa 21900
agttcatatt gtaaatttgt aattttatta caaatatttt aatttgttat atttattttt 21960
ataaatatac agttattgtt aaattaagtt caatatttat ttttatctaa gtaagatttt 22020
aactatatgg ttaatagatg tatttaaaac tttaatattt aaaagttttt tgtcatatta 22080
attttgtata ttatcttaat taatatgatg attaattata ctttaaaagt ttaatggcta 22140
aatcttgctt tactgtttga cctacaagtc tatcagtcgc gtaagcgggg catatgatca 22200
caagatgcat taaggaaagg tcttggatta tagaaaaagt tacgctaggg atttttaact 22260
gtaagtcctc caatatataa tatattggaa atatattggg taaatttcaa aaataactta 22320
tgtttataag tgtatttgaa gcgaaactta tttttagcaa tgaaatagta attataaatt 22380
acttttctaa aaaataagaa cgttcaacga ctaaaaggtg agtattgcta acaataatcc 22440
tttaatcatg cccaacatct taattaatta tttagtaata tgtataataa ttattattat 22500
tttaataata ataatgaaat agacttggtt atatatcaag cttaatatta tataataaat 22560
tatattaata ttaatacaaa taaataaata taaatgttaa atattataaa atctaaaata 22620
aaaaatacat ataaaaaaaa agcattaaat gatgagaatg taagttttta taataaaaat 22680
tttgtacctg ctgtaagaga ttgaaaaaat agtatttatg tatataataa aaatacttta 22740
agtttaatac ctgttgcaag tagattagta atgaaattaa ttaaaggtta ttttaattca 22800
tataatttaa atatagaatc taaattaaga aataaaagat tacgtcgtag atatagaaaa 22860
ttatcaacaa ataaaatttt tatcagtgaa ggtgagttta aacatactaa tgataaaatt 22920
aatataacat tatatgtata taataaacaa aaactaaact atttagctaa attaaaaaaa 22980
aaatatacaa gcttatttgg aaaagatata tttattaaaa aattacaatt aataaaatct 23040
aaagcaatag gtatattaac tcaacaacaa aaaaaaagta aaacattaac aaatgtatta 23100
cctaaatata gtacaaaagt aaataaaata caaaatattt attatagaac ttatataaaa 23160
aaatcaatta aaagattaaa atattatatg tattataaac aattacttta cataaataaa 23220
gcaaaatttg aaaattcata tttacaaggt ttaattgatt taatcaaaaa aatatataaa 23280
aaaaatatag aatttaatat aataaattta aaatacttat attttaatag tgatatctac 23340
acacaaccat tagtattaaa attaagaaaa aaaagagatt tattaagata tttaaaagat 23400
ttagtaaata aagcaaaaat agaaaaagta agtttaaata aaagatcaga atattatttt 23460
aacttagaaa atttatttac aagaaataat gtagatatta caaataattt attaaataat 23520
ttaatgcaat ataataaaaa aaattcagag tatttaaaaa aagttatatt aaatgatata 23580
aaatataaaa gagtatctgg tgttagatta gaagcagcag gaagattaac taaacgttat 23640
acagcttcta gatcacaata taaatttaaa tataaaggta atttagtaaa tacatattca 23700
tctattcaag gttatccatc atctttaata agaggaaatg ataaacctaa tttacaatat 23760
acaaaattaa attctaaatc acgtattgga tcttttggtg ttaaaggttg agtaagtgga 23820
gtttaataaa ttaatataat cctaaaaata attaattaag atgaagaaat agtctgaacc 23880
attttaagaa aagtggaaat aaaagaaaaa cttttatgat aacaaaattg aacaggctaa 23940
tttgcgcaag agagtacaaa ttgagtgcgc ggtttggcac ctcgatgtcg gcttagctca 24000
