CN113637623B - 一种提高2′-岩藻糖基乳糖产量的枯草芽孢杆菌及其构建方法与应用 - Google Patents

一种提高2′-岩藻糖基乳糖产量的枯草芽孢杆菌及其构建方法与应用 Download PDF

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CN113637623B
CN113637623B CN202110970220.7A CN202110970220A CN113637623B CN 113637623 B CN113637623 B CN 113637623B CN 202110970220 A CN202110970220 A CN 202110970220A CN 113637623 B CN113637623 B CN 113637623B
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刘龙
陈坚
吕雪芹
堵国成
李江华
刘延峰
于文文
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Abstract

本发明公开了一种提高2’‑岩藻糖基乳糖产量的枯草芽孢杆菌及其构建方法与应用，以枯草芽孢杆菌168为出发菌株，采用组成型启动子Psrfa在基因组上表达外源基因双功能酶L‑岩藻糖激酶fkp、采用组成型启动子P43在基因组上分别表达外源基因α‑1，2‑岩藻糖基转移酶futC、乳糖转运酶lac12及内源性基因单糖转运蛋白酶glcp，并通过分别优化fkp、futC、lac12及glcp的核糖体结合位点RBS序列得到。本发明通过优化RBS序列，对2’‑FL代谢途径关键基因进行系统优化，提高基因的初始翻译速率，最终2’‑FL发酵产量达到1.0g/L，操作简便且提升效果显著。

Description

一种提高2′-岩藻糖基乳糖产量的枯草芽孢杆菌及其构建方 法与应用
技术领域
本发明属于基因工程技术领域，特别涉及一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌及其构建方法与应用。
背景技术
2’-岩藻糖基乳糖(2’-fucosyllactose,2’-FL)是一种由三个单糖分子组成的寡糖，在母乳中含量为2.4g/L左右，是母乳中含量最丰富的寡糖。2’ -FL具有良好的营养价值与生理活性，实验证明其可以抑制病原菌与上皮细胞表面特定受体的粘附降低婴儿病原体感染的风险，从而作为可溶的益生元促进肠道中有益微生物的生长。当前欧盟与FDA均批准2’-FL可以添加至婴幼儿配方奶粉中，市场前景广阔。目前化学和微生物合成方法均可用于生产2’-FL。化学合成要求进行多步的保护和脱保护过程会降低收率，此外使用有毒试剂会造成污染。相比起化学合成法微生物发酵法生产2’-FL更加绿色环保，且更适应食品添加剂的生产。
微生物发酵法中，以L-岩藻糖为底物，经过双功能酶L-岩藻糖激酶FKP 的催化得到岩藻糖-1-磷酸后，经过双功能酶岩藻糖-1-磷酸鸟苷酸转移酶FKP 的催化得到GDP-L-岩藻糖。GDP-L-岩藻糖在胞内获得后，可以作为岩藻糖基的供体，在岩藻糖基转移酶FutC的催化下将胞内的乳糖岩藻糖基化，获得2’-FL。但目前2’-FL的发酵产量仍处于一个较低的水平，无法满足工业化生产的需求，因此如何提高2’-FL的发酵产量，是微生物发酵法生产2’-FL亟待解决问题。
鉴于上述原因，本发明人积极加以研究创新，以期创建一种新型的提高 2’-FL产量的枯草芽孢杆菌及其构建方法，使其更具有产业上的利用价值。
发明内容
为解决微生物发酵法生产2’-FL产量较低的问题，本发明通过代谢工程技术，提供了一种提高2’-FL发酵产量的方法。本发明的技术方案通过优化RBS序列，对2’-FL代谢途径关键基因进行系统优化，提高基因的初始翻译速率，构建了一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌。本发明主要是通过以下技术方案解决上述技术问题。
本发明提供了一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌，所述枯草芽孢杆菌是以枯草芽孢杆菌168为出发菌株，采用组成型启动子Psrfa在基因组上表达外源基因双功能酶L-岩藻糖激酶fkp、采用组成型启动子P43在基因组上分别表达外源基因α-1，2-岩藻糖基转移酶futC、乳糖转运酶lac12及内源性基因单糖转运蛋白酶glcp，并通过分别优化fkp、futC、lac12及glcp 的核糖体结合位点RBS序列得到；所述Psrfa-fkp、P43-futC、P43-glcp及P43-lac12 的基因序列如SEQ ID NO.1～SEQ ID NO.4所示。
优选的，所述优化后双功能酶L-岩藻糖激酶fkp基因的RBS序列为GTCTTATTAAAGGAGGTTTTTTTT；所述优化后α-1，2-岩藻糖基转移酶futC 基因的RBS序列为GGGGAGAAGACTCCGCTCGAAAGGAGGTTTTTATT；所述优化后单糖转运蛋白酶glcp基因的RBS序列为GCTTAAAAGGAGGTTTTTTTT；所述优化后乳糖转运酶lac12基因的RBS 序列为GAATAAAAGGAGAGTTTTTTTT。
优选的，所述双功能酶L-岩藻糖激酶fkp基因来源于脆弱拟杆菌 Bacteriodesfragilis 9343；所述α-1，2-岩藻糖基转移酶futC基因来源于幽门螺杆菌H.pylori；所述乳糖转运酶lac12基因来源于大肠杆菌E.coli。
本发明还提供了一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌的构建方法，包括以下步骤：
(1)以枯草芽孢杆菌168为出发菌株，利用组成型启动子Psrfa在基因组上过表达来源于脆弱拟杆菌Bacteriodes fragilis 9343的FKP编码基因双功能酶L-岩藻糖激酶fkp，构建枯草芽孢杆菌WCY1；
(2)以枯草芽孢杆菌WCY1为出发菌株，利用组成型启动子P43在基因组上过表达来源于幽门螺杆菌H.pylori的α-1，2-岩藻糖基转移酶FutC，构建枯草芽孢杆菌WCY2；
(3)以枯草芽孢杆菌WCY2为出发菌株，利用组成型启动子P43在基因组上过表达内源性基因单糖转运蛋白酶glcp，构建枯草芽孢杆菌WCY3；
(4)以枯草芽孢杆菌WCY2为出发菌株，选择RBS序列GCTTAAAAGGAGGTTTTTTTT，并利用组成型启动子P43在基因组上过表达内源性基因单糖转运蛋白酶glcp，构建枯草芽孢杆菌WCY4；
(5)以枯草芽孢杆菌WCY4为出发菌株，选择RBS序列 GAATAAAAGGAGAGTTTTTTTT，并利用组成型启动子P43在基因组上过表达来源于E.coli基因乳糖转运酶lac12，构建枯草芽孢杆菌WCY5；
(6)以枯草芽孢杆菌WCY5为出发菌株，选择RBS序列 GTCTTATTAAAGGAGGTTTTTTTT替换WCY5菌株双功能酶L-岩藻糖激酶fkp基因原始RBS，构建枯草芽孢杆菌WCY6；
(7)以枯草芽孢杆菌WCY6为出发菌株，选择RBS序列 GGGGAGAAGACTCCGCTCGAAAGGAGGTTTTTATT替换WCY6菌株α-1，2-岩藻糖基转移酶futC基因原始RBS，构建枯草芽孢杆菌WCY7，即得所述枯草芽孢杆菌。
本发明还提供了一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌在合成 2'-岩藻糖基乳糖中的应用。
与现有技术相比，本发明的积极进步效果在于：
本发明以枯草芽孢杆菌168为出发菌株，采用组成型启动子在基因组上分别表达fkp、futC、lac12、glcp基因，并对4个基因原RBS序列依次进行理性设计优化，通过对2’-FL代谢途径关键基因进行系统优化，最终2’-FL 发酵产量达到1.0g/L。与游离型质粒表达关键基因策略相比，本发明中的基因都整合至基因组表达，遗传性状稳定具有良好的工业化应用潜能。与优化启动子或融合酶表达等策略相比，本发明的技术方案通过优化RBS序列，提高基因的初始翻译速率，操作简便且提升效果显著。
具体实施方式
一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌，所述枯草芽孢杆菌是以枯草芽孢杆菌168为出发菌株，采用组成型启动子Psrfa在基因组上表达外源基因双功能酶L-岩藻糖激酶fkp、采用组成型启动子P43在基因组上分别表达外源基因α-1，2-岩藻糖基转移酶futC、乳糖转运酶lac12及内源性基因单糖转运蛋白酶glcp，并通过分别优化fkp、futC、lac12及glcp的核糖体结合位点RBS序列得到；所述Psrfa-fkp、P43-futC、P43-glcp及P43-lac12的基因序列如SEQ ID NO.1～SEQ ID NO.4所示。
微生物中2’-FL代谢途径为以L-岩藻糖为底物，经过双功能酶L-岩藻糖激酶FKP的催化得到岩藻糖-1-磷酸后，经过双功能酶岩藻糖-1-磷酸鸟苷酸转移酶FKP的催化得到GDP-L-岩藻糖。GDP-L-岩藻糖在胞内获得后，可以作为岩藻糖基的供体，在岩藻糖基转移酶FutC的催化下将胞内的乳糖岩藻糖基化，获得2’-FL。本发明中，通过优化2’-FL代谢途径中的fkp、futC、 glcp、lac12基因的核糖体结合位点RBS序列，提高基因的初始翻译速率，提高了2’-FL发酵产量。本发明中，双功能酶L-岩藻糖激酶fkp基因的RBS 序列优选GTCTTATTAAAGGAGGTTTTTTTT；α-1，2-岩藻糖基转移酶futC 基因的RBS序列优选为GGGGAGAAGACTCCGCTCGAAAGGAGGTTTTTATT；单糖转运蛋白酶 glcp基因的RBS序列优选为GCTTAAAAGGAGGTTTTTTTT；乳糖转运酶 lac12基因的RBS序列优选为GAATAAAAGGAGAGTTTTTTTT。
本发明中，利用组成型启动子Psrfa过表达来源于脆弱拟杆菌Bacteriodesfragilis 9343的FKP编码基因fkp；所述脆弱拟杆菌9343的fkp基因如NCBI 上GenBank:AY849806.1所示；本发明中，利用组成型启动子P43过表达来源于幽门螺杆菌H.pylori的futC基因；所述幽门螺旋杆菌futC基因如NCBI 上GenBank:KY499613所示；本发明中，利用组成型启动子P43过表达内源性基因glcp；本发明中，利用组成型启动子P43过表达来源于大肠杆菌E.coli 的lac12基因。
本发明还提供了一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌的构建方法，包括以下步骤：
(1)以枯草芽孢杆菌168为出发菌株，利用组成型启动子Psrfa在基因组上过表达来源于脆弱拟杆菌Bacteriodes fragilis 9343的FKP编码基因双功能酶L-岩藻糖激酶fkp，构建枯草芽孢杆菌WCY1；
(2)以枯草芽孢杆菌WCY1为出发菌株，利用组成型启动子P43在基因组上过表达来源于幽门螺杆菌H.pylori的α-1，2-岩藻糖基转移酶FutC，构建枯草芽孢杆菌WCY2；
(3)以枯草芽孢杆菌WCY2为出发菌株，利用组成型启动子P43在基因组上过表达内源性基因单糖转运蛋白酶glcp，构建枯草芽孢杆菌WCY3；
(4)以枯草芽孢杆菌WCY2为出发菌株，选择RBS序列 GCTTAAAAGGAGGTTTTTTTT，并利用组成型启动子P43在基因组上过表达内源性基因单糖转运蛋白酶glcp，构建枯草芽孢杆菌WCY4；
(5)以枯草芽孢杆菌WCY4为出发菌株，选择RBS序列 GAATAAAAGGAGAGTTTTTTTT，并利用组成型启动子P43在基因组上过表达来源于E.coli基因乳糖转运酶lac12，构建枯草芽孢杆菌WCY5；
(6)以枯草芽孢杆菌WCY5为出发菌株，选择RBS序列 GTCTTATTAAAGGAGGTTTTTTTT替换WCY5菌株双功能酶L-岩藻糖激酶fkp基因原始RBS，构建枯草芽孢杆菌WCY6；
(7)以枯草芽孢杆菌WCY6为出发菌株，选择RBS序列 GGGGAGAAGACTCCGCTCGAAAGGAGGTTTTTATT替换WCY6菌株α-1，2-岩藻糖基转移酶futC基因原始RBS，构建枯草芽孢杆菌WCY7，即得所述枯草芽孢杆菌。
本发明还提供了一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌在合成 2'-岩藻糖基乳糖中的应用。
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例对本发明进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1重构枯草芽孢杆菌WCY1与WCY2
(a)人工合成基因片段Psrfa-fkp，以枯草芽孢杆菌168基因组为模版，采用引物amye-U-F、amye-U-R扩增得到基因amye-U，采用引物amye-D-F、 amye-D-R扩增得到基因amye-D，以质粒F19-1(SEQ ID NO.5)为模版，采用F19-amye-F、F19-amye-R扩增得到片段F19-1H。
(b)将步骤(a)中获得的四个片段amye-U、amye-D、F19-1H和Psrfa-fkp 基因片段按照等摩尔比配置进行一步克隆连接，连接条件：50℃，60min。将连接之后的反应体系热击转入大肠杆菌JM109感受态后，采用引物amye-U-F与amye-D-R菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37℃培养16h后利用生工质粒提取试剂盒提取得到质粒F19-fkp。
(c)制备枯草芽孢杆菌168感受态，并导入质粒PHT-xcr6(SEQ ID NO.9)与质粒F19-fkp，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(d)挑选步骤(c)中平板上长出的单菌落培养至2mLLB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY1。
(e)人工合成基因片段P43-futC，以枯草芽孢杆菌168基因组为模版，采用引物npre-U-F、npre-U-R扩增得到基因npre-U，采用引物npre-D-F、 npre-D-R扩增得到基因npre-D，以质粒F19-2(SEQ ID NO.6)为模版，采用F19-npre-F、F19-npre-R扩增得到片段F19-2H。
(f)将步骤(e)中获得的四个片段npre-U、npre-D、F19-2H和P43-futC 基因片段按照等摩尔比配置进行一步克隆连接，连接条件：50℃，60min。将连接之后的反应体系热击转入大肠杆菌JM109感受态后，采用引物 npre-U-F与npre-D-R菌落PCR验证，并进行测序。测序正确的单菌落接入 2mL LB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-futC。
(g)制备枯草芽孢杆菌WCY1感受态，并导入质粒F19-futC，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(h)挑选步骤(g)中平板上长出的单菌落培养至2mLLB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY2。
引物序列：
amye-D-F taagtcggtcctaaccgcaactttagagtaaagg
amye-D-R gatctgccgttcgtgaacaggagcgttgattgatataac
amye-U-F ttagttagcttggccggttcatcctctgtctctatcaat
amye-U-R ttgcgaagagcgtctaatcatccttccagggtat
F19-amye-F atcaatcaacgctcctgttcacgaacggcagatcagaat
F19-amye-R tagagacagaggatgaaccggccaagctaactaagtttg
F19-npre-F cctgttacgaacggcaga
F19-npre-R gtgaataagtgaggcactggccaagctaactaa
npre-D-F taaatcacaaaaatataatgcataaaattctgctacaggtcacgt
npre-D-R gatctgccgttcgtaacaggggcagccatcttcaccatat
npre-U-F agttagcttggccagtgcctcacttattcacacgcacata
