CN108148797B - 一种基于CRISPRi的共利用葡萄糖和木糖生产N-乙酰氨糖的方法 - Google Patents

一种基于CRISPRi的共利用葡萄糖和木糖生产N-乙酰氨糖的方法 Download PDF

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CN108148797B
CN108148797B CN201810036701.9A CN201810036701A CN108148797B CN 108148797 B CN108148797 B CN 108148797B CN 201810036701 A CN201810036701 A CN 201810036701A CN 108148797 B CN108148797 B CN 108148797B
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刘龙
陈坚
堵国成
李江华
武耀康
陈泰驰
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Abstract

本发明公开了一种基于CRISPRi的共利用葡萄糖和木糖生产N‑乙酰氨糖的方法，属于遗传工程领域。本发明以枯草芽孢杆菌BSGNY‑Pveg‑glmS‑P43‑GNA1为出发菌株，在基因组上整合了受木糖诱导的dCas9以及三个可分别作用于zwf，pfkA和glmM三个基因的sgRNA表达片段，并利用该菌株在摇瓶发酵，使N‑乙酰氨基葡萄糖产量达到20.5g/L，N‑乙酰氨基葡萄糖得率为0.612g/g葡萄糖，同时实现了重组枯草芽孢杆菌对于葡糖糖和木糖的高效共利用，并为进一步代谢工程改造枯草芽孢杆菌生产氨基葡萄糖及其工业化奠定了基础。

Description

一种基于CRISPRi的共利用葡萄糖和木糖生产N-乙酰氨糖的 方法
技术领域
本发明涉及一种基于CRISPRi的共利用葡萄糖和木糖生产N-乙酰氨糖的方法，属于遗传工程领域。
背景技术
乙酰氨基葡萄糖是生物体内的一种单糖，广泛存在于细菌、酵母、霉菌、植物以及动物体内。在人体中，乙酰氨基葡萄糖是糖胺聚糖二糖单元的合成前体，其对修复和维持软骨及关节组织功能具有重要作用。因此，乙酰氨基葡萄糖被广泛作为药物和营养膳食添加来治疗和修复关节损伤。此外，乙酰氨基葡萄糖在化妆品领域也具有诸多应用。目前，乙酰氨基葡萄糖主要采用酸解虾壳或蟹壳中甲壳素生产，此方法产生的废液对环境污染较为严重，而且得到的产品易引起过敏反应，不适宜海鲜过敏的人群服用。
枯草芽孢杆菌(Bacillus subtilis)是一种被广泛用作食品酶制剂及重要营养化学品的生产宿主，其产品被FDA认证为“generally regarded as safe”(GRAS)安全级别。因此，运用代谢工程手段构建重组枯草芽孢杆菌是生产食品安全级乙酰氨基葡萄糖的有效途径。然而，重组枯草芽孢杆菌(BSGNY-Pveg-glmS-P43-GNA1)的产量与产物转化率尚不能满足工业化的要求，因此还需进一步提高重组枯草芽孢杆菌的生产能力。葡糖糖进入细胞之后会进入到糖酵解途径及磷酸戊糖途径，同时肽聚糖的合成也会利用一部分葡萄糖，想要进一步提高乙酰氨基葡萄糖的产量与得率必须减弱葡萄糖去往这些途径的流量。但是这些途径对于细胞生长都发挥着极其重要的作用，直接将其阻断势必会影响到细胞的生长，因此需要对这些途径进行动态的调控，以达到细胞生长与产物合成之间的平衡。同时，木糖作为木质纤维素水解的主要产物，是除葡萄糖以外自然界中最丰富的糖分，但是多数微生物对的其利用能力较弱，葡萄糖与木糖的共利用也存在很大的问题，若能构建一株可以同时高效利用葡萄糖与木糖的生产菌株，便可以使其利用可再生的生物质资源进行目的产物的合成。现有技术中在枯草芽孢杆菌中实现葡萄糖与木糖的共利用是通过解除木糖代谢的调控实现的，即在枯草芽孢杆菌中强化表达其转运蛋白araE并表达来自于大肠杆菌的木糖代谢途径基因xylAB(参考文献：CHEN T等.Engineering Bacillus subtilis for acetoinproduction from glucose and xylose mixtures[J].Journal of Biotechnology,Elsevier B.V.,2013,168(4):499–505.)。然而现有技术只是强化了木糖的代谢途径，并未对葡萄糖的代谢进行调控，而当细胞可以同时利用葡萄糖和木糖时，可以将更多的葡萄糖引入到目标产物的合成途径中去，以避免碳源的浪费。
发明内容
为了解决上述问题，本发明提供了一种基于CRISPRi的高效共利用葡萄糖和木糖生产N-乙酰氨基葡萄糖的方法。
本发明的第一个目的是提供一种能够高效共利用葡萄糖和木糖生产N-乙酰氨基葡萄糖的基因工程菌，所述基因工程菌利用dCas9蛋白和sgRNA调控糖酵解、磷酸戊糖及肽聚糖合成三条途径。
在本发明的一种实施方式中，所述基因工程菌以枯草芽孢杆菌为宿主。
在本发明的一种实施方式中，所述基因工程菌整合表达了dCas9蛋白，同时整合了三个可分别作用于zwf，pfkA与glmM三个基因的sgRNA表达片段。
在本发明的一种实施方式中，所述dCas9蛋白的氨基酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，所述作用于zwf的sgRNA表达片段的核苷酸序列如SEQ ID NO.2所示；所述作用于pfkA的sgRNA表达片段的核苷酸序列如SEQ ID NO.3所示；所述作用于glmM的sgRNA表达片段的核苷酸序列如SEQ ID NO.4所示；
在本发明的一种实施方式中，所述dCas9蛋白通过载体pLCx进行整合表达。
在本发明的一种实施方式中，所述载体pLCx的序列如SEQ ID NO.5所示。
在本发明的一种实施方式中，所述sgRNA的表达片段整合到重组枯草芽孢杆菌BSGNY-Pveg-glmS-P43-GNA1基因组上。
在本发明的一种实施方式中，所述sgRNA的表达片段整合到重组枯草芽孢杆菌BSGNY-Pveg-glmS-P43-GNA1基因组上amyE位点。
本发明的第二个目的是提供所述基因工程菌的构建方法，所述方法是以枯草芽孢杆菌BSGNY-Pveg-glmS-P43-GNA1为出发菌株；所述出发菌株以枯草芽孢杆菌168为基础，基因型作如下改造得到的：ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔptaΔglcKΔpckAΔpyk::lox72，并以启动子Pveg调控表达来自大肠杆菌的磷酸酶基因yqaB与枯草芽孢杆菌168自身的glmS、在质粒上以启动子P43调控GNA1的重组表达。
在本发明的一种实施方式中，所述dCas9蛋白的整合表达是通过转化线性化的载体pLCx-dCas9后得到的，dCas9的氨基酸序列为SEQ ID NO.1。
在本发明的一种实施方式中，整合载体pLCx-dCas9，序列如SEQ ID NO.5所示，是将dCas9插入到整合载体pLCx的酶切位点BamHI与PstI之间后得到。
在本发明的一种实施方式中，整合载体pLCx，序列如SEQ ID NO.6所示，是由F1、F2、F3、F4及F5五个片段通过一步克隆的方式构建而成，其中F1包含壮观霉素抗性基因aadA与大肠杆菌复制子pMB1，F2为枯草芽孢杆菌lacA基因的5′端的800个碱基片段、F3为含有lox71与lox66的氯霉素抗性片段、F4为木糖阻遏蛋白及启动子、F5为lacA基因的3′端的794个碱基片段。pLCx的木糖启动子之后为枯草芽孢杆菌的RBS序列，紧接着是两个酶切位点BamHI与PstI。
在本发明的一种实施方式中，所述枯草芽孢杆菌中三个可分别作用于zwf、pfkA、glmM基因的sgRNA表达片段的整合是通过转化线性化后的整合载体psga-zpg后得到。
在本发明的一种实施方式中，整合载体psga-zpg序列如SEQ ID NO.7所示，由整合载体psga构建而成，构建过程如下：通过反向PCR的方式分别构建带有可作用于pfkA，zwf与glmM三个基因的sgRNA的整合载体psga-zwf、psga-pfkA与psga-glmM，并利用Golden Gate的方式将三个sgRNA组装到线性化的psga载体上。
在本发明的一种实施方式中，所述整合表达载体psga序列如SEQ ID NO.8所示，由壮观霉素抗性基因aadA、大肠杆菌复制子pMB1、枯草芽孢杆菌amyE基因5′端的539个碱基片段、含有lox71与lox66的博莱霉素抗性片段、启动子Pveg、sgRNA片段及枯草芽孢杆菌amyE基因3′端的1027个碱基片段七部分组成。其构建过程如下：将amyE 3′片段插入到载体pTargetF的酶切位点EcoRI与HindIII得到载体psga0；amyE基因5′片段、含有lox71与lox66的博莱霉素抗性片段与启动子Pveg通过融合PCR进行组装，然后插入到psga0的酶切位点BamHI与BcuI之间。利用此载体可以将sgRNA表达片段整合到枯草芽孢杆菌基因组的amyE位点；载体pTargetF的构建见文献JIANG Y等.Multigene editing in the Escherichiacoli genome via the CRISPR-Cas9system[J].Applied and EnvironmentalMicrobiology,2015,81(7):2506–2514.
