CN109749976B - 一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用 - Google Patents

一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用 Download PDF

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CN109749976B
CN109749976B CN201910093694.0A CN201910093694A CN109749976B CN 109749976 B CN109749976 B CN 109749976B CN 201910093694 A CN201910093694 A CN 201910093694A CN 109749976 B CN109749976 B CN 109749976B
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bacillus subtilis
kinase
fucose
recombinant bacillus
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刘龙
邓洁莹
陈春梅
吕雪芹
李江华
堵国成
陈坚
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Bright Dairy and Food Co Ltd
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Abstract

本发明提供了一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用，所述重组芽孢杆菌是通过在枯草芽孢杆菌168的基因组中强化表达鸟苷酸激酶和核苷二磷酸激酶基因，并表达外源岩藻糖激酶和磷酸鸟嘌呤基转移酶基因得到的。本发明通过改造枯草芽孢杆菌168获得了可高效合成鸟苷二磷酸岩藻糖的菌株，其胞内积累量高达196.15g/L。本发明通过强化表达鸟苷酸激酶和核苷二磷酸激酶基因，强化胞内GDP‑L‑fucose合成辅因子GTP的供应，促进了鸟苷二磷酸岩藻糖的合成。本发明重组枯草芽孢杆菌的构建方法简单，便于使用，具有很好的应用前景。

Description

一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构 建方法与应用
技术领域
本发明属于遗传工程的技术领域，具体涉及一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用。
背景技术
母乳中含有重要营养成分，生物活性物质，刺激肠道菌群生长的因子。其中，母乳寡糖(Human Milk Oligosacchrides,hMOs)在许多生理功能上起到了关键的作用，例如促进双歧杆菌生长，病原体感染的抑制作用，和提高免疫反应。母乳寡糖中的藻糖基化的寡糖(FOSs)由于其生理功能如其作为肠道致病菌的受体类似物的能力、促进免疫调节的能力和减少炎症的能力，已经获得了极大的关注。因为藻糖基化的寡糖是由岩藻糖基转移酶催化进行糖类岩藻糖基化而成，这就需要鸟苷二磷酸岩藻糖 (GDP-L-fucose)作为岩藻糖基的供体。随着岩藻糖基化的寡糖的热度越来越高，许多制药公司试图通过化学方法和生物方法高效合成充足的 GDP-L-fucose。在化学高效合成中，GDP-L-fucose以L-岩藻吡喃糖基四乙酸为起始原料，由HBr、Ag2CO3、N-四丁铵磷酸二甲苯酯等物质引发的化学反应。GDP-L-fucose是可拉酸的前体，可拉酸是革兰氏阴性菌细胞壁的主要成分，因此，一些肠道细菌如大肠杆菌和沙门氏菌能在体内高效合成GDP-L-岩藻糖。在生物中发现了两种高效合成GDP-L-fucose的代谢途径：补救途径和从头途径。
补救途径是在人类的代谢途径中发现的，外源的岩藻糖转移到胞内，由岩藻糖激酶消耗ATP磷酸化(EC 2.7.1.52)形成岩藻糖-1-磷酸 (Fuc-1-P)。Fuc-1-P结合鸟苷三磷酸(GTP)在岩藻糖-1-磷酸鸟苷酰基转移酶(L-fucose-1-phosphate guanylyltransferase)(EC 2.7.7.30)的催化下生成GDP-L-岩藻糖。从头高效合成途径普遍存在于原核生物和真核生物中，其中GDP-L-fucose是由GDP-甘露糖通过甘露糖脱氢酶(GMD， EC4.2.1.47)和GDP-岩藻糖高效合成酶(WCAG，EC 1.1.1.271)催化高效合成的。
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，如何利用枯草芽孢杆菌，通过生物方法高效高效合成鸟苷二磷酸岩藻糖，仍是本领域亟待解决的问题。
发明内容
为了解决上述技术问题，本发明的目的在于提供一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用，构建的重组枯草芽孢杆菌可以高效高效合成鸟苷二磷酸岩藻糖。
具体的，一方面，本发明提供了所述重组芽孢杆菌是通过在枯草芽孢杆菌168的基因组中强化表达鸟苷酸激酶和核苷二磷酸激酶基因，并表达外源岩藻糖激酶和磷酸鸟嘌呤基转移酶基因得到的。
本发明提供了一种强化表达鸟苷酸激酶和核苷二磷酸激酶基因促进枯草芽孢杆菌合成鸟苷二磷酸盐藻糖的方法。由于回补途径合成 GDP-L-fucose的过程需要GTP和ATP提供能量和GDP基团，所以GMP 到GDP，GDP到GTP的可逆反应中，酶促反应方向向合成GTP方向移动，但是反应速度受到了鸟苷酸激酶和核苷二磷酸激酶自身表达强度的限制，如图1所示。本发明通过强化基因组中的鸟苷酸激酶gmk和核苷二磷酸激酶基因ndk，提高了鸟苷一磷酸(GMP)到鸟苷三磷酸(GTP)的转化效率，强化了GTP的胞内供应，促进了鸟苷二磷酸盐藻糖积累。
进一步的，将P43启动子替换枯草芽孢杆菌168中鸟苷酸激酶和核苷二磷酸激酶基因的启动子，来强化表达鸟苷酸激酶和核苷二磷酸激酶基因。
进一步的，所述鸟苷酸激酶基因如NCBI上Gene ID:938821基因所示，核苷二磷酸激酶如NCBI上Gene ID:938997基因所示。
进一步的，所述岩藻糖激酶和磷酸鸟嘌呤基转移酶基因为来源于脆弱拟杆菌的fkp基因。
第二方面，本发明还提供了所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌的构建方法，该构建方法包括以下步骤：
(1)构建包含鸟苷酸激酶和核苷二磷酸激酶基因的上下游序列、P43 启动子和壮观霉素抗性基因序列的替换框，将构建好的替换框转化重组枯草芽孢杆菌168，通过验证，确认鸟苷酸激酶和核苷二磷酸激酶强化表达成功，得到重组枯草芽孢杆菌BSGN；
(2)构建包含岩藻糖激酶和磷酸鸟嘌呤基转移酶基因的重组质粒，将构建好的重组质粒转化枯草芽孢杆菌BSGN，通过验证，确认岩藻糖激酶和磷酸鸟嘌呤基转移酶表达成功，得到重组枯草芽孢杆菌BSGNF。
进一步的，步骤(1)中，替换框的序列如SEQ ID NO.1和SEQ ID NO.2 所示。
进一步的，步骤(1)中，将构建好的替换框电转化枯草芽孢杆菌168 的感受态细胞。
进一步的，步骤(2)中，重组质粒的序列如SEQ ID NO.3所示。
第三方面，本发明提供了所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌的应用，使用所述重组枯草芽孢杆菌发酵生成鸟苷二磷酸岩藻糖。
进一步的，所述发酵是将重组芽孢杆菌种子液以OD值为0.1-0.3的接种量接入发酵培养基中，在35-40℃、200-250rpm条件下培养18-20h。
本发明的有益效果为：
本发明的重组枯草芽孢杆菌，是在枯草芽孢杆菌168的基础上强化表达了鸟苷酸激酶和核苷二磷酸激酶基因，并在此基础上表达岩藻糖激酶和磷酸鸟嘌呤基转移酶基因得到的，通过改造获得了可高效合成鸟苷二磷酸岩藻糖的菌株，其胞内积累量高达196.15g/L。本发明通过强化表达鸟苷酸激酶和核苷二磷酸激酶基因，强化胞内GDP-L-fucose合成辅因子GTP 的供应，促进了鸟苷二磷酸岩藻糖的合成。本发明重组枯草芽孢杆菌的构建方法简单，便于使用，具有很好的应用前景。
附图说明
图1为本发明鸟苷二磷酸岩藻糖合成过程中GTP参与的反应示意图；
图2为本发明实施例1中强化表达gmk和ndk基因的整合PCR验证琼脂糖凝胶电泳图；
图3为本发明实施例2中质粒转化表达fkp基因的PCR验证琼脂糖凝胶电泳图。
具体实施方式