npre-U-R tcaagcgaaaacataccacctatcattcattcaagagttatgttttttacatatca
实施例2重构枯草芽孢杆菌WCY3与WCY4
(a)人工合成基因片段P43-glcp，以枯草芽孢杆菌168基因组为模版，采用引物apre-U-F、apre-U-R扩增得到基因apre-U，采用引物apre-D-F、 apre-D-R扩增得到基因apre-D，以质粒F19-3(SEQ ID NO.7)为模版，采用F19-apre-F、F19-apre-R扩增得到片段F19-3H。
(b)将步骤(a)中获得的四个片段apre-U、apre-D、F19-3H和P43-glcp 基因片段按照等摩尔比配置进行一步克隆连接，连接条件：50℃，60min。将连接之后的反应体系热击转入大肠杆菌JM109感受态后，采用引物 apre-U-F与apre-D-R菌落PCR验证，并进行测序。测序正确的单菌落接入 2mL LB液体培养基内37℃培养16h后利用生工质粒提取试剂盒提取得到质粒F19-glcp。
(c)制备枯草芽孢杆菌WCY2感受态，并导入质粒F19-glcp，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(d)挑选步骤(c)中平板上长出的单菌落培养至2mLLB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY3。
(e)偶然发现RBS序列GCTTAAAAGGAGGTTTTTTTT更适合基因 glcp的翻译表达，即以质粒F19-glcp为模版，采用引物glcpR-F、glcpR-R扩增得到片段F19-glcpr，之后将片段F19-glcpr热击转入大肠杆菌JM109感受态后，采用引物glcpR-F与glcpR-YZ-R菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-glcpR。
(g)制备枯草芽孢杆菌WCY2感受态，并导入质粒F19-glcpR，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(h)挑选步骤(g)中平板上长出的单菌落培养至2mLLB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY4。
引物序列：
apre-D-F atcaaaaacacagtagacgatggcgttcagcaac
apre-D-R ccgttcgacgatctccaagtcgggtgcttagaa
apre-U-F tagttagcttggccgtgacattcggcacactccttt
apre-U-R ataccacctatcagctgatccacaattttttgct
F19-apre-F tttctaagcacccgacttggagatcgtcgaacggcagatcaga
F19-apre-R agtgtgccgaatgtcacggccaagctaactaagtt
glcpR-F cgcttaaaaggaggttttttttatgttaagagggacatatttatttggat
glcpR-R ataaaaaaaacctccttttaagcgctatcactttatattttacataatcgcg
glcpR-YZ-R gtactctcggttcttagacaagaaa
实施例3重构枯草芽孢杆菌WCY5
(a)人工合成基因片段P43-lac12，以枯草芽孢杆菌168基因组为模版，采用引物ganA-U-F、ganA-U-R扩增得到基因ganA-U，采用引物ganA-D-F、 ganA-D-R扩增得到基因ganA-D，以质粒F19-4(SEQ ID NO.8)为模版，采用F19-ganA-F、F19-ganA-R扩增得到片段F19-4H。
(b)将步骤(a)中获得的四个片段ganA-U、ganA-D、F19-4H和P43-lac12 基因片段按照等摩尔比配置进行一步克隆连接，连接条件：50℃，60min。将连接之后的反应体系热击转入大肠杆菌JM109感受态后，采用引物 ganA-U-F与ganA-D-R菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37℃培养16h后利用生工质粒提取试剂盒提取得到质粒F19-lac12。
(c)偶然发现RBS序列GAATAAAAGGAGAGTTTTTTTT更适合基因 lac12的翻译表达，即以质粒F19-lac12为模版，采用引物lac12R-F、lac12R-R 扩增得到片段F19-lac12r，之后将片段F19-lac12r热击转入大肠杆菌JM109 感受态后，采用引物lac12R-F与lac12R-YZ-R菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-lac12R。
(c)制备枯草芽孢杆菌WCY4感受态，并导入质粒F19-lac12R，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(d)挑选步骤(c)中平板上长出的单菌落培养至2mL LB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY5。
引物序列：
F19-ganA-R gttgccatatccaagactctagaggcactggc
F19H-ganA-F ttacagccggacctccgcagagtcgacctgttacgaacg
ganA-D-F gaaaacataccacctatcacacattctcctccttgttctc
ganA-D-R taacaggtcgactctgcggaggtccggctgtaaccgg
ganA-U-F gtgcctctagagtcttggatatggcaacgtctgc
ganA-U-R aggctgaaagcgtataataggctgatgctccgctc
lac12R-F aaaaaaaactctccttttattcggtaccgctatcactttatattttaca
lac12R-R accgaataaaaggagagttttttttatggctgatcactctagca
lac12R-YZ-R accggttgccgtcatcttt
实施例4重构枯草芽孢杆菌WCY6与WCY7
(a)偶然发现RBS序列GTCTTATTAAAGGAGGTTTTTTTT更适合基因fkp的翻译表达，即以质粒F19-fkp为模版，采用引物fkpR-F、fkpR-R扩增得到片段F19-fkpr，之后将片段F19-fkpr热击转入大肠杆菌JM109感受态后，采用引物fkpR-F与fkpR-YZ-R菌落PCR验证，并进行测序。测序正确的单菌落接入2mLLB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-fkpR。
(b)以质粒F19-fkpR为模版采用引物fkpR-F2、fkpR-R2扩增得到片段 F19-fkpr2，之后将片段F19-fkpr2热击转入大肠杆菌JM109感受态后，采用引物fkpR-F2与fkpR-YZ-R2菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-fkpR2。
(c)制备枯草芽孢杆菌WCY5感受态，并导入质粒F19-fkpR2，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(d)挑选步骤(c)中平板上长出的单菌落培养至2mLLB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY6。
(e)偶然发现RBS序列
GGGGAGAAGACTCCGCTCGAAAGGAGGTTTTTATT更适合基因futC的翻译表达，即以质粒F19-futc为模版，采用引物futcR-F、futcR-R扩增得到片段F19-futcr，之后将片段F19-futcr热击转入大肠杆菌JM109感受态后，采用引物futcR-F与futcR-YZ-R菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-futcR。
(f)以质粒F19-futcR为模版采用引物futcR-F2、futcR-R2扩增得到片段F19-futcr2，之后将片段F19-futcr2热击转入大肠杆菌JM109感受态后，采用引物futcR-F2与fkpR-YZ-R2菌落PCR验证，并进行测序。测序正确的单菌落接入2mL LB液体培养基内37培养16h后利用生工质粒提取试剂盒提取得到质粒F19-futcR2。
(g)制备枯草芽孢杆菌WCY6感受态，并导入质粒F19-futcR2，之后涂板于添加卡那霉素、氯霉素与木糖的固体培养基平板上，30℃培养18h。
(h)挑选步骤(g)中平板上长出的单菌落培养至2mL LB液体培养基内，50℃培养12h，划线至添加氯霉素的固体培养基平板上，之后长出的单菌落即为枯草芽孢杆菌WCY7。
引物序列：
fkpR-F2 acctcctttgagctcggtacataaatttctactgttgtagatcgtct
fkpR-R2 tatgtaccgagctcaaaggaggtatctacaacagtagaaattatttaaagtt
fkpR-YZ-R2 aggggacaggatgtcccaggcgaagg
fkpR-F gtcttattaaaggaggttttttttatgcaaaagttactgtctctc
fkpR-R gcataaaaaaaacctcctttaataagacgagctcggtacatattttttatcttt
fkpR-YZ-R acatctccgctggcgata
futcR-F aagactccgctcgaaaggaggtttttattatggcatttaaggttgttcagatt
futcR-R aacctcctttcgagcggagtcttctccccgctatcactttatattttacataatcgcgc
futcR-YZ-R gttggcatgattgcatcag
futcR-F2 taggtaagagaggaatgtacacaaatttctactgttgtagatcgtct
futcR-R2 attTgtgtacattcctctcttacctaatctacaacagtagaaattatttaaagtt
实施例5枯草芽孢杆菌的发酵培养
在2ml LB培养基中接种固体LB平板上的枯草芽孢杆菌单菌落，37℃， 220rpm培养8-10h后，按接种量5％接入含有20g/L甘油、2g/L岩藻糖、 5g/L乳糖的OSF液体培养基内，37℃，220rpm培养96h。发酵至24h时，加入2mL甘油。发酵结束后，取1mL菌液超声破碎离心，取上清液进行高效液相色谱检测。枯草芽孢杆菌WCY2的2’-FL发酵产量达到25mg/L，枯草芽孢杆菌WCY3的2’-FL发酵产量达到100mg/L，枯草芽孢杆菌WCY4 的2’-FL发酵产量达到350mg/L，枯草芽孢杆菌WCY5的2’-FL发酵产量达到651mg/L，枯草芽孢杆菌WCY6的2’-FL发酵产量达到836mg/L，枯草芽孢杆菌WCY7的2’-FL发酵产量达到1.0g/L。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、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> 一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌及其构建方法与应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3181
<212> DNA
<213> （人工序列）
<400> 1
gacgctcttc gcaagggtgt ctttttttgc ctttttttcg gtttttgcgc ggtacacata 60
gtcatgtaaa gattgtaaat tgcattcagc aataaaaaaa gattgaacgc agcagtttgg 120
tttaaaaatt tttatttttc tgtaaataat gtttagtgga aatgattgcg gcatcccgca 180
aaaaatattg ctgtaaataa actggaatct ttcggcatcc cgcatgaaac ttttcaccca 240
tttttcggtg ataaaaacat ttttttcatt taaactgaac ggtagaaaga taaaaaatat 300
gtaccgagct caaaggaggt gaaatgtaca catgcaaaag ttactgtctc tcccatctaa 360
cttagtccag agctttcatg aattagaaag agtaaatcgg actgactggt tttgcacatc 420
cgacccggtc ggaaaaaagc ttggcagtgg cggtggcaca tcatggctgc tcgaggaatg 480
ctataatgaa tactctgacg gcgcaacttt cggagagtgg ctcgaaaaag agaaaagaat 540
actgctccac gccggtgggc agtcccgccg tttgccgggt tacgctccta gtggtaagat 600
ccttacgccg gtcccagtat ttcgctggga aagaggacag cacctgggcc agaacctttt 660
gagtttacag cttccacttt atgagaaaat aatgagctta gctcctgaca agcttcatac 720
ccttatcgcc agcggagatg tctatatacg ctctgaaaaa ccgttgcagt ccatacctga 780
ggccgacgtt gtttgttacg ggctgtgggt cgacccttcc ttggcgacgc accatggggt 840
attcgccagt gaccgcaaac atccggagca gctcgacttc atgctccaga agccttcttt 900
ggcagaactt gaaagtctct ctaagactca tcttttcctg atggacatag ggatttggtt 960
attgtccgat cgcgcggtag agattttgat gaagcggtcc cataaggagt cctctgaaga 1020
attgaaatac tatgatcttt acagcgactt tgggcttgcc ctgggcaccc acccacggat 1080
agaagatgag gaagtgaata ctctctcagt tgcaattctg cctctcccgg gcggggagtt 1140
ttatcattac ggtactagca aagagctgat ttcctccacc ctgtcagtcc agaataaggt 1200
ttatgaccaa cgccggatca tgcatagaaa ggtcaaaccg aaccctgcca tgtttgtcca 1260
aaatgcggta gtacgtatcc cactgtgcgc tgagaacgca gacctctgga ttgaaaactc 1320
acatataggt ccgaagtgga agatagcgag ccggcatatt attactgggg tgccggagaa 1380
cgactggagt ttagcagtgc cagcaggggt atgtgtcgac gttgtcccga tgggtgataa 1440
aggttttgtg gcacggcctt acgggctgga cgatgtattt aaaggagacc tgagagattc 1500
aaaaaccact ttgaccggaa tcccattcgg tgaatggatg tccaaacggg gactctccta 1560
cactgatctg aaaggtcgga ctgacgacct tcaagccgtg agcgtgtttc cgatggtcaa 1620
ttccgttgag gaattgggcc tcgtactccg ctggatgctt tcagaacctg agttggaaga 1680
gggtaagaac atctggcttc gctccgagca cttctctgcg gatgaaataa gcgctggagc 1740
gaacctgaag cgcctctatg ctcaacggga agaattccgg aaggggaact ggaaagcatt 1800
