CN109735480A - 一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用 - Google Patents
一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种合成乳酰‑N‑新四糖产量的重组枯草芽孢杆菌,所述重组枯草芽孢杆菌是通过在宿主菌Bacillus subtilis 168 △amyE:P43‑lacY,P43‑lgtB,PxylA‑comK的基因组上整合两个β‑1,4‑半乳糖转移酶基因,并外源表达β‑1,3‑N‑葡糖氨基转移酶基因得到的。本发明的重组枯草芽孢杆菌与改造前菌株相比,其合成乳酰‑N‑新四糖的产量从720mg/L提高到1300mg/L,为进一步代谢工程改造枯草芽孢杆菌生产乳酰‑N‑新四糖奠定了基础。
Description
技术领域
本发明属于遗传工程的技术领域,具体涉及一种合成乳酰-N-新四糖的重组枯草芽孢杆菌及其构建方法与应用。
背景技术
母乳是婴儿天然和理想的食品。母乳中,除了婴儿成长和发育所需的营养素,它还含有数千种具有生物活性的物质。人乳低寡糖(Human Milk Oligosaccharides HMOs)在母乳中的含量大约在5-15g/L,是母乳中最重要的双歧因子。乳酰-N-新四糖(LNnT)是其中重要的组成成分,同时也是其他复杂母乳寡糖的核心结构元件,例如岩藻糖基化,唾液酸化的寡糖。由于乳酰-N-新四糖及其衍生物具有促进细胞分化、提高肠道上皮细胞免疫调节功能、增加肠道益生菌的相对丰度等重要生理功能,因此,合成乳酰-N-新四糖具有重要的意义。采用生物合成技术中的代谢工程的手段在枯草芽孢杆菌中构建高效合成***,不仅可以通过工业化大量生产LNnT以满足需求量,同时构建了食品级安全菌株枯草芽孢杆菌产母乳寡糖的底盘细胞,为后期在枯草芽孢杆菌中合成其他母乳寡糖提供支持。
枯草芽孢杆菌(Bacillus subtilis)是GRAS(generally regard as safe)菌株,遗传背景清晰,易于进行异源基因表达,培养周期短和不产生内毒素。因此,运用代谢工程手段构建枯草芽孢杆菌工程菌是生产乳酰-N-新四糖的有效策略。但是由于合成途径中存在关键酶的活力不足,限制最终产量的合成。如何提高关键酶的催化活力,解除限制因素,是亟需解决的问题。
发明内容
为了解决上述技术问题,本发明提供了一种合成乳酰-N-新四糖的重组枯草芽孢杆菌及其构建方法与应用。
具体的,第一方面,本发明提供了一种合成乳酰-N-新四糖产量的重组枯草芽孢杆菌。所述重组枯草芽孢杆菌是通过在宿主菌Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK的基因组上整合两个β-1,4-半乳糖转移酶基因,并外源表达β-1,3-N-葡糖氨基转移酶基因得到的。
本发明通过在基因组中增加两个β-1,4-半乳糖转移酶基因的拷贝数,增加β-1,4-半乳糖转移酶的表达量,从而提高酶活,加快前体的转化,使乳酰-N-新四糖的产量明显提高。
需要进一步说明的,所述宿主菌是以枯草芽孢杆菌168为出发菌株,通过以启动子PxylA调控基因comK的表达,乳糖透性酶编码基因整合到基因组amyE位点,β-1,4-半乳糖转移酶编码基因整合到基因组ydeS位点和ydzO位点中间得到的。
第二方面,本发明还提供了所述的合成乳酰-N-新四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)通过融合PCR构建包含ydaH,ydzA基因同源臂、β-1,4-半乳糖转移酶基因、P43启动子、博来霉素抗性基因序列的重组片段;
(2)通过融合PCR构建包含yszA,ysxE基因同源臂、β-1,4-半乳糖转移酶基因、P43启动子、氯霉素抗性基因序列的重组片段;
(3)将步骤(1)构建的重组片段转化Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK,通过验证获得重组枯草芽孢杆菌BY03a;
(4)将步骤(2)构建的重组片段转化步骤(3)的重组枯草芽孢杆菌BY03a,通过验证获得重组枯草芽孢杆菌BY03b;
(5)将pP43NMK-lgtA转化重组枯草芽孢杆菌BY03b,得到合成乳酰-N-新四糖的重组枯草芽孢杆菌BY05。
本发明构建方法是在基因组中增加两个β-1,4-半乳糖转移酶基因的拷贝数,所述增加第一个拷贝数的β-1,4-半乳糖转移酶编码基因lgtB整合在ydaH位点和ydzA位点中间。所述增加第二个拷贝数的β-1,4-半乳糖转移酶编码基因lgtB整合在yszA位点和ysxE位点中间。
进一步的,步骤(1)中,使用的是ydaH基因左侧同源臂,ydzA基因右侧同源臂。
进一步的,步骤(2)中,使用的是yszA基因左侧同源臂,ysxE基因右侧同源臂。
进一步的,步骤(1)构建的重组片段序列如SEQ ID NO.47所示,步骤(2)构建的重组片段序列如SEQ ID NO.58所示。
进一步的,步骤(3)中,步骤(1)构建的重组片段中的β-1,4-半乳糖转移酶基因、P43启动子、博来霉素抗性基因序列通过同源重组整合到基因组ydaH位点和ydzA位点中间。
进一步的,步骤(4)中,步骤(2)构建的重组片段中β-1,4-半乳糖转移酶基因、P43启动子、氯霉素抗性基因序列通过同源重组整合到基因组yszA位点和ysxE位点中间。
第三方面,本发明还提供了所述重组枯草芽孢杆菌在发酵生产乳酰-N-新四糖中的应用。
本发明的有益效果为:
本发明通过增加两个拷贝数的β-1,4-半乳糖转移酶基因,增加β-1,4-半乳糖转移酶的表达量,提高酶活,加快前体的转化,使乳酰-N-新四糖的产量明显提高。本发明的重组枯草芽孢杆菌合成乳酰-N-新四糖在胞外积累量达到1300mg/L,比改造前菌株提高了80%,为进一步代谢工程改造枯草芽孢杆菌生产乳酰-N-新四糖奠定了基础。本发明提供的发酵法合成乳酰-N-新四糖操作简便,便于工业化,具有很好的应用前景。
附图说明
图1为本发明重组BY03a菌株的PCR琼脂糖凝胶电泳图;
图2为本发明重组BY03b菌株的PCR琼脂糖凝胶电泳图;
图3为本发明增加lgtB基因拷贝数对乳酰-N-新四糖产量的影响。
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。
实施例1重组PZL(P43-lacY)片段的构建