下面结合实施例和附图对本发明进行详细的解释说明。
鸟苷二磷酸岩藻糖的测定方法：
高效液相色谱(HPLC)检测法：Agilent 1200，VWD检测器，C18柱(250 ×4.6mm，5μm)，流动相A：20mM PH6.0三乙胺乙酸缓冲液(TEAA)，流动相B：乙腈，流速0.6mL/min，柱温35℃，进样体积为10μL。
实施例1
加强表达鸟苷酸激酶gmk和核苷二磷酸激酶ndk
根据NCBI上公布的枯草芽孢杆菌(Bacillus subtilis 168购自美国典型微生物保藏中心，ATCC No.27370)的鸟苷酸激酶gmk和核苷二磷酸激酶 ndk的上下游序列、P43启动子以及壮观霉素抗性基因的序列。构建包含鸟苷酸激酶gmk的上下游序列、P43启动子以及壮观霉素抗性基因的序列的替换框1，如SEQ ID NO.1所示；及核苷二磷酸激酶ndk的上下游序列、 P43启动子以及壮观霉素抗性基因的序列的替换框2，如SEQ ID NO.2所示。
将构建好的替换框1电转化枯草芽孢杆菌168的感受态细胞，替换框添加量1为100-300ng，电转化条件：电压2.5kV，电击试剂5ms。再将将构建好的替换框2电转化枯草芽孢杆菌168的感受态细胞，替换框添加量2为100-300ng，电转化条件：电压2.5kV，电击试剂5ms。然后，37℃复苏5h涂布终浓度为10μg/mL壮观霉素抗性的LB平板，37℃厌氧培养 48h，挑选单克隆若干。
由于替换框中存在鸟苷酸激酶gmk和核苷二磷酸激酶ndk的上下游序列，与枯草芽孢杆菌168的转运蛋白基因同源，通过同源重组，替换框中的P43启动子替换枯草芽孢杆菌168鸟苷酸激酶gmk和核苷二磷酸激酶ndk的启动子。
通过壮观霉素抗性平板筛选，菌落PCR验证，测序后确认鸟苷酸激酶gmk和核苷二磷酸激酶ndk是否强化表达成功，壮观霉素抗性呈阳性的为替换框转化成功的枯草芽孢杆菌，琼脂糖凝胶电泳图见图2，菌落PCR 验证有特殊条带，且测序结果与理论结果一致的为替换框转化重组成功的枯草芽孢杆菌，即鸟苷酸激酶gmk和核苷二磷酸激酶ndk强化表达成功的枯草芽孢杆菌。
确认鸟苷酸激酶gmk和核苷二磷酸激酶ndk强化表达成功后，得到重组枯草芽孢杆菌BSGN。
实施例2
外源表达脆弱拟杆菌外源基因
根据NCBI上公布的脆弱拟杆菌(Bacteroidesfragilis，ATCC No.25285) 的岩藻糖激酶和磷酸鸟嘌呤基转移酶基因fkp的序列，通过基因fkp和质粒pP43NMK的PCR线性扩增以及使用ClonExpress II One Step Cloning Kit(Vazyme)一步克隆连接，构建序列如SEQID NO.3所示的重组质粒 pP43-Fkp。
将构建好的重组质粒电转化实施例1得到的重组枯草芽孢杆菌BSGN 的感受态细胞，重组质粒的添加量为50-300ng，电转化条件：电压2.5kV，电击试剂5ms，37℃复苏5h涂布终浓度为10μg/mL卡那霉素抗性的LB 平板，37℃厌氧培养48h，挑选单克隆若干。
通过卡那霉素抗性平板筛选，菌落PCR验证，测序后确认岩藻糖激酶和磷酸鸟嘌呤基转移酶基因fkp是否表达成功。卡那霉素抗性呈阳性的为转化成功的枯草芽孢杆菌，琼脂糖凝胶电泳图见图3，菌落PCR验证有特殊条带，且测序结果与理论结果一致的为转化重组成功的枯草芽孢杆菌，即岩藻糖激酶和磷酸鸟嘌呤基转移酶表达成功。
确认脆弱拟杆菌的岩藻糖激酶和磷酸鸟嘌呤基转移酶表达成功，得到重组枯草芽孢杆菌BSGNF。
实施例3
发酵生产鸟苷二磷酸岩藻糖
将上述重组枯草芽孢杆菌BSGNF制成种子液，种子液培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L；种子液制作方法为：挑取新鲜平板上的单菌落在种子培养基中，培养8-10h。
将种子液以OD值为0.1的接种量接入到发酵培养基中，发酵培养基的配方为：初始甘油20g/L、蛋白胨6g/L、酵母粉12g/L、(NH4)SO46g/L、 K2HPO4·3H2O 12.5g/L、KH2PO42.5g/L、CaCO35g/L、微量元素溶液10ml/L；微量元素溶液含有：MnSO4·5H2O 1.0g/L、CoCl2·6H2O0.4g/L、 NaMoO4·2H2O 0.2g/L、ZnSO4·7H2O 0.2g/L、AlCl3·6H2O 0.1g/L、CuCl2·H2O0.1g/L、H3BO40.05g/L、5M hCl，于35℃、200rpm条件下培养18h。
发酵结束时，使用气质联用色谱仪测定发酵上清液中的鸟苷二磷酸岩藻糖的含量，鸟苷二磷酸岩藻糖的气质联用色谱图如图1所示，测定的鸟苷二磷酸岩藻糖的含量达到196.15mg/L。
实施例4
发酵生产鸟苷二磷酸岩藻糖
将上述重组枯草芽孢杆菌BSGNF制成种子液，种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L；种子液制作方法为：挑取新鲜平板上的单菌落在种子培养基中，培养8-10h。
将种子液以OD值为0.3的接种量接入到发酵培养基中，发酵培养基的配方为：初始甘油20g/L、蛋白胨6g/L、酵母粉12g/L、(NH4)SO46g/L, K2HPO4·3H2O 12.5g/L、KH2PO42.5g/L、CaCO35g/L、微量元素10ml/L；微量元素溶液含有：MnSO4·5H2O 1.0g/L、CoCl2·6H2O0.4g/L、 NaMoO4·2H2O 0.2g/L、ZnSO4·7H2O 0.2g/L、AlCl3·6H2O 0.1g/L、CuCl2·H2O0.1g/L、H3BO40.05g/L、5M HCl，于40℃、250rpm条件下培养20h。
发酵结束时，发酵上清液中鸟苷二磷酸岩藻糖含量达到187.21mg/L。
对比例1
根据NCBI上公布的枯草芽孢杆菌(Bacillus subtilis 168购自美国典型微生物保藏中心，ATCC No.27370)的鸟苷酸激酶gmk和核苷二磷酸激酶 ndk的上下游序列、P43启动子以及壮观霉素抗性基因的序列。构建包含鸟苷酸激酶gmk的上下游序列、P43启动子以及壮观霉素抗性基因的序列的替换框1，如SEQ ID NO.1所示；及核苷二磷酸激酶ndk的上下游序列、 P43启动子以及壮观霉素抗性基因的序列的替换框2，如SEQ ID NO.2所示。
将构建好的替换框1电转化枯草芽孢杆菌168的感受态细胞，替换框添加量1为100-300ng，电转化条件：电压2.5kV，电击试剂5ms。再将将构建好的替换框2电转化枯草芽孢杆菌168的感受态细胞，替换框添加量2为100-300ng，电转化条件：电压2.5kV，电击试剂5ms。然后，37℃复苏5h涂布终浓度为10μg/mL壮观霉素抗性的LB平板，37℃厌氧培养 48h，挑选单克隆若干。
由于替换框中存在鸟苷酸激酶gmk和核苷二磷酸激酶ndk的上下游序列，与枯草芽孢杆菌168的转运蛋白基因同源，通过同源重组，替换框中的P43启动子替换枯草芽孢杆菌168鸟苷酸激酶gmk和核苷二磷酸激酶ndk的启动子。
通过壮观霉素抗性平板筛选，菌落PCR验证，测序后确认鸟苷酸激酶gmk和核苷二磷酸激酶ndk是否强化表达成功，壮观霉素抗性呈阳性的为替换框转化成功的枯草芽孢杆菌，菌落PCR验证有特殊条带的，且测序结果与理论结果一致的为替换框转化重组成功的枯草芽孢杆菌，即鸟苷酸激酶gmk和核苷二磷酸激酶ndk强化表达成功的枯草芽孢杆菌。
确认鸟苷酸激酶gmk和核苷二磷酸激酶ndk强化表达成功后，得到重组枯草芽孢杆菌BSGN。
用重组枯草芽孢杆菌BSGN发酵生产鸟苷二磷酸岩藻糖，发酵条件与实施例3相同，发酵结束时，离心收集获得细胞，超声破碎获得胞内可溶性混合液，其中未检测到鸟苷二磷酸岩藻糖。
对比例2
根据NCBI上公布的脆弱拟杆菌(Bacteroidesfragilis，ATCC No.25285) 的岩藻糖激酶和磷酸鸟嘌呤基转移酶基因fkp的序列，构建序列如SEQ ID NO.3所示的重组质粒。
将构建好的重组质粒电转化枯草芽孢杆菌168的感受态细胞，重组质粒的添加量为50-300ng，电转化条件：电压2.5kV，电击试剂5ms，37℃复苏5h涂布终浓度为25μg/mL卡那霉素抗性的LB平板，37℃好氧培养 12h，挑选单克隆若干。
通过卡那霉素抗性平板筛选、菌落PCR验证，测序后确认岩藻糖激酶和磷酸鸟嘌呤基转移酶基因是否表达成功，卡那霉素抗性呈阳性的为转化成功的枯草芽孢杆菌，菌落PCR验证有特殊条带的，且测序结果与理论结果一致的为替换框重组成功的枯草芽孢杆菌，即岩藻糖激酶和磷酸鸟嘌呤基转移酶表达成功。
确认脆弱拟杆菌的岩藻糖激酶和磷酸鸟嘌呤基转移酶表达成功，得到重组枯草芽孢杆菌BSF。