agccgttaac catgagaagt ccgtatttta ccaactcgat ttggccgatg cggcggagga 1860
tttcgtacgg ttgggactcg acatgcctga attactccct gaggacgccc tgcagatgag 1920
tcgcatccat aatagaatgc ttcgcgctcg catcctcaag ctcgacggca aagattaccg 1980
gcctgaggaa caggcagcgt tcgatctgct gcgcgacggt ttgctcgatg gaatctctaa 2040
ccgtaaaagc acaccaaagc tggacgttta ttctgaccaa atagtgtggg gccggagccc 2100
agtacggatt gatatggccg gcggctggac cgacacccca ccttatagct tatattccgg 2160
aggcaatgtt gtgaaccttg cgattgaatt aaatggtcaa cctccattgc aggtatacgt 2220
taagccgtgt aaggacttcc atatcgtcct gcggtccatt gacatggggg ctatggagat 2280
agtctcaact tttgacgagc tgcaagacta caaaaagatc ggttccccgt tttccatacc 2340
aaaagccgct ctctctcttg ctggattcgc cccggcattt agcgctgtgt catatgcatc 2400
attggaggaa cagttgaaag attttggagc tggtattgag gttacgttat tagccgccat 2460
tccggctggt tctggtcttg gtacatcatc aatactggct tccacagtgc tcggggcaat 2520
taatgatttt tgtggactcg catgggacaa aaatgaaatt tgtcagagaa cattagtttt 2580
agagcaactg ctgactacgg gaggaggctg gcaggaccag tacggcgggg tcttgcaagg 2640
tgttaagctt ctgcagaccg aggccgggtt cgcgcaatcc ccattagtta gatggctgcc 2700
agaccacctc tttacgcacc ctgaatacaa ggactgccat ttgttatatt acacaggaat 2760
tacgcgtacg gcaaagggaa tactggcgga gatcgtctct agcatgtttc tcaatagtag 2820
cctgcacctg aatttactct cagaaatgaa agcgcatgcc ctcgacatga acgaggctat 2880
acagcgcggc tcctttgttg agttcggtcg ccttgtcgga aagacatggg agcaaaacaa 2940
ggcactcgac agtggtacca acccaccagc agtcgaagcc ataatcgact taattaagga 3000
ttatacatta gggtataaac tcccaggggc tggaggaggc gggtacctct acatggttgc 3060
taaagatccg caggcagcag tgcgcatccg caaaatttta acagagaacg cgccaaaccc 3120
tcgtgcccgg tttgtggaaa tgactctgtc cgacaaaggc tttcaagtaa gtcggtccta 3180
a 3181
<210> 2
<211> 1197
<212> DNA
<213> （人工序列）
<400> 2
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcat tataggtaag agaggaatgt acacatggca 300
tttaaggttg ttcagatttg cggggggtta gggaatcaga tgtttcaata tgcgtttgcg 360
aaaagcctgc aaaaacactc aaatacgccg gttctgctgg atattacgtc gtttgattgg 420
tcagatagaa aaatgcaact ggaactgttt ccgatcgatc tgccatatgc aagcgaaaaa 480
gaaattgcaa tcgcaaaaat gcaacatctg cctaaactgg tgagagatgc gctgaaatgc 540
atgggattcg accgcgtttc aaaagaaatt gtttttgaat acgagccgga actgctgaaa 600
ccgtcaagac tgacatactt ctatggttac ttccaagatc cgagatattt tgatgcgatc 660
agtcctctga ttaaacaaac atttacactg ccgccgccgc cgcctgaaaa tggcaacaat 720
aaaaagaaag aagaggaata tcaccgcaaa ttagcactga ttctggcagc aaaaaattca 780
gtttttgttc atattagaag aggcgattat gttggcattg gctgccaact gggcattgat 840
tatcaaaaaa aagcactgga atatatggca aaaagagttc cgaatatgga actgtttgtt 900
ttttgcgaag atctgacatt tacacaaaat ctggatctgg gctatccgtt tatggatatg 960
acaacaagag ataaagaaga agaagcatat tgggatatgc tgctgatgca atcatgccaa 1020
catggcatta ttgcaaattc aacatattca tggtgggcag catatctgat taataatccg 1080
gaaaaaatta ttattggccc gaaacattgg ctgtttggcc atgaaaatat tctgtgcaaa 1140
gaatgggtta aaattgaatc acattttgaa gttaaatcac aaaaatataa tgcataa 1197
<210> 3
<211> 1494
<212> DNA
<213> （人工序列）
<400> 3
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcaa aggaggtgaa atgtacacat gttaagaggg 300
acatatttat ttggatatgc tttctttttt acagtaggta ttatccatat atcaacaggg 360
agtttgacac catttttatt agaggctttt aacaagacaa cagatgatat ttcggtcata 420
atcttcttcc agtttaccgg atttctaagc ggagtattaa tcgcaccttt aatgattaag 480
aaatacagtc attttaggac acttacttta gctttgacaa taatgcttgt agcgttaagt 540
atcttttttc taaccaagga ttggtattat attattgtaa tggcttttct cttaggatat 600
ggagcaggca cattagaaac gacagttggt tcatttgtta ttgctaattt cgaaagtaat 660
gcagaaaaaa tgagtaagct ggaagttctc tttggattag gcgctttatc tttcccatta 720
ttaattaatt ccttcataga tatcaataac tggtttttac catattactg tatattcacc 780
tttttattcg tcctattcgt agggtggtta attttcttgt ctaagaaccg agagtacgct 840
aagaatgcta accaacaagt gacctttcca gatggaggag catttcaata ctttatagga 900
gatagaaaaa aatcaaagca attaggcttt tttgtatttt tcgctttcct atatgctgga 960
attgaaacaa attttgccaa ctttttacct tcaatcatga taaaccaaga caatgaacaa 1020
attagtctta taagtgtctc ctttttctgg gtagggatca tcataggaag aatattgatt 1080
ggtttcgtaa gtagaaggct tgatttttcc aaataccttc tttttagctg tagttgttta 1140
attgttttgt tgattgcctt ctcttatata agtaacccaa tacttcaatt gagtggtaca 1200
tttttgattg gcctaagtat agcggggata tttcccattg ctttaacact agcatcaatc 1260
attattcaga agtacgttga cgaagttaca agtttattta ttgcctcggc aagtttcgga 1320
ggagcgatca tctctttctt aattggatgg agtttaaacc aggatacgat cttattaacc 1380
atgggaatat ttacaactat ggcggtcatt ctagtaggta tttctgtaaa gattaggaga 1440
actaaaacag aagaccctat ttcacttgaa aacaaagcat caaaaacaca gtag 1494
<210> 4
<211> 2058
<212> DNA
<213> （人工序列）
<400> 4
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcgg taccaaagga ggtgaaatgt acacatggct 300
gatcactcta gcagctcctc ctcattgcag aaaaaaccga taaacacgat agaacataaa 360
gacactctcg gaaatgacag agaccacaag gaggccctga actccgataa cgataacact 420
agtggtctta aaataaacgg tgtccctatt gaggacgcgc gcgaggaagt actcctccct 480
ggttatttaa gcaagcaata ctataaatta tacggtttgt gctttattac ctacttgtgt 540
gcaaccatgc aagggtatga tggtgcatta atgggtagca tttatactga agatgcgtat 600
ttgaaatatt accacctcga tataaattcc tcctcaggca cgggcctggt atttagcatc 660
ttcaatgtcg ggcaaatatg cggagcgttc ttcgtccctt taatggactg gaaagggcgg 720
aaacctgcta tcttgatcgg ttgtctgggc gttgtcatag gtgcgatcat atctagtttg 780
accaccacca agagtgcatt gataggaggc cggtggtttg tcgcgttttt cgccacaatc 840
gcgaatgcgg cggctcctac ctactgcgcg gaggtagctc cggcacacct ccggggaaag 900
gtagctggtc tctataacac attatggagt gtaggctcta ttgttgcggc gttttcaacc 960
tacgggacca ataaaaactt cccaaattcc tccaaggctt tcaagatacc attatatctc 1020
caaatgatgt ttcctgggct tgtctgtata tttggttggc tcataccaga atctcctaga 1080
tggctggtcg gggtcgggcg cgaggaagag gctcgggaat ttattatcaa gtaccatctt 1140
aacggtgatc gcacgcatcc attgttagac atggaaatgg ccgaaattat cgagagtttc 1200
cacggaactg atctgtctaa tccattagaa atgttagatg tccgtagtct gtttcgtaca 1260
cggtccgatc gctaccgcgc aatgttggtt atactgatgg cttggtttgg tcagtttagt 1320
gggaataatg tttgttcata ttatcttcct acaatgctgc gtaacgtagg catgaaatct 1380
gtcagtctga acgtgctgat gaatggtgtc tattctattg tgacgtggat cagctccata 1440
tgcggtgcat tttttatcga taaaataggg agaagagaag gtttcctcgg ctccatctct 1500
ggagctgccc tcgcgttaac gggactgtca atctgtactg ctagatacga gaaaacgaaa 1560
aaaaagtctg cctcaaacgg agcgctggtg ttcatatacc tgtttggtgg gattttctct 1620
tttgctttca ctccgatgca atctatgtac agtacagaag tctctaccaa tctcacccgt 1680
tcaaaggcac agttattgaa tttcgtcgtg agtggtgtcg cccaatttgt gaaccagttc 1740
gctactccaa aagctatgaa gaatatcaag tattggtttt atgtctttta cgtcttcttc 1800
gatatctttg agtttattgt gatttatttt ttcttcgtcg agactaaagg ccggagtctc 1860
gaagagcttg aggttgtatt tgaagctcca aacccgcgga aagctagtgt agaccaagcg 1920
ttccttgcac aagtgcgtgc gaccctggtt caacgtaacg acgtccgtgt cgcgaacgca 1980
caaaatttga aagaacagga gcctcttaaa tctgatgctg accatgtaga gaagctctcc 2040
gaggctgaaa gcgtataa 2058
<210> 5
<211> 4268
<212> DNA
<213> （人工序列）
<400> 5
atgtcatgac attggtgtac agaatggcga atggccatga ccaaaatccc ttaacgtgag 60
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 120
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 180
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 240
cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 300
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 360
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 420
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 480