本发明的第三个目的是提供一种高效共利用葡萄糖与木糖生产N-乙酰氨基葡萄糖的方法，具体操作如下：将整合表达了dCas9蛋白与三个可分别作用于zwf，pfkA与glmM三个基因的sgRNA表达片段的基因工程菌接种入发酵培养基，发酵生产N-乙酰氨基葡萄糖。
在本发明的一种实施方式中，所述基因工程菌是重组枯草芽孢杆菌。
在本发明的一种实施方式中，所述发酵培养基含有葡萄糖和木糖。
在本发明的一种实施方式中，所述发酵培养基含有能够水解为葡萄糖和木糖的原料。
在本发明的一种实施方式中，所述方法将重组枯草芽孢杆菌接种入含葡萄糖的发酵培养基，在3-9h内添加浓度为5-20g/L的木糖。
本发明的第四个目的是提供上述重组枯草芽孢杆菌在药物、营养保健品或者化妆品中的应用。
在本发明的一种实施方式中，所述应用是利用所述重组枯草芽孢杆菌生产乙酰氨基葡萄糖。
在本发明的一种实施方式中，所述应用是将生产菌株活化后，以5-10％的接种量转入发酵培养基，35-38℃、150-250rpm条件下培养24-60h。
本发明还提供所述基因工程菌在食品、日化领域或制备药物方面的应用。
本发明的有益效果：
(1)本发明提供了一种高效共利用葡萄糖和木糖的方法，通过利用CRISPRi系统对枯草芽孢杆菌中氨基葡萄糖合成的三个主要竞争途径糖酵解途径，磷酸戊糖途径以及肽聚糖合成途径进行动态的调控，即通过在发酵的对数中后期添加木糖诱导dCas9蛋白表达来抑制枯草芽孢杆菌中zwf，pfkA与glmM这三个基因的表达，使得去往乙酰氨基葡萄糖合成途径的葡糖糖进一步提高，同时添加的木糖也有一部分被消耗，补充了细胞生长所需要的碳源，实现了细胞对葡萄糖与木糖的高效共利用。
(2)本发明构建的重组枯草芽孢杆菌可高效共利用葡萄糖与木糖合成乙酰氨基葡萄糖，其在摇瓶上产量可达到20.5g/L，发酵生产乙酰氨基葡萄糖的得率为0.612/g葡萄糖，实现了乙酰氨基葡萄糖在重组枯草芽孢杆菌胞外产量的提高，并为进一步代谢工程改造枯草芽孢杆菌生产氨基葡萄糖及其工业化奠定了基础。
附图说明
图1为整合载体pLCx；
图2为整合载体pLCx-dCas9；
图3为整合载体psga；
图4为整合载体psga-zpg；
图5为木糖添加时间对细胞生长及N-乙酰氨基葡萄糖合成的影响；
图6为木糖添加浓度对细胞生长及N-乙酰氨基葡萄糖合成的影响。
具体实施方式
种子培养基(g/L)：胰蛋白胨10，酵母粉5，NaCl 10。
发酵培养基(g/L)：胰蛋白胨6，酵母粉12，(NH4)SO46，K2HPO4·3H2O 12.5，KH2PO42.5，CaCO3 5，微量元素10ml/L；微量元素溶液按g/L计含有：MnSO4·5H2O 1.0，CoCl2·6H2O0.4，NaMoO4·2H2O 0.2，ZnSO4·7H2O 0.2，AlCl3·6H2O 0.1，CuCl2·H2O 0.1，H3BO4 0.05，含5M HCl。
乙酰氨基葡萄糖的测定方法：高效液相色谱(HPLC)检测法：Agilent 1260，RID检测器，HPX-87H柱(Bio-Rad Hercules,CA)，流动相：5mM H2SO4，流速0.6mL/min，柱温35℃，进样体积为10μL。
乙酰氨基葡萄糖得率的计算：
乙酰氨基葡萄糖得率(YGlcNAc/Glc)＝乙酰氨基葡萄糖产量(g/L)/消耗的葡萄糖(g/L)。
实施例1：CRISPRi系统的构建
本发明构建的CRISPRi系统由两个整合载体组成，分别为pLCx-dCas9(SEQ IDNO.5所示)与psga(SEQ ID NO.8所示)。其中pLCx-dCas9用于将受木糖诱导表达的dCas9蛋白整合到枯草芽孢杆菌基因组的lacA位点，psga可以将sgRNA表达整合到枯草芽孢杆菌基因组的amyE位点。
在pLCx-dCas9构建过程中，首先构建了一个可以用于枯草芽孢杆菌的整合表达载体pLCx，其是由F1、F2、F3、F4及F5五个片段通过一步克隆的方式构建而成，F1-F5的序列如SEQ ID NO.9～SEQ ID NO.13所示。其中F1包含壮观霉素抗性基因aadA与大肠杆菌复制子pMB1，F2为枯草芽孢杆菌lacA基因的5′端的800个碱基片段、F3为含有lox71与lox66的氯霉素抗性片段、F4为木糖阻遏蛋白及启动子、F5为lacA基因的3′端的794个碱基片段。pLCx的RBS之后有两个酶切位点BamHI与Pst，用于目的基因的插入。将dCas9插入到整合载体pLCx的酶切位点BamHI与PstI之间后，便得到载体pLCx-dCas9。
载体psga由壮观霉素抗性基因aadA、大肠杆菌复制子pMB1、枯草芽孢杆菌amyE基因5′端的539个碱基片段、含有lox71与lox66的博莱霉素抗性片段、启动子Pveg、sgRNA片段及枯草芽孢杆菌amyE基因3′端的1027个碱基片段七部分组成。其构建过程如如下：将amyE3′片段插入到载体pTargetF的酶切位点EcoRI与HindIII得到载体psga0；amyE基因5′片段、含有lox71与lox66的博莱霉素抗性片段与启动子Pveg通过融合PCR进行组装，然后插入到psga0的酶切位点BamHI与BcuI之间。
实施例2：利用CRISPRi系统调控zwf、pfkA与glmM的表达
根据zwf、pfkA与glmM这三个基因的序列设计引物，以载体psga为模板进行反向PCR，得到三个分别含有作用于zwf、pfkA与glmM三个基因的sgRNA的载体psga-zwf、psga-pfkA与psga-glmM。其中psga-zwf所用引物为sg-F:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG与sg-zwf-R：
TTTCTAGCTCTAAAACTGGTCTAATGAGGATCTTCGACATTTATTGTACAACACGAGCC，psga-pfkA所用引物为sg-F:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG与sg-pfkA-R：TCTAGCTCTAAAACCGGGAATGAACGCAGCAGTTACATTTATTGTACAACACGAGCC，psga-glmM所用引物为sg-F:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG与sg-glmM-R：
TTTCTAGCTCTAAAACATAGTGAGCTTACACCTGAGACATTTATTGTACAACACGAGCC。
分别以载体psga-zwf、psga-pfkA与psga-glmM为模板，并利用Golden Gate的方式将三个sgRNA组装到线性化的psga载体上，得到同时含有三个sgRNA的载体psga-zpg(Golden Gate 组装方法参见文献ENGLER C等.Golden gate shuffling:Aone-pot DNAshuffling method based on type ils restriction enzymes[J].PLoS ONE,PublicLibrary ofScience,2009,4(5):e5553.)。
将载体pLCx-dCas9及psga-zpg利用限制性内切酶Eco91I进行线性化后，转化重组枯草芽孢杆菌BSGNY-Pveg-glmS-P43-GNA1，得到重组枯草芽孢杆菌BSGNX-dCas9-zpg，通过在其发酵培养基中添加一定量的木糖诱导dCas9表达，便可实现对zwf、pfkA与glmM三个基因表达的调控。
实施例3：重组枯草芽孢杆菌BSGNX-dCas9-zpg发酵生产乙酰氨基葡萄糖
采用实施例2构建得到的重组芽孢杆菌进行摇瓶发酵，以枯草芽孢杆菌BSGNY-Pveg-glmS-P43-GNA1作为对照，在相同条件下培养和发酵。将37℃、220rpm下培养12h的种子以5％的接种量转入发酵培养基，并于接种后6h加入终浓度为15g/L的木糖，37℃、220rpm条件下培养48h。最终发酵上清液中乙酰氨基葡萄糖含量达到20.5g/L，较出发菌株(BSGNY-Pveg-glmS-P43-GNA1)提高了32.2％；同时本发明提供的重组枯草芽孢杆菌发酵乙酰氨基葡萄糖得率为0.612g/g葡萄糖，较出发菌株提高了96.8％(结果如表1所示)，实现了乙酰氨基葡萄糖在重组枯草芽孢杆菌胞外产量与得率的提高，同时实现了重组枯草芽孢杆菌对于葡糖糖与木糖的高效共利用，并为进一步代谢工程改造枯草芽孢杆菌生产氨基葡萄糖及其工业化奠定了基础。
表1过表达yqaB与glmS前后细胞生长和乙酰氨基葡萄糖合成情况
Figure BDA0001548185080000061
对照例1：木糖添加量对于细胞生长与乙酰氨基葡萄糖合成的影响
本发明的方法中，木糖具有两个作用，(1)诱导dCas9蛋白表达，从而降低zwf、pfkA与glmM这三个基因的表达，减少葡萄糖去往糖酵解途径、磷酸戊糖途径与肽聚糖合成途径的流量，促使更多的葡萄糖去往N-乙酰氨基葡萄糖合成途径；(2)作为碳源被细胞吸收利用，用以补充弱化葡萄糖代谢后细胞生长所需的碳源。因此，木糖的添加时间很大程度上决定了细胞对葡萄糖及木糖两种碳源的利用。
分别在接种后的0h、3h、6h、9h及12h加入终浓度为15g/L的葡萄糖，发酵结果如附图5所示。当在接种后的6h及9h时乙酰氨基葡萄糖产量均能达到20g/L，只是9h添加木糖时对葡萄糖的得率为0.562，低于6h添加时的0.612g/g，而在0h，3h与12h添加时乙酰氨基葡萄糖产量及其对葡萄糖的得率均有不同程度的提高，但都达不到最佳水平。木糖在被细胞利用时需要与磷酸戊糖途径中的多种中间代谢物发生反应，过早的阻断葡萄糖的利用途径可能影响到了磷酸戊糖途径中间代谢物的积累，细胞因此无法高效利用木糖；而木糖添加过晚时由于细胞已达到稳定期，此时弱化葡萄糖的利用途径对于乙酰氨基葡萄糖产量以及得率促进作用不大。
对照例2：木糖添加时间对于细胞生长与乙酰氨基葡萄糖合成的影响
木糖的添加量会影响dCas9的表达强度，因此也会影响到zwf，pfkA及glmM这三个基因的表达强度；而且葡萄糖与木糖添加比例的变化也会影响到细胞对这两种碳源的利用。分别在接种6h后加入终浓度为0、5、10、15、20、25g/L的木糖，发酵结果如附图6所示，加入10g/L和15g/L的木糖产量分别为20.2g/L与20.5g/L，但是加入10g/L时乙酰氨基葡萄糖的得率只有0.51g/g，低于加入15g/L时的0.612g/g，而加入0、5、20和25g/L的木糖时乙酰氨基葡萄糖产量分别为16.6、18.6、19.0和19.1g/L，均低于20g/L。木糖添加量过低时对于葡萄糖利用途径的抑制效果较弱，因此对于N-乙酰氨基葡萄糖合成的促进作用也较为有限；而当木糖添加量超过15g/L时，虽然乙酰氨基葡萄糖的得率会有进一步的提高，但是此时葡萄糖利用速率较慢，因此乙酰氨基葡萄糖的产量反而不如加入15g/L木糖时高。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种基于CRISPRi的共利用葡萄糖和木糖生产N-乙酰氨糖的方法
<160> 18
<170> PatentIn version 3.3
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Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
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Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
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Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
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ccgattgtaa aatcagtgcc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 102
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tatcctcagg ttcgtccatc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 102