用重组枯草芽孢杆菌BSF发酵生产鸟苷二磷酸岩藻糖，发酵条件与实施例3相同，发酵结束时，离心收集获得细胞，超声破碎获得胞内可溶性混合液，其中未检测到鸟苷二磷酸岩藻糖。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明实质内容上所作的任何修改、等同替换和简单改进等，均应包含在本发明的保护范围之内。
序列表
<110> 光明乳业股份有限公司
<120> 一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用
<160> 3
<170> SIPOSequenceListing 1.0
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ggcttgaaat tgattggccg ctgctcgatg agtttgtcgc agccgcgaga gatatgaaga 60
agcggtatca attgtctgca gagcccgatg taatggattt ctttaagctt gatcatgtcg 120
tccaggttca tgaagaacag acgcaaaatg acaagctgga ggcgttgatc atcgatgctg 180
cagaagaagc ggtaaaaggt ctctgtgaaa tgcgtgaaaa agaggggctg ctcttagcga 240
aagactgcct gatgcatatt gatcagcttg aagagctggt gagagaaact gaattgctgg 300
ctgcagatgt cgtatctcgt taccgggaac ggctgtatgc cagaatcaaa gaatggactg 360
aagacgtatt agacgagagc cggcttgtaa cagaatgcgc tatttttgca gaccgctctg 420
atatcactga agagattaca aggctgaaga gccacttcgc ccagtttcgt gatatattag 480
ctagtggcgg agctgtcggg cgcaaactcg actttcttgt tcaggagctc aatcgggaag 540
caaatacgat cggctcaaaa gcaaatgatc atcagatcac aaagcttgtc gttgaaatga 600
aaagttctat tgaaaaaata aaagaacaag tgcaaaatat agaatagtga ctgtgcgtat 660
tgtttacaga cggtcttact aggctaaact agagacgtct attttacagg gggaacgtag 720
aagatgacga ttaaactgat taatatcgga tttggcaata tcatctccgc caatcggatg 780
atttcgattg tcagcccgga gtctgcgcca atcaaacgga tgattcagga tgcaagagac 840
cgcggaatgc taattgacgc tacatacgga cgaagaaccc gtgcagttgt cgtcatggat 900
agtgatcaca ttatcttatc tgccgtccag cctgagacag ttgcacacag actttctgtt 960
aaagaagaaa ttatggatga agggcagggg taattgccgc tacccgggga tcctctagag 1020
attgtaccgt tcgtatagca tacattatac gaagttatcg attttcgttc gtgaatacat 1080
gttataataa ctataactaa taacgtaacg tgactggcaa gagatatttt taaaacaatg 1140
aataggttta cacttacttt agttttatgg aaatgaaaga tcatatcata tataatctag 1200
aataaaatta actaaaataa ttattatcta gataaaaaat ttagaagcca atgaaatcta 1260
taaataaact aaattaagtt tatttaatta acaactatgg atataaaata ggtactaatc 1320
aaaatagtga ggaggatata tttgaataca tacgaacaaa ttaataaagt gaaaaaaata 1380
cttcggaaac atttaaaaaa taaccttatt ggtacttaca tgtttggatc aggagttgag 1440
agtggactaa aaccaaatag tgatcttgac tttttagtcg tcgtatctga accattgaca 1500
gatcaaagta aagaaatact tatacaaaaa attagaccta tttcaaaaaa aataggagat 1560
aaaagcaact tacgatatat tgaattaaca attattattc agcaagaaat ggtaccgtgg 1620
aatcatcctc ccaaacaaga atttatttat ggagaatggt tacaagagct ttatgaacaa 1680
ggatacattc ctcagaagga attaaattca gatttaacca taatgcttta ccaagcaaaa 1740
cgaaaaaata aaagaatata cggaaattat gacttagagg aattactacc tgatattcca 1800
ttttctgatg tgagaagagc cattatggat tcgtcagagg aattaataga taattatcag 1860
gatgatgaaa ccaactctat attaacttta tgccgtatga ttttaactat ggacacgggt 1920
aaaatcatac caaaagatat tgcgggaaat gcagtggctg aatcttctcc attagaacat 1980
agggagagaa ttttgttagc agttcgtagt tatcttggag agaatattga atggactaat 2040
gaaaatgtaa atttaactat aaactattta aataacagat taaaaaaatt ataaataact 2100
tcgtatagca tacattatac gaacggtaga atcgtcgact gataggttgg tgtggttggt 2160
gataggtggt atgttttcgc ttgaactttt aaatacagcc attgaacata cggttgattt 2220
aataactgac aaacatcacc ctcttgctaa agcggccaag gacgctgccg ccggggctgt 2280
ttgcgttttt gccgtgattt cgtgtatcat tggtttactt atttttttgc caaagctgta 2340
atggctgaaa attcttacat ttattttaca tttttagaaa tgggcgtgaa aaaaagcgcg 2400
cgattatgta aaatataaag tgatagcatg aaagaaagag ggttattaat cgttctctca 2460
ggtccctcag gagttggtaa aggaacggtt cgacaagcga tcttttcgca ggaagacaca 2520
aaatttgaat attcgatttc agtaaccaca agaagtccaa gagagggcga agtgaacgga 2580
gtcgattatt ttttcaaaac aagagacgaa ttcgagcaaa tgattgcgga caacaagctg 2640
cttgaatggg cagagtatgt cggcaattat tacggcacgc cagtcgatta tgttgaacag 2700
acgcttcaag atggaaaaga cgtcttttta gaaattgaag ttcaaggggc tcttcaagtg 2760
agaaatgctt tcccggaagg cctgtttatt ttccttgcgc ctccaagcct ttctgaactg 2820
aaaaacagaa tcgtgacacg aggaacagaa acagacgctc tgattgaaaa tcgaatgaaa 2880