以枯草芽孢杆菌(Bacillus subtilis 168)基因组为模板,根据NCBI上公布的amyE基因(Gene ID:938356),设计两侧同源臂引物,序列分别为SEQ ID NO:1和SEQ ID NO:2的左侧同源臂引物:
amyE-1F:5’-TATTCCGTATGTCAAGTGGCTGCGGTTTAT-3’(SEQ ID NO:1),
amyE-1R:5’-AATTGTTATCCGCTCTCTTGACACTCCTTATTTGATTTTTTGAAGACTTACTTCGG-3’(SEQ ID NO:2),
序列分别为SEQ ID NO:3和SEQ ID NO:4的右侧同源臂引物:
amyE-2F:5’-CTTAAGGGCAAGGCTAGACGGGACTTA-3’(SEQ ID NO:3),
amyE-2R:5’-GGCACACCGATGTACACGTCATC-3’(SEQ ID NO:4),
使用上述引物从枯草芽孢杆菌基因组中扩增amyE两侧同源臂基因序列;以质粒pP43NMK为模板,分别设计序列为SEQ ID NO:5和SEQ ID NO:6的引物:
P43-F:5’-CATTATACGAACGGTAAATCTGATAGGTGGTATGTTTTCGCTTGAACTTTTAAATACAG-3’(SEQ ID NO:5),
P43-R:5’-AGTTTGTGTTTTTTAAATAGTACATGTGTACATTCCTCTCTTACCTATAATGGTACCGC-3’(SEQ ID NO:6),
使用上述引物,获得P43的扩增片段。以P7Z6质粒为模板,设计序列分别为SEQ IDNO:7和SEQ ID NO:8的博来霉素抗性基因扩增引物:
zeo-F:5’-TCAAATAAGGAGTGTCAAGAGAGCGGATAACAATTTCACACAGGAAACAG-3’(SEQ IDNO:7),
zeo-R:5’-AACATACCACCTATCAGATTTACCGTTCGTATAATGTATGCTATACGAAGTTATTCAGT-3’(SEQ ID NO:8),
使用上述引物,获得博来霉素抗性基因的扩增片段。将P43的扩增片段和博来霉素抗性基因扩增片段跑胶验证正确后进行柱回收。将P43的扩增片段和博来霉素抗性基因扩增片段通过融合PCR技术进行融合。第一轮PCR条件为:等摩尔量柱回收的P43和博来霉素抗性DNA,总量大于1000ng,primer star酶量25μL,加ddH2O定容50μL,PCR条件55℃,11个循环。第二轮PCR条件为:上述PCR产物为模板,zeo-F,P43-R分别为上下游引物,按照常规PCR设置条件,获得ZP43片段。
以大肠杆菌(Escherichia coli K-12)基因组为模板,根据NCBI上公布的乳糖透性酶基因lacY(Gene ID:949083),设计序列分别为SEQ ID NO:9和SEQ ID NO:10的引物:
lacY-F:5’-GTAAGAGAGGAATGTACACATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGTT-3’(SEQ ID NO:9),
lacY-R:5’-TAAGTCCCGTCTAGCCTTGCCCTTAAGCGACTTCATTCACCTGACGACG-3’(SEQ IDNO:10),
使用上述引物扩增乳糖透性酶基因片段。将amyE基因左侧同源臂、amyE基因右侧同源臂、ZP43片段、乳糖透性酶基因片段跑胶验证正确后进行柱回收。通过融合PCR技术将amyE基因左侧同源臂、amyE基因右侧同源臂、ZP43片段、乳糖透性酶基因片段进行融合。第一轮PCR条件为:等摩尔量柱回收的DNA,总量大于1000ng,primer star酶量25μL,加ddH2O定容50μL,PCR条件55℃,11个循环。第二轮PCR条件为:上述PCR产物为模板,以SEQ ID NO:11和SEQ ID NO:12所示的序列:
zong-F:5’-CATGTAAGCCATAAGCCATTCGTAA-3’(SEQ ID NO:11),
zong-R:5’-AGCAAGACTCATCGCAACCC-3’(SEQ ID NO:12)分别为上下游引物,按照常规PCR设置条件,获得PZL片段。重组片段PZL的序列如SEQ ID NO:13所示。
实施例2重组PxylA-comk片段的构建
以枯草芽孢杆菌(Bacillus subtilis 168)基因组为模板,设计两侧同源臂引物,序列分别为SEQ ID NO:14和SEQ ID NO:15的左侧同源臂引物:
yhzC-F:5’-CATACATAGGAAGCAGGCATTGTTCATAAC-3’(SEQ ID NO:14),
yhzC-R:5’-atacgggatcaaatccgatgaaagagaaaaaatcgtacactgagctc-3’(SEQ IDNO:15),
序列分别为SEQ ID NO:16和SEQ ID NO:17的右侧同源臂引物:
comK-F:5’-aagggggaaatgggatccatgagtcagaaaacagacgcacct-3’(SEQ ID NO:16),
comK-R:5’-ACTACCTCAGTTGAAGGCTATAATCCAAG-3’(SEQ ID NO:17),
使用上述引物从枯草芽孢杆菌基因组中两侧同源臂基因序列;以质粒pLCx-dcas9为模板,分别设计序列为SEQ ID NO:18和SEQ ID NO:19的引物:
PxylA-F:5’-tttctctttcatcggatttgatcccgtataccgttcgtatagcatacattat-3’(SEQID NO:18),
PxylA-R:5’-gactcatggatcccatttccccctttgatttttagatatcac-3’(SEQ ID NO:19),
使用上述引物,获得带有氯霉素抗性的PxylA启动子的扩增片段。通过融合PCR,将重组同源臂以及带抗性基因的启动子融合,第一轮PCR条件为:等摩尔量柱回收的DNA,总量大于1000ng,primer star酶量25μL,加ddH2O定容50μL,PCR条件55℃,11个循环。第二轮PCR条件为:上述PCR产物为模板,以SEQ ID NO:20和SEQ ID NO:21所示的序列:
zong-F:5’-gacccgcgtccggtttttgataagccgaagaaag-3’(SEQ ID NO:20),