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<212> DNA
<213> 人工序列
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tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacggg aattccatat ggattccgtg atgtcaaagc 1920
ttgaaaaaac gcacgtaaca aaagcaaaat ttatgctcca tgggggagac tacaaccccg 1980
atcagtggct ggatcggccc gatattttag ctgacgatat caaactgatg aagctttctc 2040
atacgaatac gttttctgtc ggcatttttg catggagcgc acttgagccg gaggagggcg 2100
tatatcaatt tgaatggctg gatgatattt ttgagcggat tcacagtata ggcggccggg 2160
tcatattagc aacgccgagc ggagcccgtc cggcctggct gtcgcaaacc tatccggaag 2220
ttttgcgcgt caatgcctcc cgcgtcaaac agctgcacgg cggaaggcac aaccactgcc 2280
tcacatctaa agtctaccga gaaaaaacac ggcacatcaa ccgcttatta gcagaacgat 2340
acggacatca cccggcgctg ttaatgtggc acatttcaaa cgaatacggg ggagattgcc 2400
actgtgattt atgccagcat gctttccggg agtggctgaa atcgaaatat gacaacagcc 2460
tcaagacatt gaaccatgcg tggtggaccc ctttttggag ccatacgttc aatgactggt 2520
cacaaattga aagcccttcg ccgatcggtg aaaatggctt gcatggcctg aatttagatt 2580
ggcgccggtt cgtcaccgat caaacgattt cgttttatga aaatgaaatc attccgctga 2640
aagaattgac gcctgatatc cctatcacaa cgaattttat ggctgacaca ccggatttga 2700
tcccgtatac cgttcgtata gcatacatta tacgaagtta tgccatagtg actggcgatg 2760
ctgtcggaat ggacgatcgg caatagttac ccttattatc aagataagaa agaaaaggat 2820
ttttcgctac gctcaaatcc tttaaaaaaa cacaaaagac cacatttttt aatgtggtct 2880
tttattcttc aactaaagca cccattagtt caacaaacga aaattggata aagtgggata 2940
tttttaaaat atatatttat gttacagtaa tattgacttt taaaaaagga ttgattctaa 3000
tgaagaaagc agacaagtaa gcctcctaaa ttcactttag ataaaaattt aggaggcata 3060
tcaaatgaac tttaataaaa ttgatttaga caattggaag agaaaagaga tatttaatca 3120
ttatttgaac caacaaacga cttttagtat aaccacagaa attgatatta gtgttttata 3180
ccgaaacata aaacaagaag gatataaatt ttaccctgca tttattttct tagtgacaag 3240
ggtgataaac tcaaatacag cttttagaac tggttacaat agcgacggag agttaggtta 3300
ttgggataag ttagagccac tttatacaat ttttgatggt gtatctaaaa cattctctgg 3360
tatttggact cctgtaaaga atgacttcaa agagttttat gatttatacc tttctgatgt 3420
agagaaatat aatggttcgg ggaaattgtt tcccaaaaca cctatacctg aaaatgcttt 3480
ttctctttct attattccat ggacttcatt tactgggttt aacttaaata tcaataataa 3540
tagtaattac cttctaccca ttattacagc aggaaaattc attaataaag gtaattcaat 3600
atatttaccg ctatctttac aggtacatca ttctgtttgt gatggttatc atgcaggatt 3660
gtttatgaac tctattcagg aattgtcaga taggcctaat gactggcttt tataatatga 3720
gataatgccg actgtacttt ttacagtcgg ttttctaaaa cgatacatta ataggtacga 3780
aaaagcaact ttttttgcgc ttaaaaccag tcataccaat aaataacttc gtatagcata 3840
cattatacga acggtacgga attccgttaa ttcagaacgc tcggttgccg ccgggcgttt 3900
tttatgcagc aatggcaaga acgtcccggg gagctcctaa cttatagggg taacacttaa 3960
aaaagaatca ataacgatag aaaccgctcc taaagcaggt gcattttttc ctaacgaaga 4020
aggcaatagt tcacatttat tgtctaaatg agaatggact ctagaagaaa cttcgttttt 4080
aatcgtattt aaaacaatgg gatgagattc aattatatga tttctcaaga taacagcttc 4140
tatatcaaat gtattaagga tattggttaa tccaattccg atataaaagc caaagttttg 4200
aagtgcattt aacatttcta catcattttt atttgcgcgt tccacaatct cttttcgaga 4260
aatattcttt tcttctttag agagcgaagc cagtaacgct ttttcagaag catataattc 4320
ccaacagcct cgatttccac agctgcattt gggtccatta aaatctatcg tcatatgacc 4380
catttcccca gaaaaaccct gaacaccttt atacaattcg ttgttaataa caagtccagt 4440
tccaattccg atattaatac tgatgtaaac gatgttttca tagttttttg tcataccaaa 4500
tactttttca ccgtatgctc ctgcattagc ttcattttca acaaaaaccg gaacattaaa 4560
ctcactctca attaaaaact gcaaatcttt gatattccaa tttaagttag gcatgaaaat 4620
aatttgctga tgacgatcta caaggcctgg aacacaaatt cctattccga ctagaccata 4680
aggggactca ggcatatggg ttacaaaacc atgaataagt gcaaataaaa tctcttttac 4740
ttcactagcg gaagaactag acaagtcaga agtcttctcg agaataatat ttccttctaa 4800
gtcggttaga attccgttaa gatagtcgac tcctatatca ataccaatcg agtagcctgc 4860
attcttatta aaaacaagca ttacaggtct tctgccgcct ctagattgcc ctgccccaat 4920
ttcaaaaata aaatcttttt caagcagtgt atttacttga gaggagacag tagacttgtt 4980
taatcctgta atctcagaga gagttgccct ggagacaggg gagttcttca aaatttcatc 5040
taatattaat ttttgattca ttttttttac taaagcttga tctgcaattt gaataataac 5100
cactcctttg tttatccacc gaactaagtt ggtgtttttt gaagcttgaa ttagatattt 5160
aaaagtatca tatctaatat tataactaaa ttttctaaaa aaaacattga aataaacatt 5220
tattttgtat atgatgagat aaagttagtt tattggataa acaaactaac tcaattaaga 5280
tagttgatgg ataaacttgt tcacttaaat caaaggggga aatgacaaat ggtccaaact 5340
agtgatatct aaaaatcaaa gggggaaatg ggatccatgg ataagaaata ctcaataggc 5400
ttagctatcg gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaggttccg 5460
tctaaaaagt tcaaggttct gggaaataca gaccgccaca gtatcaaaaa aaatcttata 5520
ggggctcttt tatttgacag tggagagaca gcggaagcga ctcgtctcaa acggacagct 5580
cgtagaaggt atacacgtcg gaagaatcgt atttgttatc tacaggagat tttttcaaat 5640
gagatggcga aagtagatga tagtttcttt catcgacttg aagagtcttt tttggtggaa 5700
gaagacaaga agcatgaacg tcatcctatt tttggaaata tagtagatga agttgcttat 5760
catgagaaat atccaactat ctatcatctg cgaaaaaaat tggtagattc tactgataaa 5820
gcggatttgc gcttaatcta tttggcctta gcgcatatga ttaagtttcg tggtcatttt 5880
ttgattgagg gagatttaaa tcctgataat agtgatgtgg acaaactatt tatccagttg 5940
gtacaaacct acaatcaatt atttgaagaa aaccctatta acgcaagtgg agtagatgct 6000
aaagcgattc tttctgcacg attgagtaaa tcaagacgat tagaaaatct cattgctcag 6060
ctccccggtg agaagaaaaa tggcttattt gggaatctca ttgctttgtc attgggtttg 6120
acccctaatt ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca gctttcaaaa 6180
gatacttacg atgatgattt agataattta ttggcgcaaa ttggagatca atatgctgat 6240