gccgcaaaag ctgagatcga aatgatggat gcttatgact atgtcgttga aaacgataat 2940
gtcgaaacgg cttgcgataa aatcaaagca atcgttcttg ctgaacattt gaagcgtgaa 3000
cgcgttgcac caagatataa gaaaatgctg gaggttgaat aatcaatgtt agatccgtca 3060
attgattctt taatgaataa attagattca aaatatacgc tggtgactgt ttctgcgaga 3120
cgtgcccgtg aaatgcaaat caaaaaagac caaatgattg aacatacgat ttcacacaaa 3180
tatgtaggca aagctttaga agaaattgat gcaggcctgc tttcgtttga aaaggaagac 3240
cgcgaatagt agcacaagta gcaacctata tcatgtaggt tgttattttt ttccgtgcgg 3300
ttttgtaaag tgcagagggg gagaattcat tgcttaacaa tcgaaatgtg ttactttgcg 3360
tgagtggagg catcgctgtt tataaagcct gtgcgttaac gagcaagctg gttcaggcag 3420
gagcaaatgt caaagtgatt atgactgaat ccgcttgccg attcgtttca ccgctgacat 3480
ttcaggcatt gagccgccac gaagtctata cagatacatt taaagaacaa aatccaagcg 3540
tcatttctca tattgatgcc gcagactggg ccgacttgat tatcgtagcg ccggctacgg 3600
ctaatgtgat tggaaaactg gcaaacggca tcgctgatga tatgctgacg acaacattgt 3660
tggcagcaac ggctcccgtt tggatcgcgc cggcaatgaa cgttcatatg tatgaccacc 3720
cggcagtcaa acgaaacatt tctgttcttt atcaggacgg ttattgtttt attgagccaa 3780
gtgagggcta tttagcatgc ggttacgtag gaaaaggcag attggaagag ccggaaaaca 3840
tcgtaaagct ggccgaaaaa cattttgctg aagaaacatc cgctccttta gaaggcaagc 3900
atgtggtcat tacagccggc ccaacgcggg aagcgattga tcctgtccgg ttttttacca 3960
ataaatcgac gggcaaaatg ggctacgcac tggcggaggc tgctgttcag ctcggcgcac 4020
gggtaatctt aatttctggg cc 4042
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<212> DNA
<213> 人工序列(未知)
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tccgattacc cgcttttaag cgaggcaggt cttcacctgc tgcaggccgg agggaaacgt 60
attcggcctg ttttcgtgct gctttctggc atgtttggcg attatgatat taataagatt 120
aaatatgtcg ccgtcactct ggaaatgatt cacatggcat ctttggttca tgatgatgtc 180
attgatgatg cagagcttcg ccgaggaaaa ccgacaatca aagcaaagtg ggacaatcgt 240
attgcgatgt acacaggcga ttatatgctt gcgggatctc ttgaaatgat gacgagaatt 300
aacgaaccga aagcccatag gattttgtca cagacgatcg ttgaagtttg tctaggggaa 360
attgagcaga tcaaagacaa atacaacatg gaacaaaatc tcagaacgta tctccgccgt 420
atcaaaagaa aaacagctct cttgatcgcg gtcagctgcc agcttggtgc cattgcgtct 480
ggagctgatg agaagattca taaggcattg tactggtttg ggtattacgt cggcatgtct 540
tatcagatta ttgatgatat tcttgatttt acttcaactg aggaagagct gggtaaaccc 600
gtaggaggag atttgcttca aggaaacgtc acattgccag tgctgtatgc cctgaaaaat 660
cctgcattaa aaaaccagct taaattgatt aacagtgaga caacgcagga acagcttgaa 720
ccaatcattg aagaaatcaa aaaaacagat gcaattgaag catctatggc agtaagcgaa 780
atgtatctgc agaaagcttt tcagaaatta aacacgcttc ctcgagggcg cgcacgctcg 840
tctcttgcag ccatcgcaaa atatatcggt aaaagaaaat tttaattttt gtagatatta 900
agaaatttct ccgccccgga ttgattaatt atcagaaaga tgttacgatt tcggtgtgca 960
gtgtgaaagc ctatacatac ataatgatgg ggtggagaat atgatggaaa agacttttat 1020
catggtgaaa ccagacggtg tccaacgtca gctcattggg gacattttat ctagattcga 1080
acgtaagggc ttacaattag ctggcgccaa gttaatgaga gtgactgaac aaatggctga 1140
gaaacactac gccgaacatc aaggtaagcc tttcttcgga gagctcgttg agtttattac 1200
ttcaggacct gtattcgcaa tggtgtggga aggcgaaaat gtcattgaag tgacgagaca 1260
gctgatcggg aaaacaaacc ctaaagaagc tttacctggt acgattcgtg gggattatgg 1320
catgtttgtc ggaaaaaaca tcatccacgg ctctgattct ctcgaaagtg cagaacgcga 1380
gattaacatt ttctttaaga atgaagaatt agtatcatat cagcagctta tggcaggctg 1440
gatctattaa tcattgtgaa aataagcaac agagccggca acatataacg ccgccggttt 1500
ttgttctgtt ttgtaaaggt ttacattaaa gtgaataaag ggttgaatgg cagtgtgcat 1560
cagacctctg cctttcccaa cgatctaaaa tcagctgaaa agctgatttt tttattgaaa 1620
caatacaatt tcctgataaa acaagctata ctaggacaaa aagacagcga gactgggaga 1680
gtagagatgg atacatacag cgtatttaca acgaaatgga aacaattaac cggagtcgat 1740
ttaacactat ataaagaagc acaaatgaag agaaggctaa cgtcacttta tgagaaaaag 1800
gggttccaaa gctttaagga ctttgctgcg gcattggaaa aggatcaagc tctcttaaat 1860