zong-R:5’-agtaaattcagttgcgcccggaccaatttggacac-3’(SEQ ID NO:21),
分别为上下游引物,按照常规PCR设置条件,获得PxylA-comk片段,片段PxylA-comk的序列如SEQ ID NO:22所示。
实施例3 p7S6P43-lgtB片段的构建
以枯草芽孢杆菌(Bacillus subtilis 168)基因组为模板,设计两侧同源臂引物,序列分别为SEQ ID NO:23和SEQ ID NO:24的左侧同源臂引物:
ydeS-F:5’-gggacaaggaatagtaagccggcaa-3’(SEQ ID NO:23),
ydeS-R:5’-tcctgtgtgaaattgttatccgctcctacatactctctgtagcagaggtagcttga’(SEQ ID NO:24),
序列分别为SEQ ID NO:25和SEQ ID NO:26的右侧同源臂引物:
ydzO-F:5’-tgaagcccgcctaatgagcgggcttttttctgataagaactgcaaaagctgcggatt at-3’(SEQ ID NO:25),
ydzO-R:5’-ccaccctatagataaatttttcggctgccatat-3’(SEQ ID NO:26),
使用上述引物从枯草芽孢杆菌基因组中扩增两侧同源臂基因序列。
以质粒p7S6P43为模板,分别设计序列为SEQ ID NO:27和SEQ ID NO:28的引物:
p7S6P43-F:5’-agagtatgtaggagcggataacaatttcacacagga-3’(SEQ ID NO:27),
p7S6P43-R:5’-AAAGAAATGACATGATTTTGCATgtgtacattcctctcttacctataatggtacc gc-3’(SEQ ID NO:28),
以重组质粒pP43NMK-lgtA-lgtB为模板(在专利CN108410787A中已经有公开),分别设计序列为SEQ ID NO:29和SEQ ID NO:30的引物:
lgtB-F:5’-aggtaagagaggaatgtacacATGCAAAATCATGTCATTTCTTTAGCATCAGCAG-3’(SEQ ID NO:29),
lgtB-R:5’-cccgctcattaggcgggcttcatcaTTACTGAAACGGAACGATAAACTGTTCGCG-3’(SEQ ID NO:30),
使用上述引物,将获得的扩增片段跑胶验证正确后进行柱回收。通过融合PCR,将重组同源臂,β-1,4-半乳糖转移酶编码基因以及带抗性基因的启动子融合,第一轮PCR条件为:等摩尔量柱回收的DNA,总量大于1000ng,primer star酶量25μL,加ddH2O定容50μL,PCR条件55℃,11个循环。第二轮PCR条件为:上述PCR产物为模板,以SEQ ID NO:31和SEQ IDNO:32所示的序列:
zong-F:5’-CGTATCCGATTTGGGTGAGTGTGA-3’(SEQ ID NO:31),
zong-R:5’-TGATGTCAATCTAATGCCTCCTTACTGG-3’(SEQ ID NO:32)分别为上下游引物,按照常规PCR设置条件,获得p7S6P43-lgtB片段。p7S6P43-lgtB片段的序列如SEQ IDNO:33所示。
实施例4重组质粒pP43NMK-lgtA的构建
以重组质粒pP43NMK-lgtA-lgtB为模板,设计反向重组引物,序列分别为SEQ IDNO:34和SEQ ID NO:35:
fz-lgtA-F:5’-tgaagcccgcctaatgagcgggcttttttaagcttggcgtaatcatggtcatagc tgtt-3’(SEQ ID NO:34),
fz-lgtA-R:5’-CTGTGTGAAATTGTTATCCGCTCCCAGCCTTTCTTATTAAAACCACTTTGTCAG CC-3’(SEQ ID NO:35),
使用上述引物将获得的扩增片段跑胶验证正确后,取10μL的PCR反应体系,转化大肠杆菌JM109感受态,通过测序,验证重组质粒pP43NMK-lgtA构建成功,重组质粒pP43NMK-lgtA的基因序列如SEQ ID NO:36所示。
实施例5重组p7Z6P43-lgtB片段的构建
以枯草芽孢杆菌(Bacillus subtilis 168)基因组为模板,设计两侧同源臂引物,序列分别为SEQ ID NO:37和SEQ ID NO:38的左侧同源臂引物:
ydaH-F:5’-GGCTTTCGTTGTCTTGTGTTCAAGAAATTTCCA-3’(SEQ ID NO:37),
ydaH-R:5’-CTGTGTGAAATTGTTATCCGCTCCCAGCCTTTCTTATTAAAACCACTTTGTCAGCC-3’(SEQ ID NO:38),
序列分别为SEQ ID NO:39和SEQ ID NO:40的右侧同源臂引物:
ydZA-F:5’-AAGCCCGCCTAATGAGCGGGCTTTTTTATCATAAAGATCCAGCCTTTTTGCGCT-3’(SEQ ID NO:39),
ydZA-R:5’-CTAAAGCCCAAGTCACAATATATTGATCGCCT-3’(SEQ ID NO:40),
以质粒p7Z6P43为模板,分别设计序列为SEQ ID NO:41和SEQ ID NO:42的引物:
p7Z6P43-F:5’-GTTTTAATAAGAAAGGCTGGGAGCGGATAACAATTTCACACAGGAAACAGC-3’(SEQ ID NO:41),
p7Z6P43-R:5’-AAAGAAATGACATGATTTTGCATGTGTACATTCCTCTCTTACCTATAATGGTACC GC-3’(SEQ ID NO:42),
以出发菌株Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK的基因组为模板,分别设计序列为SEQ ID NO:43和SEQ ID NO:44的引物:
lgtB-1F:5’-AGGTAAGAGAGGAATGTACACATGCAAAATCATGTCATTTCTTTAGCATCAGCAG-3’(SEQ ID NO:43),
lgtB-1R:5’-CCCGCTCATTAGGCGGGCTTCATCATTACTGAAACGGAACGATAAACTGTTCGCG-3’(SEQ ID NO:44),