tccacatgaa tgcaaaaatc atgaagggtt cgactgttcg tcgattaaag agctacatga 24060
gctgggttta atacgtcgtg agacagtatg gtttctatct tctagaggga attaaagtag 24120
aataaagata gccccttcgt acgaaaggag ttgggtatat tgacctatgg tttacctatt 24180
gtttatgtta tatatatatt ttatatatat atcatttact tatacttaag taaattgttc 24240
gcataggcat ggtaggaagg ctacgtcagt taaagataag tgctgaaagc atttaagcgc 24300
gaagcttact ttaggatact ttaaaacgta gtagaacact acgagtatca ttaaaaaaat 24360
attatttttt tttttaatgt aataggcttt agttgtacta atcttaagat ttttagacat 24420
aaagtactaa tttataaaaa actaaatata atatatatcg aaaaaagccc ggttagcata 24480
aaagtaatgt atccgttttg taatcggaat aaacaagtgc gatacttgta ctgggctata 24540
agacttaaaa ataaagttgg ttaagaccca atggtcaaga tggttaagac ataacatttt 24600
cactgttagt gcgggagttc aatcctccct tgggttgaaa gaaattagct cagttggtag 24660
agcgatcgtt ttacacacga aaggtcaggt gttcaaatca cctatttctt aaaaacgaga 24720
ttgtaaataa agtatataag cgggttgatg taatagtaac atatatggct catgcccata 24780
atatttaggt gcaactccta aatccgctaa aagactatag cttaatcggt aaagcgaacc 24840
actcatgatg gtttgagtaa atgttcaagt cattttagtc ttatataatc cgagtgctgg 24900
aattggtaga cagtcttagc ttaagtttaa gtgacgcaag tcgtaaacgt tcgaatcgtt 24960
tctcggatag gggctatagt ttaactggta aaacggcgat tttgcatatc gttatttcag 25020
gatcgagtcc tgataactcc atattagctt gagaagctca attggtagag cgggtcagtg 25080
aagttgatta ggttgtaagt tcaagtctta tctcgagcat gattttatgg gtatgctgaa 25140
ataaggtaaa caggttccgc ttaggacgga atagtcaaac tttgcaagtt caagtcttgt 25200
tacccgtatt tttatatgtt gtcgactaat cggtaagtca taaatttttg gtatttacta 25260
ttgggtgttc gagtcgcccc aacataatat acttaaatta tattctaata attctattta 25320
atttcttttt tatctctaga gccaaagtag tttaatggta gaacaataat ttcatgaatt 25380
aagaatgaga attcgatttt ctcctttggc taataggaat aaatttatat atatatatag 25440
tttatatatt gtgtttagta aaattaaaac actagtttaa taatatgata tgatagtttt 25500
atataataaa tactttataa gtaaaaaaaa aattcaattg ttaagcctat aaaaaattta 25560
tgaaaattat ttattaaata aggttaatgt aaaaaaaaaa aaataagtat aaaagtggta 25620
taaataggtg agttaataaa ataaataata taagttatat aggtagttcg gtttatttat 25680
ataatagatt tgtattttat tactataaaa attatttaaa gattacttaa tatgtatagt 25740
tatatttata aagcattatt atatcatggt tatgagaaat ttgatttaga tatttgagaa 25800
tattttaata aataatctgt tattaaaaaa taacaatatt atatagattt attaaaacct 25860
gattataata tattatctaa agcggctctt agtttagtat ttataaacct gtttgatcaa 25920
tatattacag ttaataattt gtaaatctat cattataata gattaattat ttaatagagt 25980
tattagatga aaaagcaaaa tctaaataag taacgttact ataggctagt aatgttaatt 26040
attaataaag attatattgg aataatctat aggtgggtgt agttcaaagg tagaacagct 26100