gaaacattgg acagaatgac gatcaatgtt tcagaatttt atcgtaatta taaaagatgg 1920
gaagttcttg agactgcaat tctgccgtta atcaagacct ctaggccttt aaaaatttgg 1980
agtgccgcct gctcaacggg tacccgggga tcctctagag attgtaccgt tcgtatagca 2040
tacattatac gaagttatcg attttcgttc gtgaatacat gttataataa ctataactaa 2100
taacgtaacg tgactggcaa gagatatttt taaaacaatg aataggttta cacttacttt 2160
agttttatgg aaatgaaaga tcatatcata tataatctag aataaaatta actaaaataa 2220
ttattatcta gataaaaaat ttagaagcca atgaaatcta taaataaact aaattaagtt 2280
tatttaatta acaactatgg atataaaata ggtactaatc aaaatagtga ggaggatata 2340
tttgaataca tacgaacaaa ttaataaagt gaaaaaaata cttcggaaac atttaaaaaa 2400
taaccttatt ggtacttaca tgtttggatc aggagttgag agtggactaa aaccaaatag 2460
tgatcttgac tttttagtcg tcgtatctga accattgaca gatcaaagta aagaaatact 2520
tatacaaaaa attagaccta tttcaaaaaa aataggagat aaaagcaact tacgatatat 2580
tgaattaaca attattattc agcaagaaat ggtaccgtgg aatcatcctc ccaaacaaga 2640
atttatttat ggagaatggt tacaagagct ttatgaacaa ggatacattc ctcagaagga 2700
attaaattca gatttaacca taatgcttta ccaagcaaaa cgaaaaaata aaagaatata 2760
cggaaattat gacttagagg aattactacc tgatattcca ttttctgatg tgagaagagc 2820
cattatggat tcgtcagagg aattaataga taattatcag gatgatgaaa ccaactctat 2880
attaacttta tgccgtatga ttttaactat ggacacgggt aaaatcatac caaaagatat 2940
tgcgggaaat gcagtggctg aatcttctcc attagaacat agggagagaa ttttgttagc 3000
agttcgtagt tatcttggag agaatattga atggactaat gaaaatgtaa atttaactat 3060
aaactattta aataacagat taaaaaaatt ataaataact tcgtatagca tacattatac 3120
gaacggtaga atcgtcgact gataggttgg tgtggttggt gataggtggt atgttttcgc 3180
ttgaactttt aaatacagcc attgaacata cggttgattt aataactgac aaacatcacc 3240
ctcttgctaa agcggccaag gacgctgccg ccggggctgt ttgcgttttt gccgtgattt 3300
cgtgtatcat tggtttactt atttttttgc caaagctgta atggctgaaa attcttacat 3360
ttattttaca tttttagaaa tgggcgtgaa aaaaagcgcg cgattatgta aaatataaag 3420
tgatagcaaa ggaggtgaaa tgtacacatg atggaaaaga cttttatcat ggtgaaacca 3480
gacggtgtcc aacgtcagct cattggggac attttatcta gattcgaacg taagggctta 3540
caattagctg gcgccaagtt aatgagagtg actgaacaaa tggctgagaa acactacgcc 3600
gaacatcaag gtaagccttt cttcggagag ctcgttgagt ttattacttc aggacctgta 3660
ttcgcaatgg tgtgggaagg cgaaaatgtc attgaagtga cgagacagct gatcgggaaa 3720
acaaacccta aagaagcttt acctggtacg attcgtgggg attatggcat gtttgtcgga 3780
aaaaacatca tccacggctc tgattctctc gaaagtgcag aacgcgagat taacattttc 3840
tttaagaatg aagaattagt atcatatcag cagcttatgg caggctggat ctattaatca 3900
ttgtgaaaat aagcaacaga gccggcaaca tataacgccg ccggtttttg ttctgttttg 3960
taaaggttta cattaaagtg aataaagggt tgaatggcag tgtgcatcag acctctgcct 4020
ttcccaacga tctaaaatca gctgaaaagc tgattttttt attgaaacaa tacaatttcc 4080
tgataaaaca agctatacta ggacaaaaag acagcgagac tgggagagta gagatggata 4140
catacagcgt atttacaacg aaatggaaac aattaaccgg agtcgattta acactatata 4200
aagaagcaca aatgaagaga aggctaacgt cactttatga gaaaaagggg ttccaaagct 4260
ttaaggactt tgctgcggca ttggaaaagg atcaagctct cttaaatgaa acattggaca 4320
gaatgacgat caatgtttca gaattttatc gtaattataa aagatgggaa gttcttgaga 4380
ctgcaattct gccgttaatc aagacctcta ggcctttaaa aatttggagt gccgcctgct 4440
caacggg 4447
<210> 3
<211> 9577
<212> DNA
<213> 人工序列(未知)
<400> 3
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccttaaggaa cgtacagacg 420
gcttaaaagc ctttaaaaac gtttttaagg ggtttgtaga caaggtaaag gataaaacag 480
cacaattcca agaaaaacac gatttagaac ctaaaaagaa cgaatttgaa ctaactcata 540
accgagaggt aaaaaaagaa cgaagtcgag atcagggaat gagtttataa aataaaaaaa 600
gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg ttctttctta tcttgataca 660
tatagaaata acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt 720
atgtgtttta aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta 780
aagttataag tgactaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt 840