使用上述引物,将获得的扩增片段跑胶验证正确后进行柱回收。通过融合PCR,将重组同源臂,β-1,4-半乳糖转移酶编码基因以及带抗性基因的启动子融合。第一轮PCR条件为:等摩尔量柱回收的DNA,总量大于1000ng,primer star酶量25μL,加ddH2O定容50μL,PCR条件55℃,11个循环。第二轮PCR条件为:上述PCR产物为模板,融合PCR的引物分别为SEQ IDNO:45和SEQ ID NO:46:
zong-1F:5’-AACCTCCTTGTCTTCTTTCCAGTCTTATCTC-3’(SEQ ID NO:45),
zong-1R:5’-ATCACTTCTCTTTCTTTCACGCTCATCCT-3’(SEQ ID NO:46),按照常规PCR设置条件,获得p7Z6P43-lgtB片段,p7Z6P43-lgtB片段的序列如SEQ ID NO:47所示。
实施例6重组p7C6P43-lgtB片段的构建
以枯草芽孢杆菌(Bacillus subtilis 168)基因组为模板,设计两侧同源臂引物,序列分别为SEQ ID NO:48和SEQ ID NO:49的左侧同源臂引物:
yszA-F:5’-CATCCAGCGTAAAACGTTCACGGGAATAATCTAGG-3’(SEQ ID NO:48),
yszA-R:5’-TTATCCGCTCTGGCGCGGACTTGTTTGTTTATATCCATTCTAAATGAAGG-3’(SEQID NO:49),
序列分别为SEQ ID NO:50和SEQ ID NO:51的右侧同源臂引物:
ysxE-F:5’-GCCCGCCTAATGAGCGGGCTTTTTTTTAAGACGTGGACTCGTTTTCAGCCTGAAATT TT-3’(SEQ ID NO:50),
ysxE-R:5’-CATCCCAGCAGCTGATCAGGATGAATTCT-3’(SEQ ID NO:51),
以质粒p7C6P43为模板,分别设计序列为SEQ ID NO:52和SEQ ID NO:53的引物:
p7C6P43-F:5’-ACAAGTCCGCGCCAGAGCGGATAACAATTTCACACAGGAAACAGCTATG-3’(SEQID NO:52),
p7C6P43-R:5’-AGAAATGACATGATTTTGCATGTGTACATTCCTCTCTTACCTATAATGGTACCG CT-3’(SEQ ID NO:53),
以出发菌株基因组为模板,分别设计序列为SEQ ID NO:54和SEQ ID NO:55的引物:
lgtB-2F:5’-ATAGGTAAGAGAGGAATGTACACATGCAAAATCATGTCATTTCTTTAGCATCAGCAG CG-3’(SEQ ID NO:54),
lgtB-2R:5’-GCCCGCTCATTAGGCGGGCTTCATCATTACTGAAACGGAACGATAAACTGTTCGCGT-3’(SEQ ID NO:55),
使用上述引物,将获得的扩增片段跑胶验证正确后进行柱回收。通过融合PCR,将重组同源臂,β-1,4-半乳糖转移酶编码基因以及带抗性基因的启动子融合。第一轮PCR条件为:等摩尔量柱回收的DNA,总量大于1000ng,primer star酶量25μL,加ddH2O定容50μL,PCR条件55℃,11个循环。第二轮PCR条件为:上述PCR产物为模板,融合PCR的引物分别为SEQ IDNO:56和SEQ ID NO:57:
zong-2F:5’-GCCAGGAAGCCACAGCACATCATAGC-3’(SEQ ID NO:56),
zong-2R:5’-TTCGTTCATTCGTTCCCGTTTACAGAACAAATAGC-3’(SEQ ID NO:57),按照常规PCR设置条件,获得p7C6P43-lgtB片段,p7C6P43-lgtB片段的序列如SEQ ID NO:58所示。
实施例7出发菌株枯草芽孢杆菌的构建
将构建好的重组片段PxylA-comK转化枯草芽孢杆菌感受态细胞(Bacillussubtilis 168),重组片段的添加量为100-300ng,电转条件:电压2.5kV,电击试剂5ms,37℃复苏5h后涂布氯霉素抗性平板,37℃培养24h,获得重组枯草芽孢杆菌BS168comk。
BS168comk重组枯草芽孢杆菌通过木糖诱导制作感受态。在37℃,220rpm条件下,LB液体培养12h后,用LB液体培养基将OD600稀释至1,并添加终浓度为2%的木糖,继续培养2h,获得BS168comk感受态,重组片段PZL的添加量为500ng,37℃培养1h后涂布博来霉素抗性平板,37℃培养过夜,获得重组枯草芽孢杆菌BY00。
BY00通过木糖诱导制作成感受态细胞,重组片段p7S6P43-lgtB的添加量为500ng,37℃培养1h后涂布壮观霉素抗性平板,37℃培养过夜,获得重组枯草芽孢杆菌BY01。通过Cre/lox重组***,敲除抗性基因,获得BY01.2菌。
BY01.2通过木糖诱导制作成感受态细胞,重组质粒pP43NMK-lgtA的添加量为500ng,37℃培养1h后涂布卡那霉素抗性平板,37℃培养过夜,获得菌株BY03。
实施例8
将构建好的重组p7Z6P43-lgtB片段转化感受态细胞BY01.2,挑选转化子进行菌落PCR验证,分别设计验证引物序列SEQ ID NO:59和SEQ ID NO:60的引物:
YZ-1F:5’-CGAAGGAATGCCGGTGTGCCAACTGAATC-3’(SEQ ID NO:59),
YZ-1R:5’-CGACCTCCAGCGTGTATTGCTTGATGATGC-3’(SEQ ID NO:60),验证正确后获得重组枯草芽孢杆菌BY03a,菌落PCR琼脂糖凝胶电泳图如图1所示。
将重组质粒pP43NMK-lgtA转化上述重组枯草芽孢杆菌BY03a,37℃培养1h后涂布卡那霉素抗性平板,37℃培养过夜,获得重组枯草芽孢杆菌BY04。
将构建好的重组p7C6P43-lgtB片段转化重组枯草芽孢杆菌BY03a感受态细胞,挑选转化子进行菌落PCR验证,分别设计验证引物序列SEQ ID NO:61和SEQ ID NO:62的引物:
YZ-2F:5’-GGGCACAGCGGGATACATCATCTCAAGAAA-3’(SEQ ID NO:61),
YZ-2R:5’-TGAACCACGGCAGCCTGTCTATACACCAT-3’(SEQ ID NO:62)。