gtatgtggca tagtatatcc tagttcaatt ctaggtatcc accctaacgt acttaatgta 26160
aaaggttata acctagtttg gttaaaggta accacattaa ctaaataata aaaaagatta 26220
tacactattt attattcata taaataaaag ttcatgtata gcatacgtaa agtatgttat 26280
taatgtaagg taaagttatt tttataatta attattgatc tcaatacaaa taatgtatta 26340
ggtaaaaaat tttcaacctc aactaaaaaa gaaaatatta aacaaataga aagttcatcg 26400
ttcttaacgt ttaaacaacc tacagaatga caagaaagat gatatttatc atcaaatgct 26460
aaagatattg gaacattata tttaatgttt gcattatttt caggtttatt aggtacagct 26520
ttttctgtac ttataagatt agaattatca gggcctggtg ttcaatatat agcagataac 26580
caattatata atagtataat aacagctcat gccataatga tgattttctt tatggttatg 26640
ccagctttaa taggaggttt cggaaatttt ttattaccat tattagtagg aggtccagat 26700
atggcaaaat aataagtctt aaataataaa gactttttgg aactattata aataataatt 26760
atttattata atgacttaaa aatatactta gcaggtttat tttaaggaaa tatttgaatc 26820
aataatctag aagaacataa ttcaagattt tagataaaaa aattaatgga tataatagga 26880
tcaggattta ttagatttaa atataattat ataataatta ttattattaa tataattcca 26940
gaattaatgc cgttaaaaag tgtaaacttt ttaattatta cttcgctgta tgcatggaaa 27000
atcccgaaat aacaaataca tagacaccta attggagttt atatacttat aaagttatat 27060
ttccaactga attagtcgta atacttatgt attcatggga ataatatgca ggaaaccaaa 27120
gtaattttga ataaaattat tagtaggatc ctcagagact acacgcgagg ctccattata 27180
atataatgga tgaagacata gtccgttgtc cagttctaac tattaactgg acctaaaaaa 27240
aaaaattctt actcttaaat ttttttgaag taacgaattt aggactaaac aaagagagat 27300
aagggggaaa tacgacccag atgtagggtg atttagctat ttttttagat cattaataac 27360
gtgtatactg acgttaaacc aaaattatgt ggttacaatt tcacaaattt taggtaatga 27420
tttagaaagt agattagcta ataaagtatt acaagattat gtttttaata gcttaagttt 27480
accaaataaa tataatttta ttatgtcaat tggtaattat aattcaatac cccataatcc 27540
gactatcaca tagaaaattt tagaattagg tttcctagat taaataatat aagtttctga 27600
ttattagtac ctagtttact attatttgta ttctctgcaa caatagaaaa tggagcaggt 27660
acaggttgaa ctctttaccc tcctttatca ggaatacaat cacacagtgg gccaagtgtt 27720
gatttagcta tttttggttt acacttaagt ggaattagta gtatgttagg agctatgaac 27780
ttcataacaa caatattaaa tatgagaagt ccaggtatac gtttacacaa attagcttta 27840
tttggttgag cagtaattat aacagctgtg ttattattat tatcattacc tgtattagcc 27900
ggtaaacata gaaaactgcc ggcattaaat ttggctatat actggaaact gttatatatt 27960
tatagatata tatcacaatc agtaggaaac catctaggtt taaacctaaa tgggttcttc 28020
agagactata cgccaaagta ttttcattgt aaattcttac attcgacgaa caataataat 28080
ttaccttttg ctgcttattt agcaggtctt attgaaggtg acggtactat aatagtacca 28140
aatactttaa gatcacctaa aggtaaattg aattatcctt caattcaaat tgtttttcat 28200