ttgtttttgg cagctaagaa tttatcagat gctattttac tttcagatat cctaagagta 6300
aatactgaaa taactaaggc tcccctatca gcttcaatga ttaaacgcta cgatgaacat 6360
catcaagact tgactctttt aaaagcttta gttcgacaac aacttccaga aaagtataaa 6420
gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg gggagctagc 6480
caagaagaat tttataaatt tatcaaacca attttagaaa aaatggatgg tactgaggaa 6540
ttattggtga aactaaatcg tgaagatttg ctgcgcaagc aacggacctt tgacaacggc 6600
tctattcccc atcaaattca cttgggtgag ctgcatgcta ttttgagaag acaagaagac 6660
ttttatccat ttttaaaaga caatcgtgag aagattgaaa aaatcttgac ttttcgaatt 6720
ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag 6780
tctgaagaaa caattacccc atggaatttt gaagaagttg tcgataaagg tgcttcagct 6840
caatcattta ttgaacgcat gacaaacttt gataaaaatc ttccaaatga aaaagtacta 6900
ccaaaacata gtttgcttta tgagtatttt acggtttata acgaattgac aaaggtcaaa 6960
tatgttactg aaggaatgcg aaaaccagca tttctttcag gtgaacagaa gaaagccatt 7020
gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa agaagattat 7080
ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag gagttgaaga tagatttaat 7140
gcttcattag gtacctacca tgatttgcta aaaattatta aagataaaga ttttttggat 7200
aatgaagaaa atgaagatat cttagaggat attgttttaa cattgacctt atttgaagat 7260
agggagatga ttgaggaaag acttaaaaca tatgctcacc tctttgatga taaggtgatg 7320
aaacagctta aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa attgattaat 7380
ggtattaggg ataagcaatc tggcaaaaca atattagatt ttttgaaatc agatggtttt 7440
gccaatcgca attttatgca gctgatccat gatgatagtt tgacatttaa agaagacatt 7500
caaaaagcac aagtgtctgg acaaggcgat agtttacatg aacatattgc aaatttagct 7560
ggtagccctg ctattaaaaa aggtatttta cagactgtaa aagttgttga tgaattggtc 7620
aaagtaatgg ggcggcataa gccagaaaat atcgttattg aaatggcacg tgaaaatcag 7680
acaactcaaa agggccagaa aaattcgcga gagcgtatga aacgaatcga agaaggtatc 7740
aaagaattag gaagtcagat tcttaaagag catcctgttg aaaatactca attgcaaaat 7800
gaaaagctct atctctatta tctccaaaat ggaagagaca tgtatgtgga ccaagaatta 7860
gatattaatc gtttaagtga ttatgatgtc gatgccattg ttccacaaag tttccttaaa 7920
gacgattcaa tagacaataa ggtcttaacg cgttctgata aaaatcgtgg taaatcggat 7980
aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact attggagaca acttctaaac 8040
gccaagttaa tcactcaacg taagtttgat aatttaacga aagctgaacg tggaggtttg 8100
agtgaacttg ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact 8160
aagcatgtgg cacaaatttt ggatagtcgc atgaatacta aatacgatga aaatgataaa 8220
cttattcgag aggttaaagt gattacctta aaatctaaat tagtttctga cttccgaaaa 8280
gatttccaat tctataaagt acgtgagatt aacaattacc atcatgccca tgatgcgtat 8340
ctaaatgccg tcgttggaac tgctttgatt aagaaatatc caaaacttga atcggagttt 8400
gtctatggtg attataaagt ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa 8460
ataggcaaag caaccgcaaa atatttcttt tactctaata tcatgaactt cttcaaaaca 8520
gaaattacac ttgcaaatgg agagattcgc aaacgccctc taatcgaaac taatggggaa 8580
actggagaaa ttgtctggga taaagggcga gattttgcca cagtgcgcaa agtattgtcc 8640
atgccccaag tcaatattgt caagaaaaca gaagtacaga caggcggatt ctccaaggag 8700
tcaattttac caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga ctgggatcca 8760
aaaaaatatg gtggttttga tagtccaacg gtagcttatt cagtcctagt ggttgctaag 8820
gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag agttactagg gatcacaatt 8880
atggaaagaa gttcctttga aaaaaatccg attgactttt tagaagctaa aggatataag 8940
gaagttaaaa aagacttaat cattaaacta cctaaatata gtctttttga gttagaaaac 9000
ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa aaggaaatga gctggctctg 9060
ccaagcaaat atgtgaattt tttatattta gctagtcatt atgaaaagtt gaagggtagt 9120
ccagaagata acgaacaaaa acaattgttt gtggagcagc ataagcatta tttagatgag 9180
attattgagc aaatcagtga attttctaag cgtgttattt tagcagatgc caatttagat 9240
aaagttctta gtgcatataa caaacataga gacaaaccaa tacgtgaaca agcagaaaat 9300
attattcatt tatttacgtt gacgaatctt ggagctcccg ctgcttttaa atattttgat 9360
acaacaattg atcgtaaacg atatacgtct acaaaagaag ttttagatgc cactcttatc 9420
catcaatcca tcactggtct ttatgaaaca cgcattgatt tgagtcagct aggaggtgac 9480
taactcgagt aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 9540
cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac 9600
cttcgggtgg gcctttctgc gtttatacct agggatatat tccgcttcct cgctcactgc 9660
agcgtcatca cgaaagaaca agacttttca ccatataaac tgctgatcgt cccgatgctg 9720
tatttaatca gcgaggacac cgtttcccgt ttaaaagcgt ttacggctga cggcggcacc 9780
ttagtcatga cgtatatcag cggggttgtg aatgagcatg acttaacata cacaggcgga 9840
tggcatccgg atcttcaagc tatatttgga gttgagcctc ttgaaacgga caccctgtat 9900
ccgaaggatc gaaacgctgt cagctaccgc agccaaatat atgaaatgaa ggattatgca 9960
accgtgattg atgtaaagac agcttcagtg gaagcggtgt atcaagaaga tttttatgcg 10020
cgcacgccag cggtcacaag ccatgagtat cagcagggca aggcgtattt tatcggcgcg 10080
cgtttggagg atcaatttca gcgtgatttc tatgagggtc tgatcacaga cctgtctctc 10140
tctccagttt ttccggttcg gcacggaaaa ggcgtctccg tacaagcgag gcaggatcag 10200
gacaatgatt atatttttgt catgaatttc acggaagaaa aacagctggt cacgtttgat 10260
cagagtgtga aggacataat gacaggagac atattgtcag gcgacctgac gatggaaaag 10320
tatgaagtga gaattgtcgt aaacacacat tagggggtac ccc 10363
<210> 6
<211> 6179
<212> DNA
<213> 人工序列
<400> 6
tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacggg aattccatat ggattccgtg atgtcaaagc 1920
ttgaaaaaac gcacgtaaca aaagcaaaat ttatgctcca tgggggagac tacaaccccg 1980
atcagtggct ggatcggccc gatattttag ctgacgatat caaactgatg aagctttctc 2040
atacgaatac gttttctgtc ggcatttttg catggagcgc acttgagccg gaggagggcg 2100
tatatcaatt tgaatggctg gatgatattt ttgagcggat tcacagtata ggcggccggg 2160