验证正确后获得重组枯草芽孢杆菌BY03b,得到的菌落PCR琼脂糖凝胶电泳图如图2所示。
将重组质粒pP43NMK-lgtA转化重组枯草芽孢杆菌BY03b,37℃培养1h后涂布卡那霉素抗性平板,37℃培养过夜,获得一种合成乳酰-N-新四糖的重组枯草芽孢杆菌BY05。
所用pLCx-dcas9,p7S6P43,p7Z6P43,p7C6P43质粒来自江苏省无锡市滨湖区蠡湖大道1800号,生物工程学院,生物***与生物加工工程研究室,上述p7S6P43,p7Z6P43,p7C6P43质粒在Modular pathway engineering of key carbon-precursor supply-pathways for improved N-acetylnuraminic acid production in Bacillus subtilis(Zhang XL,Liu YF,Liu L,Wang M,Li JH,Du GC,Chen J.2018)中已经公开。上述pLCx-dcas9质粒在CRISPRi allows optimal temporal control of Nacetylglucosaminebioproduction by a dynamic coordination of glucose and xylose metabolism inBacillus subtilis(Wu YK,Chen TC,Liu YF,Lv XQ,Li JH,Du GC,Amaro RL,Liu L.2018)中已经公开。
实施例9重组枯草芽孢杆菌发酵法产乳酰-N-新四糖
将实施例8的重组枯草芽孢杆菌BY05制成种子液,种子液的制作方法为:挑取平板上的单菌落在种子培养基中(种子培养基:胰蛋白胨10g/L,酵母粉5g/L,NaCl 10g/L)。同时添加终浓度为30μg/mL的卡那霉素,在37℃、220rpm条件下,培养10-12h。
将种子液按10%的接种量转入发酵培养基中(发酵培养基:胰蛋白胨6g/L,酵母粉12g/L,(NH4)SO46g/L,K2HPO4·3H2O 12.5g/L,KH2PO42.5g/L,MgSO4·7H2O 1.2g/L,葡萄糖60g/L,乳糖5g/L),于37℃、220rpm条件下培养48h。在发酵培养液的上清测得乳酰-N-新四糖,含量为1300mg/L。较菌株(Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK,pP43NMK-lgtA)提高了80.6%,实现了乳酰-N-新四糖在重组枯草芽孢杆菌的高效合成,为进一步代谢工程改造枯草芽孢杆菌生产乳酰-N-新四糖及以乳酰-N-新四糖为核心元件的复杂寡糖奠定了基础。
对照实施例1 BY03和BY04菌株发酵法生产乳酰-N-新四糖
将Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK,pP43NMK-lgtA和重组枯草芽孢杆菌BY04制成种子液,种子液的制作方法为:挑取平板上的单菌落在种子培养基中,同时添加终浓度为30μg/mL的卡那霉素,在37℃、220rpm条件下,培养10-12h。
将种子液按照10%的接种量转入发酵培养基中,于37℃、220rpm条件下培养48h。在发酵培养液的上清测得乳酰-N-新四糖,BY03上清液中的含量大约是720mg/L,重组枯草芽孢杆菌BY04上清液中的含量大约是1090mg/L。重组枯草芽孢杆菌BY04较出发菌株(Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK,pP43NMK-lgtA)提高了51.3%,比BY05菌中乳酰-N-新四糖的产量为1300mg/L低了16%,如图3所示。已知BY05菌的基因组上有2个拷贝数的β-1,4-半乳糖转移酶编码基因,BY05菌的基因组上有3个拷贝数的β-1,4-半乳糖转移酶编码基因,说明增加关键基因的拷贝数,有效提高了乳酰-N-新四糖在重组枯草芽孢杆菌的合成效率。
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> 一种合成乳酰-N-新四糖的重组枯草芽孢杆菌及其构建方法与应用
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<170> SIPOSequenceListing 1.0
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tattccgtat gtcaagtggc tgcggtttat 30
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<212> DNA
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aattgttatc cgctctcttg acactcctta tttgattttt tgaagactta cttcgg 56
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<212> DNA
<213> 人工序列(未知)
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cttaagggca aggctagacg ggactta 27
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ggcacaccga tgtacacgtc atc 23
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<212> DNA
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<400> 5
cattatacga acggtaaatc tgataggtgg tatgttttcg cttgaacttt taaatacag 59
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<212> DNA
<213> 人工序列(未知)
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agtttgtgtt ttttaaatag tacatgtgta cattcctctc ttacctataa tggtaccgc 59