cctaatagta gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt 900
ggaaaagtga gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg 960
catattcttg aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat 1020
aaacaaaatc gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg 1080
ctttgtccaa tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt 1140
gttgctgaag ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt 1200
aaaaatgttt atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt 1260
cgccgaatga tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg 1320
gaagtgacaa taaataataa agataattct tataatcagc acatgcatgt attggtatgt 1380
gtggaaccaa cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa 1440
ttttggaaaa aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga 1500
ccgaaaaata aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct 1560
gtaaaggata cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat 1620
ttggaggaag gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata 1680
cataaaaaat taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac 1740
gaaaaagccg atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat 1800
tattttatta aagagtagtt caacaaacgg gccagtttgt tgaagattag atgctataat 1860
tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct gaataagaac 1920
ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga aaagggaatg 1980
agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat tgttcatgaa 2040
attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg tgtttatggc 2100
tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg tgtcatgtca 2160
acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt ggaagtgaat 2220
tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga ttggccgctt 2280
acacatggtc aatttttctc tattttgccg atttatgatt caggtggata cttagagaaa 2340
gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat ttgtgccctt 2400
atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt gcaaggaccg 2460
acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat gttgattggt 2520
ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc agttaagcaa 2580
tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg tcaactttcc 2640
gactctgaga aacttctgga atcgctagag aatttctgga atgggattca ggagtggaca 2700
gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac gatgacctct 2760
aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt agtaattatc 2820
atggctgtca tggcgcatta acggaataaa gggtgtgctt aaatcgggcc attttgcgta 2880
ataagaaaaa ggattaatta tgagcgaatt gaattaataa taaggtaata gatttacatt 2940
agaaaatgaa aggggatttt atgcgtgaga atgttacagt ctatcccggc attgccagtc 3000
ggggatatta aaaagagtat aggtttttat tgggataaag taggtttcac tttggttcac 3060
catgaagatg gattcgcagt tctaatgtgt aatgaggttc ggattcatct atgggaggca 3120
agtgatgaag gctggcgcct cgtagtaatg attcaccggt ttgtacaggt gcggagtcgt 3180
ttattgctgg tactgctagt tgccgcattg aagtagaggg aattgatgaa ttatatcaac 3240
atattaagcc tttgggcatt ttgcacccca atacatcatt aaaagatcag tggtgggatg 3300
aacgagactt tgcagtaatt gatcccgaca acaatttgat tagctttttt caacaaataa 3360
aaagctaaaa tctattatta atctgttcag caatcgggcg cgattgctga ataaaagata 3420
cgagagacct ctcttgtatc ttttttattt tgagtggttt tgtccgttac actagaaaac 3480
cgaaagacaa taaaaatttt attcttgctg agtctggctt tcggtaagct agacaaaacg 3540
gacaaaataa aaattggcaa gggtttaaag gtggagattt tttgagtgat cttctcaaaa 3600
aatactacct gtcccttgct gatttttaaa cgagcacgag agcaaaaccc ccctttgctg 3660
aggtggcaga gggcaggttt ttttgtttct tttttctcgt aaaaaaaaga aaggtcttaa 3720
aggttttatg gttttggtcg gcactgccgc gcctcgcaga gcacacactt tatgaatata 3780
aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 3840
atttgtgcca cctaaaaagg agcgatttac atatgagtta tgcagtttgt agaatgcaaa 3900
aagtgaaatc agctggacta aaaggcatgc aatttcataa tcaaagagag cgaaaaagta 3960