ttaaaagatt tacctctagc tttaatgatt caaaaagaat taggttttgg ttctctttca 28260
agaaaaaaag gtgttaatgc ttatattttg actataaata ataaggaggg tatattattt 28320
gttatatctt tattaaatgg taatatgaga acacctaaga taaatagttt atataaattg 28380
attgattttt atcaagataa tataaatatt gaaaaaaagc ctttaaataa tgaaccttta 28440
gaatctaatg cctgattatc aggatttata gaagcagatg gttcttttca agttagaagt 28500
actcttagtg gtaaatatcc taaattcgaa tgtaagttgg aaatatctca aagacaaaca 28560
gatcataaag gttttagtaa tcaggagttt ttaaataaaa ttgctgattt ctttcacact 28620
gaagttaaag aaacaagatt aaatagatct actccagagt atagagttag aactactaat 28680
ttacaaggta ataatcaagc aaagagttat tttattaaat atccattgtt tggaactaag 28740
tatttagatt ctatagattg aatgaaagtt gtagatttat ttaataatgg tgaacataaa 28800
actgaattag gtaaagaaaa aattttaaat ataaaatcta atatgaatga taaaagaact 28860
gtttttactt gagatcattt acaaaatttt tacaaattga aaatataaaa tatagtccaa 28920
acaatttcga gagaaattga gtgtctacct taaataactt acccctatag ggtaagttat 28980
ttttgaaact aagttttata cttatagtta tgggatttta tccagtagac caataataat 29040
agggtattac tatggtttta acagatagaa actttaatac atcattcttc gaagtagctg 29100
gtggtggtga tcctatctta ttccaacatt tattctgatt cttcgggcac ccagaggtca 29160
aaaatatagg cttcttaacg ttgctgtatg ctgggaccac ttcaatatta agttttaaat 29220
actcaatctt aaatgacaca gtaaaaaagt taaaacgatg aagtatatca gcaggtaaca 29280
ttttaaatgg aacctcagag actttatgca acgaaattgt agtaaacaca gaaaatataa 29340
aatctatttc tgttcatgta cctaaacatt taaaaccagt tagtgatgat caatttggac 29400
attatttagc tggtttaata gatggtgatg gtcattttaa tagtaaacaa caattagtta 29460
ttgcatttca ctcattagat gtttctttag cctattatat aaaaaaaaga ttaggttatg 29520
gaagtgttaa aaaagttaaa aataaaaatg cttttattct agttgtaaca gctagagaag 29580
gtatagaaaa agtaattaat ttaataaatg gcaaaattag aacagaaaat aaatttaatc 29640
aaataactaa taatatatta aatcatgaga attatgctga atttagtaaa aaaattagtt 29700
taaaattaaa tttaagtaat aattttaaaa atcattgatt agctggtttt tctgatgctg 29760
atgctagcgg agctggcctt caccccggct gcgtcagccc aaaggctagt tttcaaataa 29820
aaatacttaa tcgtaataaa aaaattgaag ttagattaaa ttttcaaatt gatcaaaaaa 29880
aagaatatat tttactatta attaaagaat ttttaggtgg taatattggc tatagaaaaa 29940
gtcaagatac atattactat ggatctacta gttttggttc agctaaaaat gttattaatt 30000
attttgatta ctttcattta ttatctagta aacatgttaa ctatttaaaa tgaagaaaag 30060
catatttaat aattcaaaat aaagaccatt taaacaatga tggacttgaa aaaattataa 30120
aattaaaaag tacaatgaac agagtaaatg atactacaat ttaagataaa gtcctaacaa 30180
ggatgtttaa ttcttgagta ttaattagaa tagattatta ttattattat taactattcg 30240
attaatcaaa atttttgtta tattttaatt ataccaggtt ttggtataat tagtacagtt 30300