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tcaaataagg agtgtcaaga gagcggataa caatttcaca caggaaacag 50
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aacataccac ctatcagatt taccgttcgt ataatgtatg ctatacgaag ttattcagt 59
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gtaagagagg aatgtacaca tgtactattt aaaaaacaca aacttttgga tgttcggtt 59
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<212> DNA
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taagtcccgt ctagccttgc ccttaagcga cttcattcac ctgacgacg 49
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<212> DNA
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catgtaagcc ataagccatt cgtaa 25
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agcaagactc atcgcaaccc 20
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<212> DNA
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catgtaagcc ataagccatt cgtaaaagtg cgggaggaag gtcatgaata atctgcgtaa 60
tagactttca ggcgtgaatg ggaaaaataa gagagtaaaa gaaaaagaac aaaaaatctg 120
gtcggagatt gggatgatag cgggagcatt tgcgctgctt gatgtgatca tccgcggcat 180
tatgtttgaa tttccgttta aagaatgggc tgcaagcctt gtgtttttgt tcatcattat 240
cttatattac tgcatcaggg ctgcggcatc cggaatgctc atgccgagaa tagacaccaa 300
agaagaactg caaaaacggg tgaagcagca gcgaatagaa tcaattgcgg tcgcctttgc 360
ggtagtggtg cttacgatgt acgacagggg gattccccat acattcttcg cttggctgaa 420
aatgattctt ctttttatcg tctgcggcgg cgttctgttt ctgcttcggt atgtgattgt 480
gaagctggct tacagaagag cggtaaaaga agaaataaaa aagaaatcat cttttttgtt 540
tggaaagcga gggaagcgtt cacagtttcg ggcagctttt tttataggaa cattgatttg 600
tattcactct gccaagttgt tttgatagag tgattgtgat aattttaaat gtaagcgtta 660
acaaaattct ccagtcttca catcggtttg aaaggaggaa gcggaagaat gaagtaagag 720
ggatttttga ctccgaagta agtcttcaaa aaatcaaata aggagtgtca agagagcgga 780
taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct cggtacccgg 840
ggatcctcta gagataccgt tcgtatagca tacattatac gaagttatct tgatatggct 900
ttttatatgt gttactctac atacagaaag gaggaactaa acatggccaa gttgaccagt 960
gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg 1020
ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg 1080
accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg 1140
tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc 1200
cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc 1260
gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgaata 1320
acttcgtata gcatacatta tacgaacggt aaatctgata ggtggtatgt tttcgcttga 1380
acttttaaat acagccattg aacatacggt tgatttaata actgacaaac atcaccctct 1440
tgctaaagcg gccaaggacg ccgccgccgg ggctgtttgc gttcttgccg tgatttcgtg 1500
taccattggt ttacttattt ttttgccaag gctgtaatgg ctgaaaattc ttacatttat 1560
tttacatttt tagaaatggg cgtgaaaaaa agcgcgcgat tatgtaaaat ataaagtgat 1620