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 540
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 600
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 660
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 720
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 780
gattctgtgg ataaccgtat taccgccttt gagtgagctg aaattatgag gggatctctc 840
agagctcgag gtcatcgttc aaaatggtat gcgttttgac acatccacta tatatccgtg 900
tcgttctgtc cactcctgaa tcccattcca gaaattctct agcgattcca gaagtttctc 960
agagtcggaa agttgaccag acattacgaa ctggcacaga tggtcataac ctgaaggaag 1020
atctgattgc ttaactgctt cagttaagac cgaagcgctc gtcgtataac agatgcgatg 1080
atgcagacca atcaacatgg cacctgccat tgctacctgc acagtcaagg atggtagaaa 1140
tgttgtcggt ccttgcacac gaatattacg ccatttgcct gcatattcaa acagctcttc 1200
tacgataagg gcacaaatcg catcgtggaa cgtttgggct tctaccgatt tagcagtttg 1260
atacactttc tctaagtatc cacctgaatc ataaatcggc aaaatagaga aaaattgacc 1320
atgtgtaagc ggccaatctg attccacctg agatgcataa tctagtagaa tctcttcgct 1380
atcaaaattc acttccacct tccactcacc ggttgtccat tcatggctga actctgcttc 1440
ctctgttgac atgacacaca tcatctcaat atccgaatag ggcccatcag tctgacgacc 1500
aagagagcca taaacaccaa tagccttaac atcatcccca tatttatcca atattcgttc 1560
cttaatttca tgaacaatct tcattctttc ttctctagtc attattattg gtccattcac 1620
tattctcatt cccctttcag ataattttag atttgctttt ctaaataaga atatttggag 1680
agcaccgttc ttattcagct attaaaccca ttatatcggg tttttgaggg gatttcaact 1740
gcagacacct aaattcaaaa tctatcggtc agatttatac cgatttgatt ttatatattc 1800
ttgaataaca tacgccgagt tatcacataa aagcgggaac caatcatcaa atttaaactt 1860
cattgcataa tccattaaac tcttaaattc tacgattcct tgttcatcaa taaactcaat 1920
catttcttta attaatttat atctatctgt tgttgttttc tttaataatt catcaacatc 1980
tacaccgcca taaactatca tatcttcttt ttgatattta aatttattag gatcgtccat 2040
gtgaagcata tatctcacaa gacctttcac acttcctgca atctgcggaa tagtcgcatt 2100
caattcttct gtaattattt ttatctgttc ataagattta ttaccctcat acatcactag 2160
aatatgataa tgctcttttt tcatcctatc ttctgtatca gtatccctat catgtaatgg 2220
agacactaca aattgaatgt gtaactcttt taaatactct aaccactcgg cttttgctga 2280
ttctggatat aaaacaaatg tccaattacg tcctcttgaa tttttcttgt tttcagtttc 2340
ttttattaca ttttcgctca tgatataata acggtgctaa tacatttaac aaaatttagt 2400
catagatagg cagcatgcca gtgctgtcta tctttttttg tttaaaatgc accgtattcc 2460
tcctttgcat atttttttat tagaataccg gttgcatctg atttgctaat attatatttt 2520
tctttgattc tatttaatat ctcattttct tctgttgtaa gtcttaaagt aacagcaact 2580
tttttctctt cttttctatc tacaaccatc actgtacctc ccaacatctg tttttttcac 2640
tttaacataa aaaacaacct tttaacatta aaaacccaat atttatttat ttgtttggac 2700
aatggacaat ggacacctag gggggaggtc gtagtacccc cctatgtttt ctcccctaaa 2760
taaccccaaa aatctaagaa aaaaagacct caaaaaggtc tttaattaac atctcaaatt 2820
tcgcatttat tccaatttcc tttttgcgtg tgatgcgtta ttaacgttga tataatttaa 2880
attttatttg acaaaaatgg gctcgtgttg tacaataaat gtagaggtag agacgcgagg 2940
tctaagaact ttaaataatt tctactgttg tagatatcgg agcgggtttt gttggaagaa 3000
tttctactgt tgtagatcgt ctctgaactg attcaagcaa gcttaaaccc agctcaatga 3060
gctgggtttt ttgtttgttt tttcaaactt agttagcttg gccagtgcct ctagagtccc 3120
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 3180
taataactga caaacatcac cctcttgcta aagcggccaa ggacgccgcc gccggggctg 3240
tttgcgttct tgccgtgatt tcgtgtacca ttggtttact tatttttttg ccaaggctgt 3300
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 3360
gcgattatgt aaaatataaa gtgatagcgg taccctcgag aaaaggagga aaaaaaatgt 3420
ttatgaaatc tactggtatt gtacgtatgg tgagcaaggg cgaggaggat aacatggcca 3480
tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 3540
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 3600
aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 3660
acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 3720
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 3780
tgacccagga ctcctccctg caggacggcg aattcatcta caaggtgaag ctgcgcggca 3840
ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 3900
ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 3960
tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 4020
tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 4080
actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 4140
acgagctgta caagtaaaga gtcgacctgt tacgaacggc agatcagaat tttgtaataa 4200
aaaaagagcc tgctcattac actgcgggct ctttttcatg gtcagaagac gggtaaccaa 4260
gataacaa 4268
<210> 6
<211> 4268
<212> DNA
<213> （人工序列）
<400> 6
atgtcatgac attggtgtac agaatggcga atggccatga ccaaaatccc ttaacgtgag 60
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 120
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 180
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 240
cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 300
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 360
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 420
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 480
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 540
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 600
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 660
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 720
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 780
gattctgtgg ataaccgtat taccgccttt gagtgagctg aaattatgag gggatctctc 840
agagctcgag gtcatcgttc aaaatggtat gcgttttgac acatccacta tatatccgtg 900
tcgttctgtc cactcctgaa tcccattcca gaaattctct agcgattcca gaagtttctc 960
agagtcggaa agttgaccag acattacgaa ctggcacaga tggtcataac ctgaaggaag 1020
atctgattgc ttaactgctt cagttaagac cgaagcgctc gtcgtataac agatgcgatg 1080
atgcagacca atcaacatgg cacctgccat tgctacctgc acagtcaagg atggtagaaa 1140
tgttgtcggt ccttgcacac gaatattacg ccatttgcct gcatattcaa acagctcttc 1200
tacgataagg gcacaaatcg catcgtggaa cgtttgggct tctaccgatt tagcagtttg 1260
atacactttc tctaagtatc cacctgaatc ataaatcggc aaaatagaga aaaattgacc 1320
atgtgtaagc ggccaatctg attccacctg agatgcataa tctagtagaa tctcttcgct 1380
atcaaaattc acttccacct tccactcacc ggttgtccat tcatggctga actctgcttc 1440
ctctgttgac atgacacaca tcatctcaat atccgaatag ggcccatcag tctgacgacc 1500
aagagagcca taaacaccaa tagccttaac atcatcccca tatttatcca atattcgttc 1560
cttaatttca tgaacaatct tcattctttc ttctctagtc attattattg gtccattcac 1620
tattctcatt cccctttcag ataattttag atttgctttt ctaaataaga atatttggag 1680
agcaccgttc ttattcagct attaaaccca ttatatcggg tttttgaggg gatttcaact 1740
gcagacacct aaattcaaaa tctatcggtc agatttatac cgatttgatt ttatatattc 1800
ttgaataaca tacgccgagt tatcacataa aagcgggaac caatcatcaa atttaaactt 1860
cattgcataa tccattaaac tcttaaattc tacgattcct tgttcatcaa taaactcaat 1920
catttcttta attaatttat atctatctgt tgttgttttc tttaataatt catcaacatc 1980
tacaccgcca taaactatca tatcttcttt ttgatattta aatttattag gatcgtccat 2040
gtgaagcata tatctcacaa gacctttcac acttcctgca atctgcggaa tagtcgcatt 2100
caattcttct gtaattattt ttatctgttc ataagattta ttaccctcat acatcactag 2160
aatatgataa tgctcttttt tcatcctatc ttctgtatca gtatccctat catgtaatgg 2220
agacactaca aattgaatgt gtaactcttt taaatactct aaccactcgg cttttgctga 2280
ttctggatat aaaacaaatg tccaattacg tcctcttgaa tttttcttgt tttcagtttc 2340
ttttattaca ttttcgctca tgatataata acggtgctaa tacatttaac aaaatttagt 2400
catagatagg cagcatgcca gtgctgtcta tctttttttg tttaaaatgc accgtattcc 2460
tcctttgcat atttttttat tagaataccg gttgcatctg atttgctaat attatatttt 2520