attgctgctg gatcaggaaa aaatgtattc ggatatttag gtatggttta tgccatgatg 30360
tctattggtg tgttaggttt cttagtttga agtcaccaca tgtatactgt tggtttagac 30420
gttgatacaa gagcatattt cacagctgct actttaatta ttgctgttcc tactggaatt 30480
aaaatattta gttgattagc tacatgttat ggtggttcat tacatttaac acctcctatg 30540
ttatttgcat taggttttgt agttttattc actattggag gattaagtgg tgtagtttta 30600
gctaatgcat ctcttgatgt agcattccat gatacatatt acgttgtagc tcatttccac 30660
tacgtattat ctatgggagc tgtatttgct ttatttagtg gatgatattt ctgaatacct 30720
aaattattag gtttatctta tgatcaattt gcagctaaag ttcacttctg aatcttgttt 30780
attggtgtta atttaacttt cttccctcaa catttcttag gtctacaagg aatgcctaga 30840
agaataagtg attatcctga tgctttctat ggttgaaact tactaagtag tataggttca 30900
attattagtg ttgtagcaac atgatacttc ttaacaatta tatacaaaca attaacagaa 30960
ggtaaagcag tttcaagata tccttgatta acacctcaat tattcagtga tacattccaa 31020
gtgttattca ctagaaataa tagttcatta gaatgatgtt taacaagtcc acctaaacct 31080
catgcttttg ctagtttacc tttacaatcg taaagtctaa gtttttggta atagagtgtt 31140
aggcaactgt agttattttt ttttttactc tagctagtta tataacatat ttatataaaa 31200
tatatactgg gacagtaact gtcctattag gttatatgca tttacaaata aatttaaaga 31260
atctattgaa tcatttttat gatcaacgtt atttactatt tgatgcaact gctatcaaaa 31320
aatctgtgga tatatttttg taaaacatta cctaatttta gtaacttttg gtatataagg 31380
ttacgattat tcatcaactt tatacgcttt agcaataaaa agtgtaaagt ttgatcttta 31440
agatgttgta aagatagata gtagttctaa ttatgattac taattaaaat acttttacct 31500
gagaatataa aataatctat aaaataaact ggagacagta actgccctat taggttatat 31560
acatttacaa ataaatttaa attactatgt tacaatcagc tagaataata ggaacaggat 31620
tagctacaac aggtttaatc ggagcaggtg taggtatcgg tgtagtattt ggtgccttaa 31680
tcttaggagt tgcaagaaac cctgcattaa gaggtcaact gttttcatat gcaattttag 31740
gatttgcatt tgcggaagca actggattat ttgctttaat gatggctttt ttattattat 31800
atgtagctta atacaaaatc tttgttaaat ttactagata aatttaaatg tggagtgagg 31860
atattttatc cttgggtata gggtgggtaa ataaaaaaat taaacaaaaa tttaacatga 31920
caacaacaac ttttttttta tttttaatac cagtattagc aataatcttg ctagctgtta 31980
atttaatatt ttcacctcat aatccatatc aagagaaaga tagtgcattt gaatgtggtt 32040
ttcattcctt tttaggacaa aatagaacac aatttagcat atctttcttt atatttgcct 32100
tattatttct actatttgat ttagaaatat tattagtata tccgtatgtg gtaagtgcat 32160
atacaaatgg gatatatggt ttagtaataa tgttagtatt ttttctagta ttaacattag 32220
gttttgcatt tgaattaggt aaaaatgcat taaaaattga aagtagacaa gtttataatt 32280
ttaattataa atcttgaagt ggttattcat taatatataa taaataataa ctaaacaata 32340
aggaaggaag gaataaaata taatataatt tcgaattaat gaaattaata gttaataaaa 32400