agcggtacca ttataggtaa gagaggaatg tacacatgta ctatttaaaa aacacaaact 1680
tttggatgtt cggtttattc tttttctttt acttttttat catgggagcc tacttcccgt 1740
ttttcccgat ttggctacat gacatcaacc atatcagcaa aagtgatacg ggtattattt 1800
ttgccgctat ttctctgttc tcgctattat tccaaccgct gtttggtctg ctttctgaca 1860
aactcgggct gcgcaaatac ctgctgtgga ttattaccgg catgttagtg atgtttgcgc 1920
cgttctttat ttttatcttc gggccactgt tacaatacaa cattttagta ggatcgattg 1980
ttggtggtat ttatctaggc ttttgtttta acgccggtgc gccagcagta gaggcattta 2040
ttgagaaagt cagccgtcgc agtaatttcg aatttggtcg cgcgcggatg tttggctgtg 2100
ttggctgggc gctgtgtgcc tcgattgtcg gcatcatgtt caccatcaat aatcagtttg 2160
ttttctggct gggctctggc tgtgcactca tcctcgccgt tttactcttt ttcgccaaaa 2220
cggatgcgcc ctcttctgcc acggttgcca atgcggtagg tgccaaccat tcggcattta 2280
gccttaagct ggcactggaa ctgttcagac agccaaaact gtggtttttg tcactgtatg 2340
ttattggcgt ttcctgcacc tacgatgttt ttgaccaaca gtttgctaat ttctttactt 2400
cgttctttgc taccggtgaa cagggtacgc gggtatttgg ctacgtaacg acaatgggcg 2460
aattacttaa cgcctcgatt atgttctttg cgccactgat cattaatcgc atcggtggga 2520
aaaacgccct gctgctggct ggcactatta tgtctgtacg tattattggc tcatcgttcg 2580
ccacctcagc gctggaagtg gttattctga aaacgctgca tatgtttgaa gtaccgttcc 2640
tgctggtggg ctgctttaaa tatattacca gccagtttga agtgcgtttt tcagcgacga 2700
tttatctggt ctgtttctgc ttctttaagc aactggcgat gatttttatg tctgtactgg 2760
cgggcaatat gtatgaaagc atcggtttcc agggcgctta tctggtgctg ggtctggtgg 2820
cgctgggctt caccttaatt tccgtgttca cgcttagcgg ccccggcccg ctttccctgc 2880
tgcgtcgtca ggtgaatgaa gtcgcttaag ggcaaggcta gacgggactt accgaaagaa 2940
accatcaatg atggtttctt ttttgttcat aaatcagaca aaacttttct cttgcaaaag 3000
tttgtgaagt gttgcacaat ataaatgtga aatacttcac aaacaaaaag acatcaaaga 3060
gaaacatacc ctggaaggat gattaatgat gaacaaacat gtaaataaag tagctttaat 3120
cggagcgggt tttgttggaa gcagttatgc atttgcgtta attaaccaag gaatcacaga 3180
tgagcttgtg gtcattgatg taaataaaga aaaagcaatg ggcgatgtga tggatttaaa 3240
ccacggaaag gcgtttgcgc cacaaccggt caaaacatct tacggaacat atgaagactg 3300
caaggatgct gatattgtct gcatttgcgc cggagcaaac caaaaacctg gtgagacacg 3360
ccttgaatta gtagaaaaga acttgaagat tttcaaaggc atcgttagtg aagtcatggc 3420
gagcggattt gacggcattt tcttagtcgc gacaaatccg gttgatatcc tgacttacgc 3480
aacatggaaa ttcagcggcc tgccaaaaga gcgggtgatt ggaagcggca caacacttga 3540
ttctgcgaga ttccgtttca tgctgagcga atactttggc gcagcgcctc aaaacgtaca 3600
cgcgcatatt atcggagagc acggcgacac agagcttcct gtttggagcc acgcgaatgt 3660
cggcggtgtg ccggtcagtg aactcgttga gaaaaacgat gcgtacaaac aagaggagct 3720
ggaccaaatt gtagatgatg tgaaaaacgc agcttaccat atcattgaga aaaaaggcgc 3780
gacttattat ggggttgcga tgagtcttgc t 3811
<210> 14
<211> 30
<212> DNA
<213> 人工序列(未知)
<400> 14
catacatagg aagcaggcat tgttcataac 30
<210> 15
<211> 47
<212> DNA
<213> 人工序列(未知)
<400> 15
atacgggatc aaatccgatg aaagagaaaa aatcgtacac tgagctc 47