tctttgattc tatttaatat ctcattttct tctgttgtaa gtcttaaagt aacagcaact 2580
tttttctctt cttttctatc tacaaccatc actgtacctc ccaacatctg tttttttcac 2640
tttaacataa aaaacaacct tttaacatta aaaacccaat atttatttat ttgtttggac 2700
aatggacaat ggacacctag gggggaggtc gtagtacccc cctatgtttt ctcccctaaa 2760
taaccccaaa aatctaagaa aaaaagacct caaaaaggtc tttaattaac atctcaaatt 2820
tcgcatttat tccaatttcc tttttgcgtg tgatgcgtta ttaacgttga tataatttaa 2880
attttatttg acaaaaatgg gctcgtgttg tacaataaat gtagaggtag agacgcgagg 2940
tctaagaact ttaaataatt tctactgttg tagatgatat gcgcctgtcg ttaacggaaa 3000
tttctactgt tgtagatcgt ctctgaactg attcaagcaa gcttaaaccc agctcaatga 3060
gctgggtttt ttgtttgttt tttcaaactt agttagcttg gccagtgcct ctagagtccc 3120
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 3180
taataactga caaacatcac cctcttgcta aagcggccaa ggacgccgcc gccggggctg 3240
tttgcgttct tgccgtgatt tcgtgtacca ttggtttact tatttttttg ccaaggctgt 3300
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 3360
gcgattatgt aaaatataaa gtgatagcgg taccctcgag aaaaggagga aaaaaaatgt 3420
ttatgaaatc tactggtatt gtacgtatgg tgagcaaggg cgaggaggat aacatggcca 3480
tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 3540
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 3600
aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 3660
acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 3720
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 3780
tgacccagga ctcctccctg caggacggcg aattcatcta caaggtgaag ctgcgcggca 3840
ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 3900
ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 3960
tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 4020
tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 4080
actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 4140
acgagctgta caagtaaaga gtcgacctgt tacgaacggc agatcagaat tttgtaataa 4200
aaaaagagcc tgctcattac actgcgggct ctttttcatg gtcagaagac gggtaaccaa 4260
gataacaa 4268
<210> 7
<211> 4268
<212> DNA
<213> （人工序列）
<400> 7
atgtcatgac attggtgtac agaatggcga atggccatga ccaaaatccc ttaacgtgag 60
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 120
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 180
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 240
cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 300
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 360
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 420
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 480
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 540
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 600
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 660
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 720
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 780
gattctgtgg ataaccgtat taccgccttt gagtgagctg aaattatgag gggatctctc 840
agagctcgag gtcatcgttc aaaatggtat gcgttttgac acatccacta tatatccgtg 900
tcgttctgtc cactcctgaa tcccattcca gaaattctct agcgattcca gaagtttctc 960
agagtcggaa agttgaccag acattacgaa ctggcacaga tggtcataac ctgaaggaag 1020
atctgattgc ttaactgctt cagttaagac cgaagcgctc gtcgtataac agatgcgatg 1080
atgcagacca atcaacatgg cacctgccat tgctacctgc acagtcaagg atggtagaaa 1140
tgttgtcggt ccttgcacac gaatattacg ccatttgcct gcatattcaa acagctcttc 1200
tacgataagg gcacaaatcg catcgtggaa cgtttgggct tctaccgatt tagcagtttg 1260
atacactttc tctaagtatc cacctgaatc ataaatcggc aaaatagaga aaaattgacc 1320
atgtgtaagc ggccaatctg attccacctg agatgcataa tctagtagaa tctcttcgct 1380
atcaaaattc acttccacct tccactcacc ggttgtccat tcatggctga actctgcttc 1440
ctctgttgac atgacacaca tcatctcaat atccgaatag ggcccatcag tctgacgacc 1500
aagagagcca taaacaccaa tagccttaac atcatcccca tatttatcca atattcgttc 1560
cttaatttca tgaacaatct tcattctttc ttctctagtc attattattg gtccattcac 1620
tattctcatt cccctttcag ataattttag atttgctttt ctaaataaga atatttggag 1680
agcaccgttc ttattcagct attaaaccca ttatatcggg tttttgaggg gatttcaact 1740
gcagacacct aaattcaaaa tctatcggtc agatttatac cgatttgatt ttatatattc 1800
ttgaataaca tacgccgagt tatcacataa aagcgggaac caatcatcaa atttaaactt 1860
cattgcataa tccattaaac tcttaaattc tacgattcct tgttcatcaa taaactcaat 1920
catttcttta attaatttat atctatctgt tgttgttttc tttaataatt catcaacatc 1980
tacaccgcca taaactatca tatcttcttt ttgatattta aatttattag gatcgtccat 2040
gtgaagcata tatctcacaa gacctttcac acttcctgca atctgcggaa tagtcgcatt 2100
caattcttct gtaattattt ttatctgttc ataagattta ttaccctcat acatcactag 2160
aatatgataa tgctcttttt tcatcctatc ttctgtatca gtatccctat catgtaatgg 2220
agacactaca aattgaatgt gtaactcttt taaatactct aaccactcgg cttttgctga 2280
ttctggatat aaaacaaatg tccaattacg tcctcttgaa tttttcttgt tttcagtttc 2340
ttttattaca ttttcgctca tgatataata acggtgctaa tacatttaac aaaatttagt 2400
catagatagg cagcatgcca gtgctgtcta tctttttttg tttaaaatgc accgtattcc 2460
tcctttgcat atttttttat tagaataccg gttgcatctg atttgctaat attatatttt 2520
tctttgattc tatttaatat ctcattttct tctgttgtaa gtcttaaagt aacagcaact 2580
tttttctctt cttttctatc tacaaccatc actgtacctc ccaacatctg tttttttcac 2640
tttaacataa aaaacaacct tttaacatta aaaacccaat atttatttat ttgtttggac 2700
aatggacaat ggacacctag gggggaggtc gtagtacccc cctatgtttt ctcccctaaa 2760
taaccccaaa aatctaagaa aaaaagacct caaaaaggtc tttaattaac atctcaaatt 2820
tcgcatttat tccaatttcc tttttgcgtg tgatgcgtta ttaacgttga tataatttaa 2880
attttatttg acaaaaatgg gctcgtgttg tacaataaat gtagaggtag agacgcgagg 2940
tctaagaact ttaaataatt tctactgttg tagatcgtta acgttaatct ttacgatgaa 3000
tttctactgt tgtagatcgt ctctgaactg attcaagcaa gcttaaaccc agctcaatga 3060
gctgggtttt ttgtttgttt tttcaaactt agttagcttg gccagtgcct ctagagtccc 3120
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 3180
taataactga caaacatcac cctcttgcta aagcggccaa ggacgccgcc gccggggctg 3240
tttgcgttct tgccgtgatt tcgtgtacca ttggtttact tatttttttg ccaaggctgt 3300
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 3360
gcgattatgt aaaatataaa gtgatagcgg taccctcgag aaaaggagga aaaaaaatgt 3420
ttatgaaatc tactggtatt gtacgtatgg tgagcaaggg cgaggaggat aacatggcca 3480
tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 3540
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 3600
aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 3660
acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 3720
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 3780
tgacccagga ctcctccctg caggacggcg aattcatcta caaggtgaag ctgcgcggca 3840
ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 3900
ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 3960
tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 4020
tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 4080
actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 4140
acgagctgta caagtaaaga gtcgacctgt tacgaacggc agatcagaat tttgtaataa 4200
aaaaagagcc tgctcattac actgcgggct ctttttcatg gtcagaagac gggtaaccaa 4260
gataacaa 4268
<210> 8
<211> 4268
<212> DNA
<213> （人工序列）
<400> 8
atgtcatgac attggtgtac agaatggcga atggccatga ccaaaatccc ttaacgtgag 60
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 120
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 180
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 240
cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 300
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 360
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 420