tcatattagc aacgccgagc ggagcccgtc cggcctggct gtcgcaaacc tatccggaag 2220
ttttgcgcgt caatgcctcc cgcgtcaaac agctgcacgg cggaaggcac aaccactgcc 2280
tcacatctaa agtctaccga gaaaaaacac ggcacatcaa ccgcttatta gcagaacgat 2340
acggacatca cccggcgctg ttaatgtggc acatttcaaa cgaatacggg ggagattgcc 2400
actgtgattt atgccagcat gctttccggg agtggctgaa atcgaaatat gacaacagcc 2460
tcaagacatt gaaccatgcg tggtggaccc ctttttggag ccatacgttc aatgactggt 2520
cacaaattga aagcccttcg ccgatcggtg aaaatggctt gcatggcctg aatttagatt 2580
ggcgccggtt cgtcaccgat caaacgattt cgttttatga aaatgaaatc attccgctga 2640
aagaattgac gcctgatatc cctatcacaa cgaattttat ggctgacaca ccggatttga 2700
tcccgtatac cgttcgtata gcatacatta tacgaagtta tgccatagtg actggcgatg 2760
ctgtcggaat ggacgacggc aatagttacc cttattatca agataagaaa gaaaaggatt 2820
tttcgctacg ctcaaatcct ttaaaaaaac acaaaagacc acatttttta atgtggtctt 2880
ttattcttca actaaagcac ccattagttc aacaaacgaa aattggataa agtgggatat 2940
ttttaaaata tatatttatg ttacagtaat attgactttt aaaaaaggat tgattctaat 3000
gaagaaagca gacaagtaag cctcctaaat tcactttaga taaaaattta ggaggcatat 3060
caaatgaact ttaataaaat tgatttagac aattggaaga gaaaagagat atttaatcat 3120
tatttgaacc aacaaacgac ttttagtata accacagaaa ttgatattag tgttttatac 3180
cgaaacataa aacaagaagg atataaattt taccctgcat ttattttctt agtgacaagg 3240
gtgataaact caaatacagc ttttagaact ggttacaata gcgacggaga gttaggttat 3300
tgggataagt tagagccact ttatacaatt tttgatggtg tatctaaaac attctctggt 3360
atttggactc ctgtaaagaa tgacttcaaa gagttttatg atttatacct ttctgatgta 3420
gagaaatata atggttcggg gaaattgttt cccaaaacac ctatacctga aaatgctttt 3480
tctctttcta ttattccatg gacttcattt actgggttta acttaaatat caataataat 3540
agtaattacc ttctacccat tattacagca ggaaaattca ttaataaagg taattcaata 3600
tatttaccgc tatctttaca ggtacatcat tctgtttgtg atggttatca tgcaggattg 3660
tttatgaact ctattcagga attgtcagat aggcctaatg actggctttt ataatatgag 3720
ataatgccga ctgtactttt tacagtcggt tttctaacga tacattaata ggtacgaaaa 3780
agcaactttt tttgcgctta aaaccagtca taccaataaa taacttcgta tagcatacat 3840
tatacgaacg gtattcagaa cgctcggttg ccgccgggcg ttttttatgc agcaatggca 3900
agaacgtccc ggggagctcc taacttatag gggtaacact taaaaaagaa tcaataacga 3960
tagaaaccgc tcctaaagca ggtgcatttt ttcctaacga agaaggcaat agttcacatt 4020
tattgtctaa atgagaatgg actctagaag aaacttcgtt tttaatcgta tttaaaacaa 4080
tgggatgaga ttcaattata tgatttctca agataacagc ttctatatca aatgtattaa 4140
ggatattggt taatccaatt ccgatataaa agccaaagtt ttgaagtgca tttaacattt 4200
ctacatcatt tttatttgcg cgttccacaa tctcttttcg agaaatattc ttttcttctt 4260
tagagagcga agccagtaac gctttttcag aagcatataa ttcccaacag cctcgatttc 4320
cacagctgca tttgggtcca ttaaaatcta tcgtcatatg acccatttcc ccagaaaaac 4380
cctgaacacc tttatacaat tcgttgttaa taacaagtcc agttccaatt ccgatattaa 4440
tactgatgta aacgatgttt tcatagtttt ttgtcatacc aaatactttt tcaccgtatg 4500
ctcctgcatt agcttcattt tcaacaaaaa ccggaacatt aaactcactc tcaattaaaa 4560
actgcaaatc tttgatattc caatttaagt taggcatgaa aataatttgc tgatgacgat 4620
ctacaaggcc tggaacacaa attcctattc cgactagacc ataaggggac tcaggcatat 4680
gggttacaaa accatgaata agtgcaaata aaatctcttt tacttcacta gcggaagaac 4740
tagacaagtc agaagtcttc tcgagaataa tatttccttc taagtcggtt agaattccgt 4800
taagatagtc gactcctata tcaataccaa tcgagtagcc tgcattctta ttaaaaacaa 4860
gcattacagg tcttctgccg cctctagatt gccctgcccc aatttcaaaa ataaaatctt 4920
tttcaagcag tgtatttact tgagaggaga cagtagactt gtttaatcct gtaatctcag 4980
agagagttgc cctggagaca ggggagttct tcaaaatttc atctaatatt aatttttgat 5040
tcattttttt tactaaagct tgatctgcaa tttgaataat aaccactcct ttgtttatcc 5100
accgaactaa gttggtgttt tttgaagctt gaattagata tttaaaagta tcatatctaa 5160
tattataact aaattttcta aaaaaaacat tgaaataaac atttattttg tatatgatga 5220
gataaagtta gtttattgga taaacaaact aactcaatta agatagttga tggataaact 5280
tgttcactta aatcaaaggg ggaaatgaca aatggtccaa actagtgata tctaaaaatc 5340
aaagggggaa atgggatcca ttaccctgtc tgcaggggcg ctcgaggatg ctcaggggtt 5400
tgcgaaggcg acaaaacgtt atccgcaaac gcttcagcag cattaccgca cattctggga 5460
acacgatatc cctgtcgacg tcatcacgaa agaacaagac ttttcaccat ataaactgct 5520
gatcgtcccg atgctgtatt taatcagcga ggacaccgtt tcccgtttaa aagcgtttac 5580
ggctgacggc ggcaccttag tcatgacgta tatcagcggg gttgtgaatg agcatgactt 5640
aacatacaca ggcggatggc atccggatct tcaagctata tttggagttg agcctcttga 5700
aacggacacc ctgtatccga aggatcgaaa cgctgtcagc taccgcagcc aaatatatga 5760
aatgaaggat tatgcaaccg tgattgatgt aaagacagct tcagtggaag cggtgtatca 5820
agaagatttt tatgcgcgca cgccagcggt cacaagccat gagtatcagc agggcaaggc 5880
gtattttatc ggcgcgcgtt tggaggatca atttcagcgt gatttctatg agggtctgat 5940
cacagacctg tctctctctc cagtttttcc ggttcggcac ggaaaaggcg tctccgtaca 6000
agcgaggcag gatcaggaca atgattatat ttttgtcatg aatttcacgg aagaaaaaca 6060
gctggtcacg tttgatcaga gtgtgaagga cataatgaca ggagacatat tgtcaggcga 6120
cctgacgatg gaaaagtatg aagtgagaat tgtcgtaaac acacattagg gggtacccc 6179
<210> 7
<211> 4642
<212> DNA
<213> 人工序列
<400> 7
cagctccatc agcaaaaggg gatgataagt ttatcaccac cgactatttg caacagtgcc 60
gttgatcgtg ctatgatcga ctgatgtcat cagcggtgga gtgcaatgtc atgagggaag 120
cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc 180
tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc 240
cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc 300
gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc 360
gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta 420
agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc 480
cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg 540
ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg 600
aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc 660
gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc 720
cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca 780
tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag 840
atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat 900
aagatgccgc tcgccagtcg attggctgag ctcatgaagt tcctattccg aagttccgcg 960
aacgcgtaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 1020
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1080
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1140
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1200
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1260
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1320
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1380
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1440
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1500
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1560
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 1620
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 1680
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 1740
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 1800
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 1860
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatgctggat ccgcgatgtt 1920
tgcaaaacga ttcaaaacct ctttactgcc gttattcgct ggatttttat tgctgtttca 1980
tttggttctg gcaggaccgg cggctgcgag tgctgaaacg gcgaacaaat cgaatgagct 2040
tacagcaccg tcgatcaaaa gcggaaccat tcttcatgca tggaattggt cgttcaatac 2100
gttaaaacac aatatgaagg atattcatga tgcaggatat acagccattc agacatctcc 2160
gattaaccaa gtaaaggaag ggaatcaagg agataaaagc atgtcgaact ggtactggct 2220
gtatcagccg acatcgtatc aaattggcaa ccgttactta ggtactgaac aagaatttaa 2280
agaaatgtgt gcagccgctg aagaatatgg cataaaggtc attgttgacg cggtcatcaa 2340
tcataccacc agtgattatg ccgcgatttc caatgaggtt aagagtattc caaactggac 2400
acatggaaac acacaaatta aaaactggtc tgatcgatgg gatgtcacgc agaacatgcc 2460
atggcatgag attctaccgt tcgtatagca tacattatac gaagttatct tgatatggct 2520
ttttatatgt gttactctac atacagaaag gaggaactaa atatggccaa gttgaccagt 2580
gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg 2640
ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg 2700
accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg 2760
tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc 2820
cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc 2880
gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgaata 2940
acttcgtata gcatacatta tacgaacggt aaatcgtcga caggtcgtcg acctgcaggc 3000
ttattaacgt tgatataatt taaattttat ttgacaaaaa tgggctcgtg ttgtacaata 3060
aatgtcgaag atcctcatta gaccagtttt agagctagaa atagcaagtt aaaataaggc 3120
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attccgccac 3180
cgtcgacctg caggcttatt aacgttgata taatttaaat tttatttgac aaaaatgggc 3240
tcgtgttgta caataaatgt ccgattgtaa aatcagtgcc gttttagagc tagaaatagc 3300
aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 3360
tttgaattcc ggtacgtcga cctgcaggct tattaacgtt gatataattt aaattttatt 3420
tgacaaaaat gggctcgtgt tgtacaataa atgttatcct caggttcgtc catcgtttta 3480
gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc 3540
gagtcggtgc tttttttgaa ttccgccgat cgacatggat gagcgatgat gatatccgtt 3600
taggctgggc ggtgatagct tctcgttcag gcagtacgcc tcttttcttt tccagacctg 3660
agggaggcgg aaatggtgtg aggttcccgg ggaaaagcca aataggcgat cgcgggagtg 3720
ctttatttga agatcaggct atcactgcgg tcaatagatt tcacaatgtg atggctggac 3780
agcctgagga actctcgaac ccgaatggaa acaaccagat atttatgaat cagcgcggct 3840
cacatggcgt tgtgctggca aatgcaggtt catcctctgt ctctatcaat acggcaacaa 3900
aattgcctga tggcaggtat gacaataaag ctggagcggg ttcatttcaa gtgaacgatg 3960
gtaaactgac aggcacgatc aatgccaggt ctgtagctgt gctttatcct gatgatattg 4020
caaaagcgcc tcatgttttc cttgagaatt acaaaacagg tgtaacacat tctttcaatg 4080
atcaactgac gattaccttg cgtgcagatg cgaatacaac aaaagccgtt tatcaaatca 4140
ataatggacc agagacggcg tttaaggatg gagatcaatt cacaatcgga aaaggagatc 4200
catttggcaa aacatacacc atcatgttaa aaggaacgaa cagtgatggt gtaacgagga 4260
ccgagaaata cagttttgtt aaaagagatc cagcgtcggc caaaaccatc ggctatcaaa 4320
atccgaatca ttggagccag gtaaatgctt atatctataa acatgatggg agccgagtaa 4380
ttgaattgac cggatcttgg cctggaaaac caatgactaa aaatgcagac ggaatttaca 4440
cgctgacgct gcctgcggac acggatacaa ccaacgcaaa agtgattttt aataatggca 4500
gcgcccaagt gcccggtcag aatcagcctg gctttgatta cgtgctaaat ggtttatata 4560
atgactcggg cttaagcggt tctcttcccc attgacccaa gcttagatct attaccctgt 4620
tatccctact cgagttcatg tg 4642
<210> 8
<211> 4244
<212> DNA
<213> 人工序列
<400> 8
tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatgctg 1920
gatccgcgat gtttgcaaaa cgattcaaaa cctctttact gccgttattc gctggatttt 1980
tattgctgtt tcatttggtt ctggcaggac cggcggctgc gagtgctgaa acggcgaaca 2040
aatcgaatga gcttacagca ccgtcgatca aaagcggaac cattcttcat gcatggaatt 2100
ggtcgttcaa tacgttaaaa cacaatatga aggatattca tgatgcagga tatacagcca 2160
ttcagacatc tccgattaac caagtaaagg aagggaatca aggagataaa agcatgtcga 2220
actggtactg gctgtatcag ccgacatcgt atcaaattgg caaccgttac ttaggtactg 2280
aacaagaatt taaagaaatg tgtgcagccg ctgaagaata tggcataaag gtcattgttg 2340
acgcggtcat caatcatacc accagtgatt atgccgcgat ttccaatgag gttaagagta 2400
ttccaaactg gacacatgga aacacacaaa ttaaaaactg gtctgatcga tgggatgtca 2460
cgcagaacat gccatggcat gagattctac cgttcgtata gcatacatta tacgaagtta 2520