aaaaagttaa actaagttaa taggataaaa aagaagaaat aaaaaaatta atatataata 32460
taaatgtttt tagaaatatt aaaaggacac attctaatgg atgcaccaac accatgaggt 32520
atatttttcc aagatagtgc ctcaccacaa atggaaggaa tagaggaatt acataataat 32580
ataatgtttt atttagcaat aattttattt acagttactt gaatgatgat aacaattata 32640
agaaattttg tagctaaaaa atcaccaata gctcataaat atatgaatca cggtacatta 32700
atagagttaa tttgaacaat aacaccagca tttattttaa tactaatagc attcccttct 32760
ttcaaattat tatatttaat ggatgaagta atggatcctt ctttagttgt ttatgcagaa 32820
ggtcaccaat gatattgaag ttaccaatat cctgatttta caaatgaaga taatgagttt 32880
atagaatttg attcatatat agtaccagaa agtgatttag aagaaggtca atttagaatg 32940
ttagaggttg ataatagagt aattattcca gaattaactc acacaagatt tgtaatttct 33000
gcagcagatg ttatacattc atatgcttgt ccatctttag gtataaaagc ggatgcatac 33060
cctggtagat taaatcaagc atcagtttat ataaatcgtc ctggaacttt cttcggacaa 33120
tgttctgaaa tatgtggtat attacatagc tcaatgccta tagctataca atcagtatca 33180
ataaaagatt tcttattatg attaagagaa caaatggaag gataagt 33227
Claims (6)
1.一种产虫草素的基因工程菌，其为含有质粒pQF1的重组构巢曲霉菌；
所述质粒pQF1携带有pyr4序列、URA3序列、Amp抗性基因序列和AMA1序列；
所述AMA1序列包括内源性AMA1序列，以及外源性AMA1序列；所述外源性AMA1序列来源于构巢曲霉菌GO51株；所述构巢曲霉菌G051的保藏编号为ATCC 38163；所述外源性AMA1序列如SEQ No.1所示；
所述pyr4序列来源于粗糙脉孢菌OR74A株；所述粗糙脉孢菌OR74A株的保藏编号为MYA-4619D-2；所述pyr4序列如SEQ No.2所示；
所述URA3序列来源于酵母菌S288C株；所述酵母菌S288C菌株的保藏编号为ATCC20450，所述URA3序列如SEQ No.3所示；
Amp抗性基因序列来源于枯草芽孢杆菌2014-3557株质粒pGMrib，或者质粒PUC18，PBR322；所述Amp抗性基因序列如SEQ No.4所示；
用于构建重组构巢曲霉菌的模式菌为构巢曲霉菌A1145株和/或构巢曲霉菌LO8030株。
2.根据权利要求1所述的产虫草素基因工程菌的在生产虫草素中的应用。
3.根据权利要求2所述的应用，其特征在于，所述应用包括将所述基因工程菌接入发酵培养基，进行发酵培养，然后对所获得的发酵培养液进行分离纯化，制得虫草素。
4.根据权利要求3所述的应用，其特征在于，所述发酵培养基包括CD-ST培养基。
5.根据权利要求3或4所述的应用，其特征在于，所述发酵的温度为20-40℃；和/或，所述发酵的时间为5-12天。
6. 根据权利要求5所述的应用，其特征在于，所述发酵的温度为25-28 ℃；和/或，所述发酵的时间为10-12天。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107574178A (zh) * | 2016-01-25 | 2018-01-12 | 完整基因技术公司 | 真菌人工染色体、组成、方法和用途 |
| CN110367038A (zh) * | 2019-03-22 | 2019-10-25 | 山西万海澳生物科技有限责任公司 | 一种高效抗癌蛹虫草及其生产方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105441517A (zh) * | 2014-08-26 | 2016-03-30 | 中国科学院上海生命科学研究院 | 虫草素的合成基因簇的鉴定和应用 |
| CN107574178A (zh) * | 2016-01-25 | 2018-01-12 | 完整基因技术公司 | 真菌人工染色体、组成、方法和用途 |
| CN110367038A (zh) * | 2019-03-22 | 2019-10-25 | 山西万海澳生物科技有限责任公司 | 一种高效抗癌蛹虫草及其生产方法 |
Non-Patent Citations (1)
| Title |
|---|
| Neutral Lipid Content in Lipid Droplets: Potential Biomarker of Cordycepin Accumulation in Cordycepin-Producing Fungi;Peng Qin等;Molecules;第24卷(第18期);第3363页 * |
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