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 480
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 540
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 600
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 660
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 720
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 780
gattctgtgg ataaccgtat taccgccttt gagtgagctg aaattatgag gggatctctc 840
agagctcgag gtcatcgttc aaaatggtat gcgttttgac acatccacta tatatccgtg 900
tcgttctgtc cactcctgaa tcccattcca gaaattctct agcgattcca gaagtttctc 960
agagtcggaa agttgaccag acattacgaa ctggcacaga tggtcataac ctgaaggaag 1020
atctgattgc ttaactgctt cagttaagac cgaagcgctc gtcgtataac agatgcgatg 1080
atgcagacca atcaacatgg cacctgccat tgctacctgc acagtcaagg atggtagaaa 1140
tgttgtcggt ccttgcacac gaatattacg ccatttgcct gcatattcaa acagctcttc 1200
tacgataagg gcacaaatcg catcgtggaa cgtttgggct tctaccgatt tagcagtttg 1260
atacactttc tctaagtatc cacctgaatc ataaatcggc aaaatagaga aaaattgacc 1320
atgtgtaagc ggccaatctg attccacctg agatgcataa tctagtagaa tctcttcgct 1380
atcaaaattc acttccacct tccactcacc ggttgtccat tcatggctga actctgcttc 1440
ctctgttgac atgacacaca tcatctcaat atccgaatag ggcccatcag tctgacgacc 1500
aagagagcca taaacaccaa tagccttaac atcatcccca tatttatcca atattcgttc 1560
cttaatttca tgaacaatct tcattctttc ttctctagtc attattattg gtccattcac 1620
tattctcatt cccctttcag ataattttag atttgctttt ctaaataaga atatttggag 1680
agcaccgttc ttattcagct attaaaccca ttatatcggg tttttgaggg gatttcaact 1740
gcagacacct aaattcaaaa tctatcggtc agatttatac cgatttgatt ttatatattc 1800
ttgaataaca tacgccgagt tatcacataa aagcgggaac caatcatcaa atttaaactt 1860
cattgcataa tccattaaac tcttaaattc tacgattcct tgttcatcaa taaactcaat 1920
catttcttta attaatttat atctatctgt tgttgttttc tttaataatt catcaacatc 1980
tacaccgcca taaactatca tatcttcttt ttgatattta aatttattag gatcgtccat 2040
gtgaagcata tatctcacaa gacctttcac acttcctgca atctgcggaa tagtcgcatt 2100
caattcttct gtaattattt ttatctgttc ataagattta ttaccctcat acatcactag 2160
aatatgataa tgctcttttt tcatcctatc ttctgtatca gtatccctat catgtaatgg 2220
agacactaca aattgaatgt gtaactcttt taaatactct aaccactcgg cttttgctga 2280
ttctggatat aaaacaaatg tccaattacg tcctcttgaa tttttcttgt tttcagtttc 2340
ttttattaca ttttcgctca tgatataata acggtgctaa tacatttaac aaaatttagt 2400
catagatagg cagcatgcca gtgctgtcta tctttttttg tttaaaatgc accgtattcc 2460
tcctttgcat atttttttat tagaataccg gttgcatctg atttgctaat attatatttt 2520
tctttgattc tatttaatat ctcattttct tctgttgtaa gtcttaaagt aacagcaact 2580
tttttctctt cttttctatc tacaaccatc actgtacctc ccaacatctg tttttttcac 2640
tttaacataa aaaacaacct tttaacatta aaaacccaat atttatttat ttgtttggac 2700
aatggacaat ggacacctag gggggaggtc gtagtacccc cctatgtttt ctcccctaaa 2760
taaccccaaa aatctaagaa aaaaagacct caaaaaggtc tttaattaac atctcaaatt 2820
tcgcatttat tccaatttcc tttttgcgtg tgatgcgtta ttaacgttga tataatttaa 2880
attttatttg acaaaaatgg gctcgtgttg tacaataaat gtagaggtag agacgcgagg 2940
tctaagaact ttaaataatt tctactgttg tagatcatca atcacggttg cataatccaa 3000
tttctactgt tgtagatcgt ctctgaactg attcaagcaa gcttaaaccc agctcaatga 3060
gctgggtttt ttgtttgttt tttcaaactt agttagcttg gccagtgcct ctagagtccc 3120
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 3180
taataactga caaacatcac cctcttgcta aagcggccaa ggacgccgcc gccggggctg 3240
tttgcgttct tgccgtgatt tcgtgtacca ttggtttact tatttttttg ccaaggctgt 3300
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 3360
gcgattatgt aaaatataaa gtgatagcgg taccctcgag aaaaggagga aaaaaaatgt 3420
ttatgaaatc tactggtatt gtacgtatgg tgagcaaggg cgaggaggat aacatggcca 3480
tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 3540
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 3600
aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 3660
acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 3720
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 3780
tgacccagga ctcctccctg caggacggcg aattcatcta caaggtgaag ctgcgcggca 3840
ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 3900
ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 3960
tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 4020
tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 4080
actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 4140
acgagctgta caagtaaaga gtcgacctgt tacgaacggc agatcagaat tttgtaataa 4200
aaaaagagcc tgctcattac actgcgggct ctttttcatg gtcagaagac gggtaaccaa 4260
gataacaa 4268
<210> 9
<211> 14001
<212> DNA
<213> （人工序列）
<400> 9
ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa 60
tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120
gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180
aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240
tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300
ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360
gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420
ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480
tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540
cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600
aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660
tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720
cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780
tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840
ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900
ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa 960
aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc 1020
ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac 1080
tatggcgtgc tgctagccaa gcaagcttaa acccagctca atgagctggg ttttttgttt 1140
gttgcagcaa tggcaagaac gtcccgggcg gagctcaggc cttaactcac attaattgcg 1200
ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 1260
ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac 1320
cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa 1380
gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg 1440
gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac 1500
gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac 1560
cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga 1620
catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata 1680
tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag 1740
cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc 1800
atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg 1860
aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat 1920
gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgttttac aggcttcgac 1980
gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt 2040
aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat 2100
cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc 2160
cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac 2220
cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac 2280
tggtttcatc aaaatcgtct ccctccgttt gaatatttga ttgatcgtaa ccagatgaag 2340
cactctttcc actatcccta cagtgttatg gcttgaacaa tcacgaaaca ataattggta 2400
cgtacgatct ttcagccgac tcaaacatca aatcttacaa atgtagtctt tgaaagtatt 2460
acatatgtaa gatttaaatg caaccgtttt ttcggaagga aatgatgacc tcgtttccac 2520
cggaattagc ttggtaccag ctattgtaac ataatcggta cgggggtgaa aaagctaacg 2580
gaaaagggag cgaatggcaa gaacgtcccg gggagctcct aacttatagg ggtaacactt 2640