tcttgatatg gctttttata tgtgttactc tacatacaga aaggaggaac taaatatggc 2580
caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt 2640
ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt 2700
ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac 2760
cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt 2820
gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg 2880
ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga 2940
gcaggactga ataacttcgt atagcataca ttatacgaac ggtaaatcgt cgacctgcag 3000
gcttattaac gttgatataa tttaaatttt atttgacaaa aatgggctcg tgttgtacaa 3060
taaatgtact agtcctcaaa ctggcagatg cacgttttag agctagaaat agcaagttaa 3120
aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaat 3180
tccgtcgaca tggatgagcg atgatgatat ccgtttaggc tgggcggtga tagcttctcg 3240
ttcaggcagt acgcctcttt tcttttccag acctgaggga ggcggaaatg gtgtgaggtt 3300
cccggggaaa agccaaatag gcgatcgcgg gagtgcttta tttgaagatc aggctatcac 3360
tgcggtcaat agatttcaca atgtgatggc tggacagcct gaggaactct cgaacccgaa 3420
tggaaacaac cagatattta tgaatcagcg cggctcacat ggcgttgtgc tggcaaatgc 3480
aggttcatcc tctgtctcta tcaatacggc aacaaaattg cctgatggca ggtatgacaa 3540
taaagctgga gcgggttcat ttcaagtgaa cgatggtaaa ctgacaggca cgatcaatgc 3600
caggtctgta gctgtgcttt atcctgatga tattgcaaaa gcgcctcatg ttttccttga 3660
gaattacaaa acaggtgtaa cacattcttt caatgatcaa ctgacgatta ccttgcgtgc 3720
agatgcgaat acaacaaaag ccgtttatca aatcaataat ggaccagaga cggcgtttaa 3780
ggatggagat caattcacaa tcggaaaagg agatccattt ggcaaaacat acaccatcat 3840
gttaaaagga acgaacagtg atggtgtaac gaggaccgag aaatacagtt ttgttaaaag 3900
agatccagcg tcggccaaaa ccatcggcta tcaaaatccg aatcattgga gccaggtaaa 3960
tgcttatatc tataaacatg atgggagccg agtaattgaa ttgaccggat cttggcctgg 4020
aaaaccaatg actaaaaatg cagacggaat ttacacgctg acgctgcctg cggacacgga 4080
tacaaccaac gcaaaagtga tttttaataa tggcagcgcc caagtgcccg gtcagaatca 4140
gcctggcttt gattacgtgc taaatggttt atataatgac tcgggcttaa gcggttctct 4200
tccccattga cccaagctta gatctattac cctgttatcc ctac 4244
<210> 9
<211> 1928
<212> DNA
<213> 人工序列
<400> 9
cacacattag ggggtacccc tcgagttcat gtgcagctcc atcagcaaaa ggggatgata 60
agtttatcac caccgactat ttgcaacagt gccgttgatc gtgctatgat cgactgatgt 120
catcagcggt ggagtgcaat gtcatgaggg aagcggtgat cgccgaagta tcgactcaac 180
tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt 240
tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta 300
cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa 360
cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc 420
acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc 480
agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta 540
tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac 600
tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat 660
ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca 720
tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa 780
tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg 840
gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg 900
tgaaaggcga gatcaccaag gtagtcggca aataagatgc cgctcgccag tcgattggct 960
gagctcatga agttcctatt ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc 1020
ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 1080
gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 1140
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 1200
ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 1260
ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 1320
gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 1380
ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 1440
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 1500
ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 1560
agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 1620
agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 1680
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 1740
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 1800
accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 1860
gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacggg aattccatat 1920
ggattccg 1928
<210> 10
<211> 839
<212> DNA
<213> 人工序列
<400> 10
ggaattccat atggattccg tgatgtcaaa gcttgaaaaa acgcacgtaa caaaagcaaa 60
atttatgctc catgggggag actacaaccc cgatcagtgg ctggatcggc ccgatatttt 120
agctgacgat atcaaactga tgaagctttc tcatacgaat acgttttctg tcggcatttt 180
tgcatggagc gcacttgagc cggaggaggg cgtatatcaa tttgaatggc tggatgatat 240
ttttgagcgg attcacagta taggcggccg ggtcatatta gcaacgccga gcggagcccg 300
tccggcctgg ctgtcgcaaa cctatccgga agttttgcgc gtcaatgcct cccgcgtcaa 360
acagctgcac ggcggaaggc acaaccactg cctcacatct aaagtctacc gagaaaaaac 420
acggcacatc aaccgcttat tagcagaacg atacggacat cacccggcgc tgttaatgtg 480
gcacatttca aacgaatacg ggggagattg ccactgtgat ttatgccagc atgctttccg 540
ggagtggctg aaatcgaaat atgacaacag cctcaagaca ttgaaccatg cgtggtggac 600
ccctttttgg agccatacgt tcaatgactg gtcacaaatt gaaagccctt cgccgatcgg 660
tgaaaatggc ttgcatggcc tgaatttaga ttggcgccgg ttcgtcaccg atcaaacgat 720
ttcgttttat gaaaatgaaa tcattccgct gaaagaattg acgcctgata tccctatcac 780
aacgaatttt atggctgaca caccggattt gatcccgtat accgttcgta tagcataca 839
<210> 11
<211> 1166
<212> DNA
<213> 人工序列
<400> 11
taccgttcgt atagcataca ttatacgaag ttatgccata gtgactggcg atgctgtcgg 60
aatggacgac ggcaatagtt acccttatta tcaagataag aaagaaaagg atttttcgct 120
acgctcaaat cctttaaaaa aacacaaaag accacatttt ttaatgtggt cttttattct 180
tcaactaaag cacccattag ttcaacaaac gaaaattgga taaagtggga tatttttaaa 240