gaacgaatga tgatattgac catgagcgaa cacgtgaaaa ttatgatttg aaaaatgata 4020
aaaatattga ttacaacgaa cgtgtcaaag aaattattga atcacaaaaa acaggtacaa 4080
gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140
tttttgagca actggatcct gataggtggt atgttttcgc ttgaactttt aaatacagcc 4200
attgaacata cggttgattt aataactgac aaacatcacc ctcttgctaa agcggccaag 4260
gacgctgccg ccggggctgt ttgcgttttt gccgtgattt cgtgtatcat tggtttactt 4320
atttttttgc caaagctgta atggctgaaa attcttacat ttattttaca tttttagaaa 4380
tgggcgtgaa aaaaagcgcg cgattatgta aaatataaag tgatagcggt accgagctca 4440
aaggaggtga aatgtacaca tgcaaaagtt actgtctctc ccatctaact tagtccagag 4500
ctttcatgaa ttagaaagag taaatcggac tgactggttt tgcacatccg acccggtcgg 4560
aaaaaagctt ggcagtggcg gtggcacatc atggctgctc gaggaatgct ataatgaata 4620
ctctgacggc gcaactttcg gagagtggct cgaaaaagag aaaagaatac tgctccacgc 4680
cggtgggcag tcccgccgtt tgccgggtta cgctcctagt ggtaagatcc ttacgccggt 4740
cccagtattt cgctgggaaa gaggacagca cctgggccag aaccttttga gtttacagct 4800
tccactttat gagaaaataa tgagcttagc tcctgacaag cttcataccc ttatcgccag 4860
cggagatgtc tatatacgct ctgaaaaacc gttgcagtcc atacctgagg ccgacgttgt 4920
ttgttacggg ctgtgggtcg acccttcctt ggcgacgcac catggggtat tcgccagtga 4980
ccgcaaacat ccggagcagc tcgacttcat gctccagaag ccttctttgg cagaacttga 5040
aagtctctct aagactcatc ttttcctgat ggacataggg atttggttat tgtccgatcg 5100
cgcggtagag attttgatga agcggtccca taaggagtcc tctgaagaat tgaaatacta 5160
tgatctttac agcgactttg ggcttgccct gggcacccac ccacggatag aagatgagga 5220
agtgaatact ctctcagttg caattctgcc tctcccgggc ggggagtttt atcattacgg 5280
tactagcaaa gagctgattt cctccaccct gtcagtccag aataaggttt atgaccaacg 5340
ccggatcatg catagaaagg tcaaaccgaa ccctgccatg tttgtccaaa atgcggtagt 5400
acgtatccca ctgtgcgctg agaacgcaga cctctggatt gaaaactcac atataggtcc 5460
gaagtggaag atagcgagcc ggcatattat tactggggtg ccggagaacg actggagttt 5520
agcagtgcca gcaggggtat gtgtcgacgt tgtcccgatg ggtgataaag gttttgtggc 5580
acggccttac gggctggacg atgtatttaa aggagacctg agagattcaa aaaccacttt 5640
gaccggaatc ccattcggtg aatggatgtc caaacgggga ctctcctaca ctgatctgaa 5700
aggtcggact gacgaccttc aagccgtgag cgtgtttccg atggtcaatt ccgttgagga 5760
attgggcctc gtactccgct ggatgctttc agaacctgag ttggaagagg gtaagaacat 5820
ctggcttcgc tccgagcact tctctgcgga tgaaataagc gctggagcga acctgaagcg 5880
cctctatgct caacgggaag aattccggaa ggggaactgg aaagcattag ccgttaacca 5940
tgagaagtcc gtattttacc aactcgattt ggccgatgcg gcggaggatt tcgtacggtt 6000
gggactcgac atgcctgaat tactccctga ggacgccctg cagatgagtc gcatccataa 6060
tagaatgctt cgcgctcgca tcctcaagct cgacggcaaa gattaccggc ctgaggaaca 6120
ggcagcgttc gatctgctgc gcgacggttt gctcgatgga atctctaacc gtaaaagcac 6180
accaaagctg gacgtttatt ctgaccaaat agtgtggggc cggagcccag tacggattga 6240
tatggccggc ggctggaccg acaccccacc ttatagctta tattccggag gcaatgttgt 6300
gaaccttgcg attgaattaa atggtcaacc tccattgcag gtatacgtta agccgtgtaa 6360
ggacttccat atcgtcctgc ggtccattga catgggggct atggagatag tctcaacttt 6420
tgacgagctg caagactaca aaaagatcgg ttccccgttt tccataccaa aagccgctct 6480
ctctcttgct ggattcgccc cggcatttag cgctgtgtca tatgcatcat tggaggaaca 6540
gttgaaagat tttggagctg gtattgaggt tacgttatta gccgccattc cggctggttc 6600
tggtcttggt acatcatcaa tactggcttc cacagtgctc ggggcaatta atgatttttg 6660
tggactcgca tgggacaaaa atgaaatttg tcagagaaca ttagttttag agcaactgct 6720
gactacggga ggaggctggc aggaccagta cggcggggtc ttgcaaggtg ttaagcttct 6780
gcagaccgag gccgggttcg cgcaatcccc attagttaga tggctgccag accacctctt 6840
tacgcaccct gaatacaagg actgccattt gttatattac acaggaatta cgcgtacggc 6900
aaagggaata ctggcggaga tcgtctctag catgtttctc aatagtagcc tgcacctgaa 6960
tttactctca gaaatgaaag cgcatgccct cgacatgaac gaggctatac agcgcggctc 7020
ctttgttgag ttcggtcgcc ttgtcggaaa gacatgggag caaaacaagg cactcgacag 7080