aaaaaagaat caataacgat agaaaccgct cctaaagcag gtgcattttt tcctaacgaa 2700
gaaggcaata gttcacattt attgtctaaa tgagaatgga ctctagaaga aacttcgttt 2760
ttaatcgtat ttaaaacaat gggatgagat tcaattatat gatttctcaa gataacagct 2820
tctatatcaa atgtattaag gatattggtt aatccaattc cgatataaaa gccaaagttt 2880
tgaagtgcat ttaacatttc tacatcattt ttatttgcgc gttccacaat ctcttttcga 2940
gaaatattct tttcttcttt agagagcgaa gccagtaacg ctttttcaga agcatataat 3000
tcccaacagc ctcgatttcc acagctgcat ttgggtccat taaaatctat cgtcatatga 3060
cccatttccc cagaaaaacc ctgaacacct ttatacaatt cgttgttaat aacaagtcca 3120
gttccaattc cgatattaat actgatgtaa acgatgtttt catagttttt tgtcatacca 3180
aatacttttt caccgtatgc tcctgcatta gcttcatttt caacaaaaac cggaacatta 3240
aactcactct caattaaaaa ctgcaaatct ttgatattcc aatttaagtt aggcatgaaa 3300
ataatttgct gatgacgatc tacaaggcct ggaacacaaa ttcctattcc gactagacca 3360
taaggggact caggcatatg ggttacaaaa ccatgaataa gtgcaaataa aatctctttt 3420
acttcactag cggaagaact agacaagtca gaagtcttct cgagaataat atttccttct 3480
aagtcggtta gaattccgtt aagatagtcg actcctatat caataccaat cgagtagcct 3540
gcattcttat taaaaacaag cattacaggt cttctgccgc ctctagattg ccctgcccca 3600
atttcaaaaa taaaatcttt ttcaagcagt gtatttactt gagaggagac agtagacttg 3660
tttaatcctg taatctcaga gagagttgcc ctggagacag gggagttctt caaaatttca 3720
tctaatatta atttttgatt catttttttt actaaagctt gatctgcaat ttgaataata 3780
accactcctt tgtttatcca ccgaactaag ttggtgtttt ttgaagcttg aattagatat 3840
ttaaaagtat catatctaat attataacta aattttctaa aaaaaacatt gaaataaaca 3900
tttattttgt atatgatgag ataaagttag tttattggat aaacaaacta actcaattaa 3960
gatagttgat ggataaactt gttcacttaa atcaaagggg gaaatgacaa atggtccaaa 4020
ctagtgatat ctaaaaatca aagggggaaa tgggatccat gtcaatttat caagaatttg 4080
ttaataaata tagtttaagt aaaactctaa gatttgagtt aatcccacag ggtaaaacac 4140
ttgaaaacat aaaagcaaga ggtttgattt tagatgatga gaaaagagct aaagactaca 4200
aaaaggctaa acaaataatt gataaatatc atcagttttt tatagaggag atattaagtt 4260
cggtttgtat tagcgaagat ttattacaaa actattctga tgtttatttt aaacttaaaa 4320
agagtgatga tgataatcta caaaaagatt ttaaaagtgc aaaagatacg ataaagaaac 4380
aaatatctga atatataaag gactcagaga aatttaagaa tttgtttaat caaaacctta 4440
tcgatgctaa aaaagggcaa gagtcagatt taattctatg gctaaagcaa tctaaggata 4500
atggtataga actatttaaa gccaatagtg atatcacaga tatagatgag gcgttagaaa 4560
taatcaaatc ttttaaaggt tggacaactt attttaaggg ttttcatgaa aatagaaaaa 4620
atgtttatag tagcaatgat attcctacat ctattattta taggatagta gatgataatt 4680
tgcctaaatt tctagaaaat aaagctaagt atgagagttt aaaagacaaa gctccagaag 4740
ctataaacta tgaacaaatt aaaaaagatt tggcagaaga gctaaccttt gatattgact 4800
acaaaacatc tgaagttaat caaagagttt tttcacttga tgaagttttt gagatagcaa 4860
actttaataa ttatctaaat caaagtggta ttactaaatt taatactatt attggtggta 4920
aatttgtaaa tggtgaaaat acaaagagaa aaggtataaa tgaatatata aatctatact 4980
cacagcaaat aaatgataaa acactcaaaa aatataaaat gagtgtttta tttaagcaaa 5040
ttttaagtga tacagaatct aaatcttttg taattgataa gttagaagat gatagtgatg 5100
tagttacaac gatgcaaagt ttttatgagc aaatagcagc ttttaaaaca gtagaagaaa 5160
aatctattaa agaaacacta tctttattat ttgatgattt aaaagctcaa aaacttgatt 5220
tgagtaaaat ttattttaaa aatgataaat ctcttactga tctatcacaa caagtttttg 5280
atgattatag tgttattggt acagcggtac tagaatatat aactcaacaa atagcaccta 5340
aaaatcttga taaccctagt aagaaagagc aagaattaat agccaaaaaa actgaaaaag 5400
caaaatactt atctctagaa actataaagc ttgccttaga agaatttaat aagcatagag 5460
atatagataa acagtgtagg tttgaagaaa tacttgcaaa ctttgcggct attccgatga 5520
tatttgatga aatagctcaa aacaaagaca atttggcaca gatatctatc aaatatcaaa 5580
atcaaggtaa aaaagaccta cttcaagcta gtgcggaaga tgatgttaaa gctatcaagg 5640
atcttttaga tcaaactaat aatctcttac ataaactaaa aatatttcat attagtcagt 5700
cagaagataa ggcaaatatt ttagacaagg atgagcattt ttatctagta tttgaggagt 5760
gctactttga gctagcgaat atagtgcctc tttataacaa aattagaaac tatataactc 5820
aaaagccata tagtgatgag aaatttaagc tcaattttga gaactcgact ttggctaatg 5880
gttgggataa aaataaagag cctgacaata cggcaatttt atttatcaaa gatgataaat 5940
attatctggg tgtgatgaat aagaaaaata acaaaatatt tgatgataaa gctatcaaag 6000
aaaataaagg cgagggttat aaaaaaattg tttataaact tttacctggc gcaaataaaa 6060
tgttacctaa ggttttcttt tctgctaaat ctataaaatt ttataatcct agtgaagata 6120
tacttagaat aagaaatcat tccacacata caaaaaatgg tagtcctcaa aaaggatatg 6180
aaaaatttga gtttaatatt gaagattgcc gaaaatttat agatttttat aaacagtcta 6240
taagtaagca tccggagtgg aaagattttg gatttagatt ttctgatact caaagatata 6300
attctataga tgaattttat agagaagttg aaaatcaagg ctacaaacta acttttgaaa 6360
atatatcaga gagctatatt gatagcgtag ttaatcaggg taaattgtac ctattccaaa 6420
tctataataa agatttttca gcttatagca aagggcgacc aaatctacat actttatatt 6480
ggaaagcgct gtttgatgag agaaatcttc aagatgtggt ttataagcta aatggtgagg 6540
cagagctttt ttatcgtaaa caatcaatac ctaaaaaaat cactcaccca gctaaagagg 6600
caatagctaa taaaaacaaa gataatccta aaaaagagag tgtttttgaa tatgatttaa 6660
tcaaagataa acgctttact gaagataagt ttttctttca ctgtcctatt acaatcaatt 6720
ttaaatctag tggagctaat aagtttaatg atgaaatcaa tttattgcta aaagaaaaag 6780
caaatgatgt tcatatatta agtatagata gaggtgaaag acatttagct tactatactt 6840
tggtagatgg taaaggcaat atcatcaaac aagatacttt caacatcatt ggtaatgata 6900
gaatgaaaac aaactaccat gataagcttg ctgcaataga gaaagatagg gattcagcta 6960
ggaaagactg gaaaaagata aataacatca aagagatgaa agagggctat ctatctcagg 7020
tagttcatga aatagctaag ctagttatag agtataatgc tattgtggtt tttgaggatt 7080
taaattttgg atttaaaaga gggcgtttca aggtagagaa gcaggtctat caaaagttag 7140
aaaaaatgct aattgagaaa ctaaactatc tagttttcaa agataatgag tttgataaaa 7200
ctgggggagt gcttagagct tatcagctaa cagcaccttt tgagactttt aaaaagatgg 7260
gtaaacaaac aggtattatc tactatgtac cagctggttt tacttcaaaa atttgtcctg 7320
taactggttt tgtaaatcag ttatatccta agtatgaaag tgtcagcaaa tctcaagagt 7380
tctttagtaa gtttgacaag atttgttata accttgataa gggctatttt gagtttagtt 7440
ttgattataa aaactttggt gacaaggctg ccaaaggcaa gtggactata gctagctttg 7500
ggagtagatt gattaacttt agaaattcag ataaaaatca taattgggat actcgagaag 7560
tttatccaac taaagagttg gagaaattgc taaaagatta ttctatcgaa tatgggcatg 7620
gcgaatgtat caaagcagct atttgcggtg agagcgacaa aaagtttttt gctaagctaa 7680
ctagtgtcct aaatactatc ttacaaatgc gtaactcaaa aacaggtact gagttagatt 7740
atctaatttc accagtagca gatgtaaatg gcaatttctt tgattcgcga caggcgccaa 7800
aaaatatgcc tcaagatgct gatgccaatg gtgcttatca tattgggcta aaaggtctga 7860
tgctactagg taggatcaaa aataatcaag agggcaaaaa actcaatttg gttatcaaaa 7920
atgaagagta ttttgagttc gtgcagaata ggaataacta actgcagaac gctcggttgc 7980
cgccgggcgt tttttcgtca tcgcggaaaa gaatgatgta agcgtgaaaa attttttaaa 8040
aaatctcttg acattggaag ggagatatgt tattataaga attgcggaat tgtgagcgga 8100
taacaattcc caattaaagg aggtgggatc catgcatgcg atgcctgggg acgcagatat 8160
gttcattggg attgcggtct ctcggagcta ccccgaggac ggtgccagcg gccagataaa 8220
cattgccgcg acggcgaccg ccgtctacaa ggatggaact atcctcggcc actcgtccac 8280
ccgaccgcag ctcggggaga aactacagtc gacggatgtt cgtgacatta tgaagaatgc 8340
catcctcggc taccagcagg tgaccggtga gtcgccgacc catatcgtca tccaccgtgc 8400
gggcttcatg aacgaagacc tcgaccccgc cacggaattc ctcaacgaac aaggcgtcga 8460
gtacgacatc gtcgaaatcc gcaagcagcc ccagacacgc ctgctggcag tctccgatgt 8520
gcagtacgat acgcctgtga agagcatcgc cgctatcaac cagaacgagc cacgggcaac 8580
ggtcgccacc ttcggcgcac ccgaatactt agcgacacgc gatggaggcg gccttccccg 8640
cccaatccaa attgaacgag tcgccggcga aaccgacatc gagacgctca ctcgccaagt 8700