atatatattt atgttacagt aatattgact tttaaaaaag gattgattct aatgaagaaa 300
gcagacaagt aagcctccta aattcacttt agataaaaat ttaggaggca tatcaaatga 360
actttaataa aattgattta gacaattgga agagaaaaga gatatttaat cattatttga 420
accaacaaac gacttttagt ataaccacag aaattgatat tagtgtttta taccgaaaca 480
taaaacaaga aggatataaa ttttaccctg catttatttt cttagtgaca agggtgataa 540
actcaaatac agcttttaga actggttaca atagcgacgg agagttaggt tattgggata 600
agttagagcc actttataca atttttgatg gtgtatctaa aacattctct ggtatttgga 660
ctcctgtaaa gaatgacttc aaagagtttt atgatttata cctttctgat gtagagaaat 720
ataatggttc ggggaaattg tttcccaaaa cacctatacc tgaaaatgct ttttctcttt 780
ctattattcc atggacttca tttactgggt ttaacttaaa tatcaataat aatagtaatt 840
accttctacc cattattaca gcaggaaaat tcattaataa aggtaattca atatatttac 900
cgctatcttt acaggtacat cattctgttt gtgatggtta tcatgcagga ttgtttatga 960
actctattca ggaattgtca gataggccta atgactggct tttataatat gagataatgc 1020
cgactgtact ttttacagtc ggttttctaa cgatacatta ataggtacga aaaagcaact 1080
ttttttgcgc ttaaaaccag tcataccaat aaataacttc gtatagcata cattatacga 1140
acggtattca gaacgctcgg ttgccg 1166
<210> 12
<211> 1500
<212> DNA
<213> 人工序列
<400> 12
ttcagaacgc tcggttgccg ccgggcgttt tttatgcagc aatggcaaga acgtcccggg 60
gagctcctaa cttatagggg taacacttaa aaaagaatca ataacgatag aaaccgctcc 120
taaagcaggt gcattttttc ctaacgaaga aggcaatagt tcacatttat tgtctaaatg 180
agaatggact ctagaagaaa cttcgttttt aatcgtattt aaaacaatgg gatgagattc 240
aattatatga tttctcaaga taacagcttc tatatcaaat gtattaagga tattggttaa 300
tccaattccg atataaaagc caaagttttg aagtgcattt aacatttcta catcattttt 360
atttgcgcgt tccacaatct cttttcgaga aatattcttt tcttctttag agagcgaagc 420
cagtaacgct ttttcagaag catataattc ccaacagcct cgatttccac agctgcattt 480
gggtccatta aaatctatcg tcatatgacc catttcccca gaaaaaccct gaacaccttt 540
atacaattcg ttgttaataa caagtccagt tccaattccg atattaatac tgatgtaaac 600
gatgttttca tagttttttg tcataccaaa tactttttca ccgtatgctc ctgcattagc 660
ttcattttca acaaaaaccg gaacattaaa ctcactctca attaaaaact gcaaatcttt 720
gatattccaa tttaagttag gcatgaaaat aatttgctga tgacgatcta caaggcctgg 780
aacacaaatt cctattccga ctagaccata aggggactca ggcatatggg ttacaaaacc 840
atgaataagt gcaaataaaa tctcttttac ttcactagcg gaagaactag acaagtcaga 900
agtcttctcg agaataatat ttccttctaa gtcggttaga attccgttaa gatagtcgac 960
tcctatatca ataccaatcg agtagcctgc attcttatta aaaacaagca ttacaggtct 1020
tctgccgcct ctagattgcc ctgccccaat ttcaaaaata aaatcttttt caagcagtgt 1080
atttacttga gaggagacag tagacttgtt taatcctgta atctcagaga gagttgccct 1140
ggagacaggg gagttcttca aaatttcatc taatattaat ttttgattca ttttttttac 1200
taaagcttga tctgcaattt gaataataac cactcctttg tttatccacc gaactaagtt 1260
ggtgtttttt gaagcttgaa ttagatattt aaaagtatca tatctaatat tataactaaa 1320
ttttctaaaa aaaacattga aataaacatt tattttgtat atgatgagat aaagttagtt 1380
tattggataa acaaactaac tcaattaaga tagttgatgg ataaacttgt tcacttaaat 1440
caaaggggga aatgacaaat ggtccaaact agtgatatct aaaaatcaaa gggggaaatg 1500
<210> 13
<211> 846
<212> DNA
<213> 人工序列
<400> 13
aaaaatcaaa gggggaaatg ggatccatta ccctgtctgc aggggcgctc gaggatgctc 60
aggggtttgc gaaggcgaca aaacgttatc cgcaaacgct tcagcagcat taccgcacat 120
tctgggaaca cgatatccct gtcgacgtca tcacgaaaga acaagacttt tcaccatata 180
aactgctgat cgtcccgatg ctgtatttaa tcagcgagga caccgtttcc cgtttaaaag 240
cgtttacggc tgacggcggc accttagtca tgacgtatat cagcggggtt gtgaatgagc 300
atgacttaac atacacaggc ggatggcatc cggatcttca agctatattt ggagttgagc 360
ctcttgaaac ggacaccctg tatccgaagg atcgaaacgc tgtcagctac cgcagccaaa 420
tatatgaaat gaaggattat gcaaccgtga ttgatgtaaa gacagcttca gtggaagcgg 480
tgtatcaaga agatttttat gcgcgcacgc cagcggtcac aagccatgag tatcagcagg 540
gcaaggcgta ttttatcggc gcgcgtttgg aggatcaatt tcagcgtgat ttctatgagg 600
gtctgatcac agacctgtct ctctctccag tttttccggt tcggcacgga aaaggcgtct 660
ccgtacaagc gaggcaggat caggacaatg attatatttt tgtcatgaat ttcacggaag 720
aaaaacagct ggtcacgttt gatcagagtg tgaaggacat aatgacagga gacatattgt 780
caggcgacct gacgatggaa aagtatgaag tgagaattgt cgtaaacaca cattaggggg 840
tacccc 846
<210> 14
<211> 59
<212> DNA
<213> 人工序列
<400> 14
tttctagctc taaaactggt ctaatgagga tcttcgacat ttattgtaca acacgagcc 59
<210> 15
<211> 33
<212> DNA
<213> 人工序列
<400> 15
gttttagagc tagaaatagc aagttaaaat aag 33
<210> 16
<211> 57
<212> DNA
<213> 人工序列
<400> 16
tctagctcta aaaccgggaa tgaacgcagc agttacattt attgtacaac acgagcc 57
<210> 17
<211> 33
<212> DNA
<213> 人工序列
<400> 17
gttttagagc tagaaatagc aagttaaaat aag 33
<210> 18
<211> 59
<212> DNA
<213> 人工序列
<400> 18
tttctagctc taaaacatag tgagcttaca cctgagacat ttattgtaca acacgagcc 59

Claims (9)

1.一种基因工程菌，其特征在于，能够共利用葡萄糖和木糖生产N-乙酰氨基葡萄糖；所述共利用葡萄糖和木糖生产N-乙酰氨基葡萄糖是利用dCas9蛋白和sgRNA调控糖酵解、磷酸戊糖及肽聚糖合成三条途径中的zwf，pfkA与glmM基因实现的；所述基因工程菌以枯草芽孢杆菌BSGNY-Pveg-glmS-P43-GNA1为出发菌株。
2.根据权利要求1所述的基因工程菌，其特征在于，基因工程菌整合表达了dCas9蛋白，并整合了三个分别作用于zwf，pfkA与glmM基因的sgRNA表达片段。
3.根据权利要求2所述的基因工程菌，其特征在于，所述dCas9蛋白通过载体pLCx进行整合表达。
4.权利要求3所述基因工程菌的构建方法，其特征在于，以所述枯草芽孢杆菌为出发菌株，以载体pLCx将d Cas9蛋白整合表达，并将作用于zwf，pfkA与glmM基因的sgRNA表达片段用载体psga整合表达。
5.一种共利用葡萄糖与木糖生产N-乙酰氨基葡萄糖的方法，其特征在于，将权利要求1～2任一所述的基因工程菌接种入发酵培养基，发酵生产N-乙酰氨基葡萄糖。
6.根据权利要求5所述的方法，其特征在于，所述发酵培养基含有葡萄糖和木糖，或含有能够水解为葡萄糖和木糖的原料。
7.根据权利要求5所述的方法，其特征在于，所述方法是将重组枯草芽孢杆菌接种入含葡萄糖的发酵培养基，在3-9h内添加浓度为5-20g/L的木糖。
8.根据权利要求7所述的方法，其特征在于，所述方法是将生产菌株活化后，以5-10％的接种量转入发酵培养基，35-38℃、150-250rpm条件下培养24-60h。
9.权利要求1所述的基因工程菌在食品、日化领域或制备药物、保健品方面的应用。
CN201810036701.9A 2018-01-15 2018-01-15 一种基于CRISPRi的共利用葡萄糖和木糖生产N-乙酰氨糖的方法 Active CN108148797B (zh)

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