tggtaccaac ccaccagcag tcgaagccat aatcgactta attaaggatt atacattagg 7140
gtataaactc ccaggggctg gaggaggcgg gtacctctac atggttgcta aagatccgca 7200
ggcagcagtg cgcatccgca aaattttaac agagaacgcg ccaaaccctc gtgcccggtt 7260
tgtggaaatg actctgtccg acaaaggctt tcaagtaagt cggtcctaaa ctagtgattg 7320
ctagctctag actgcagaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 7380
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 7440
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 7500
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 7560
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 7620
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 7680
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 7740
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 7800
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 7860
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 7920
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 7980
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 8040
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 8100
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 8160
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 8220
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 8280
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 8340
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 8400
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 8460
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 8520
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 8580
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 8640
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 8700
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 8760
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 8820
gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 8880
tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 8940
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 9000
gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 9060
actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 9120
tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 9180
attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 9240
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 9300
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 9360
aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 9420
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 9480
cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 9540
acctataaaa ataggcgtat cacgaggccc tttcgtc 9577

Claims (7)

1.一种高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌是通过在枯草芽孢杆菌168的基因组中强化表达鸟苷酸激酶和核苷二磷酸激酶基因，并表达外源岩藻糖激酶和磷酸鸟嘌呤基转移酶基因得到的，其中，所述岩藻糖激酶和磷酸鸟嘌呤基转移酶基因为来源于脆弱拟杆菌的fkp基因。
2.根据权利要求1所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌，其特征在于，将P43启动子替换枯草芽孢杆菌168中鸟苷酸激酶和核苷二磷酸激酶基因的启动子，来强化表达鸟苷酸激酶和核苷二磷酸激酶基因。
3.根据权利要求2所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌，其特征在于，所述鸟苷酸激酶基因如NCBI上Gene ID: 938821基因所示，核苷二磷酸激酶如NCBI上Gene ID:938997基因所示。
4.一种权利要求1所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌的构建方法，其特征在于，该构建方法包括以下步骤：
（1）构建包含鸟苷酸激酶和核苷二磷酸激酶基因的上下游序列、P43启动子和壮观霉素抗性基因序列的替换框，将构建好的替换框转化重组枯草芽孢杆菌168，通过验证，确认鸟苷酸激酶和核苷二磷酸激酶强化表达成功，得到重组枯草芽孢杆菌BSGN，所述替换框的序列如SEQ ID NO.1和SEQ ID NO.2所示；
（2）构建包含岩藻糖激酶和磷酸鸟嘌呤基转移酶基因的重组质粒，将构建好的重组质粒转化枯草芽孢杆菌BSGN，通过验证，确认岩藻糖激酶和磷酸鸟嘌呤基转移酶表达成功，得到重组枯草芽孢杆菌BSGNF。
5.根据权利要求4所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌的构建方法，其特征在于，步骤（1）中，将构建好的替换框电转化枯草芽孢杆菌168的感受态细胞。
6.根据权利要求4所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌的构建方法，其特征在于，步骤（2）中，重组质粒的序列如SEQ ID NO.3所示。
7.权利要求1所述的高效合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌的应用，其特征在于，使用所述重组枯草芽孢杆菌发酵生成鸟苷二磷酸岩藻糖。
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