ctatctgctc tcccagtcgc atatccaggt ccataactcg actgcgcgcc tacccatcac 8760
caccgcatac gccgaccagg caagtactca cgcgaccaag ggttacctcg tccagaccgg 8820
agcgttcgag tctaatgtcg gattcctcta atcctctaga gtcgacgtcc ccggggcagc 8880
ccgcctaatg agcgggcttt tttcacgtca cgcgtccatg gagatctttg tctgcaactg 8940
aaaagtttat accttacctg gaacaaatgg ttgaaacata cgaggctaat atcggcttat 9000
taggaatagt ccctgtacta ataaaatcag gtggatcagt tgatcagtat attttggacg 9060
aagctcggaa agaatttgga gatgacttgc ttaattccac aattaaatta agggaaagaa 9120
taaagcgatt tgatgttcaa ggaatcacgg aagaagatac tcatgataaa gaagctctaa 9180
aactattcaa taaccttaca atggaattga tcgaaagggt ggaaggttaa tggtacgaaa 9240
attaggggat ctacctagaa agccacaagg cgataggtca agcttaaaga acccttacat 9300
ggatcttaca gattctgaaa gtaaagaaac aacagaggtt aaacaaacag aaccaaaaag 9360
aaaaaaagca ttgttgaaaa caatgaaagt tgatgtttca atccataata agattaaatc 9420
gctgcacgaa attctggcag catccgaagg gaattcatat tacttagagg atactattga 9480
gagagctatt gataagatgg ttgagacatt acctgagagc caaaaaactt tttatgaata 9540
tgaattaaaa aaaagaacca acaaaggctg agacagactc caaacgagtc tgttttttta 9600
aaaaaaatat taggagcatt gaatatatat tagagaatta agaaagacat gggaataaaa 9660
atattttaaa tccagtaaaa atatgataag attatttcag aatatgaaga actctgtttg 9720
tttttgatga aaaaacaaac aaaaaaaatc cacctaacgg aatctcaatt taactaacag 9780
cggccaaact gagaagttaa atttgagaag gggaaaaggc ggatttatac ttgtatttaa 9840
ctatctccat tttaacattt tattaaaccc catacaagtg aaaatcctct tttacactgt 9900
tcctttaggt gatcgcggag ggacattatg agtgaagtaa acctaaaagg aaatacagat 9960
gaattagtgt attatcgaca gcaaaccact ggaaataaaa tcgccaggaa gagaatcaaa 10020
aaagggaaag aagaagttta ttatgttgct gaaacggaag agaagatatg gacagaagag 10080
caaataaaaa acttttcttt agacaaattt ggtacgcata taccttacat agaaggtcat 10140
tatacaatct taaataatta cttctttgat ttttggggct attttttagg tgctgaagga 10200
attgcgctct atgctcacct aactcgttat gcatacggca gcaaagactt ttgctttcct 10260
agtctacaaa caatcgctaa aaaaatggac aagactcctg ttacagttag aggctacttg 10320
aaactgcttg aaaggtacgg ttttatttgg aaggtaaacg tccgtaataa aaccaaggat 10380
aacacagagg aatccccgat ttttaagatt agacgtaagg ttcctttgct ttcagaagaa 10440
cttttaaatg gaaaccctaa tattgaaatt ccagatgacg aggaagcaca tgtaaagaag 10500
gctttaaaaa aggaaaaaga gggtcttcca aaggttttga aaaaagagca cgatgaattt 10560
gttaaaaaaa tgatggatga gtcagaaaca attaatattc cagaggcctt acaatatgac 10620
acaatgtatg aagatatact cagtaaagga gaaattcgaa aagaaatcaa aaaacaaata 10680
cctaatccta caacatcttt tgagagtata tcaatgacaa ctgaagagga aaaagtcgac 10740
agtactttaa aaagcgaaat gcaaaatcgt gtctctaagc cttcttttga tacctggttt 10800
aaaaacacta agatcaaaat tgaaaataaa aattgtttat tacttgtacc gagtgaattt 10860
gcatttgaat ggattaagaa aagatattta gaaacaatta aaacagtcct tgaagaagct 10920
ggatatgttt tcgaaaaaat cgaactaaga aaagtgcaat aaactgctga agtatttcag 10980
cagttttttt tatttagaaa tagtgaaaaa aatataatca gggaggtatc aatatttaat 11040
gagtactgat ttaaatttat ttagactgga attaataatt aacacgtaga ctaattaaaa 11100
tttaatgagg gataaagagg atacaaaaat attaatttca atccctatta aattttaaca 11160
agggggggat taaaatttaa ttagaggttt atccacaaga aaagacccta ataaaatttt 11220
tactagggtt ataacactga ttaatttctt aatgggggag ggattaaaat ttaatgacaa 11280
agaaaacaat cttttaagaa aagcttttaa aagataataa taaaaagagc tttgcgatta 11340
agcaaaactc tttacttttt cattgacatt atcaaattca tcgatttcaa attgttgttg 11400
tatcataaag ttaattctgt tttgcacaac cttttcagga atataaaaca catctgaggc 11460
ttgttttata aactcagggt cgctaaagtc aatgtaacgt agcatatgat atggtatagc 11520
ttccacccaa gttagccttt ctgcttcttc tgaatgtttt tcatatactt ccatgggtat 11580
ctctaaatga ttttcctcat gtagcaaggt atgagcaaaa agtttatgga attgatagtt 11640
cctctctttt tcttcaactt ttttatctaa aacaaacact ttaacatctg agtcaatgta 11700
agcataagat gtttttccag tcataatttc aatcccaaat cttttagaca gaaattctgg 11760
acgtaaatct tttggtgaaa gaattttttt atgtagcaat atatccgata cagcaccttc 11820
taaaagcgtt ggtgaatagg gcattttacc tatctcctct cattttgtgg aataaaaata 11880
gtcatattcg tccatctacc tatcctatta tcgaacagtt gaacttttta atcaaggatc 11940
agtccttttt ttcattattc ttaaactgtg ctcttaactt taacaactcg atttgttttt 12000
ccagatctcg agggtaacta gcctcgccga tcccgcaaga ggcccggcag tcaggtggca 12060
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 12120
tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 12180
gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 12240
ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 12300
cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 12360
ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 12420
cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 12480
tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 12540
tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 12600
tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 12660
ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 12720
tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 12780
cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 12840
gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 12900
ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 12960
acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 13020
cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 13080
atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 13140
tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 13200
tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 13260
aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 13320
aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 13380
taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 13440
taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 13500
agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 13560
tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 13620
cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 13680
agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 13740
gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 13800
aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 13860
tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 13920
ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 13980
aagagcgccc aatacgcatg c 14001

Claims (3)

1.一种提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌，其特征在于，所述枯草芽孢杆菌是以枯草芽孢杆菌168为出发菌株，构建基因片段Psrfa-fkp，采用组成型启动子Psrfa在基因组上表达外源基因双功能酶L-岩藻糖激酶fkp；构建基因片段P43-futC、P43 -glcp及P43-lac12，采用组成型启动子P43在基因组上分别表达外源基因α-1，2-岩藻糖基转移酶futC、乳糖转运酶lac12及内源性基因单糖转运蛋白酶glcp；所述Psrfa-fkp、P43-futC、P43 -glcp及P43-lac12的基因序列如SEQ ID NO .1~SEQ ID NO .4所示。
2.一种如权利要求1所述的提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌的构建方法，其特征在于，所述构建方法包括以下步骤：
（1）以枯草芽孢杆菌168为出发菌株，利用组成型启动子Psrfa在基因组上过表达来源于脆弱拟杆菌Bacteriodes fragilis 9343的双功能酶L-岩藻糖激酶fkp，构建枯草芽孢杆菌WCY1；
（2）以枯草芽孢杆菌WCY1为出发菌株，利用组成型启动子P43在基因组上过表达来源于幽门螺杆菌H. pylori的α-1，2-岩藻糖基转移酶FutC，构建枯草芽孢杆菌WCY2；
（3）以枯草芽孢杆菌WCY2为出发菌株，利用组成型启动子P43在基因组上过表达内源性基因单糖转运蛋白酶glcp，构建枯草芽孢杆菌WCY3；
（4）以枯草芽孢杆菌WCY2为出发菌株，选择RBS序列GCTTAAAAGGAGGTTTTTTTT，并利用组成型启动子P43在基因组上过表达内源性基因单糖转运蛋白酶glcp，构建枯草芽孢杆菌WCY4；
（5）以枯草芽孢杆菌WCY4为出发菌株，选择RBS序列GAATAAAAGGAGAGTTTTTTTT，并利用组成型启动子P43在基因组上过表达乳糖转运酶lac12，构建枯草芽孢杆菌WCY5；
（6）以枯草芽孢杆菌WCY5为出发菌株，选择RBS序列GTCTTATTAAAGGAGGTTTTTTTT替换WCY5菌株双功能酶L-岩藻糖激酶fkp基因原始RBS，构建枯草芽孢杆菌WCY6；
（7）以枯草芽孢杆菌WCY6为出发菌株，选择RBS序列GGGGAGAAGACTCCGCTCGAAAGGAGGTTTTTATT替换WCY6菌株α-1，2-岩藻糖基转移酶 futC基因原始RBS，构建枯草芽孢杆菌WCY7，即得所述枯草芽孢杆菌。
3.一种如权利要求1所述的提高2’-岩藻糖基乳糖产量的枯草芽孢杆菌在合成2 '-岩藻糖基乳糖中的应用。
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