<210> 16
<211> 42
<212> DNA
<213> 人工序列(未知)
<400> 16
aagggggaaa tgggatccat gagtcagaaa acagacgcac ct 42
<210> 17
<211> 29
<212> DNA
<213> 人工序列(未知)
<400> 17
actacctcag ttgaaggcta taatccaag 29
<210> 18
<211> 52
<212> DNA
<213> 人工序列(未知)
<400> 18
tttctctttc atcggatttg atcccgtata ccgttcgtat agcatacatt at 52
<210> 19
<211> 42
<212> DNA
<213> 人工序列(未知)
<400> 19
gactcatgga tcccatttcc ccctttgatt tttagatatc ac 42
<210> 20
<211> 34
<212> DNA
<213> 人工序列(未知)
<400> 20
gacccgcgtc cggtttttga taagccgaag aaag 34
<210> 21
<211> 35
<212> DNA
<213> 人工序列(未知)
<400> 21
agtaaattca gttgcgcccg gaccaatttg gacac 35
<210> 22
<211> 4568
<212> DNA
<213> 人工序列(未知)
<400> 22
gacccgcgtc cggtttttga taagccgaag aaagcgaaaa aattagaggg caaaaccgct 60
attattactg gaggagacag cgggattgga cgcgctgtct cggtgttatt cgcaaaagaa 120
ggggctaatg tggtcattgt gtatttgaat gaacatcagg acgccgagga aacaaagcag 180
tatgtagaaa aggaaggggt aaaatgcctg ctgattgcag gagatgtcgg ggatgaagcg 240
ttttgcaatg atgtggtcgg gcaggcaagc caagtgtttc catccattga tatattggtt 300
aacaatgcag ctgagcagca tgtccagccc agcattgaaa aaatcaccag ccaccagctg 360
atcagaacct tccaaacaaa cattttttcc atgttttact taacaaaggc agtgctgcct 420
catttaaaaa aagggagctc tattattaat accgcttcaa ttaccgccta taaaggcaat 480
aaaacgctga tcgattattc agcgacaaaa ggcgcgatcg ttacgtttac aaggtccctt 540
tcccagtcgc ttgttcagca gggcatacgg gtcaatgcag tggcaccggg tcccatttgg 600
acaccgctta tcccggccag ctttgccgca aaagacgtgg aagtgtttgg ttcagacgtg 660
ccgatggaac gcccggggca gccggtcgaa gtggcgccaa gctatctata ccttgccagc 720
gacgattcca catacgtcac agggcagacg attcacgtca atggcggaac aattgtgaac 780
ggataataaa aagccaatcc ttgaaggatt ggcttattcg ctctgcttga gcgctgcata 840
ttctttagag agcgtaagaa acgcatcttt atctttttcg tccaaggctt tgttaatttg 900
gtccgtcagc atggcaagcc gacggttata caaggattca tcgagcacca tttgaatgta 960
aatatccgtc attgtgacat cgaattcttt tgttttttgc gtgttgcggg acttcatgag 1020
ctcagtgtac gattttttct ctttcatcgg atttgatccc gtataccgtt cgtatagcat 1080
acattatacg aagttatgcc atagtgactg gcgatgctgt cggaatggac gacggcaata 1140
gttaccctta ttatcaagat aagaaagaaa aggatttttc gctacgctca aatcctttaa 1200
aaaaacacaa aagaccacat tttttaatgt ggtcttttat tcttcaacta aagcacccat 1260
tagttcaaca aacgaaaatt ggataaagtg ggatattttt aaaatatata tttatgttac 1320
agtaatattg acttttaaaa aaggattgat tctaatgaag aaagcagaca agtaagcctc 1380
ctaaattcac tttagataaa aatttaggag gcatatcaaa tgaactttaa taaaattgat 1440
ttagacaatt ggaagagaaa agagatattt aatcattatt tgaaccaaca aacgactttt 1500
agtataacca cagaaattga tattagtgtt ttataccgaa acataaaaca agaaggatat 1560
aaattttacc ctgcatttat tttcttagtg acaagggtga taaactcaaa tacagctttt 1620
agaactggtt acaatagcga cggagagtta ggttattggg ataagttaga gccactttat 1680
acaatttttg atggtgtatc taaaacattc tctggtattt ggactcctgt aaagaatgac 1740
ttcaaagagt tttatgattt atacctttct gatgtagaga aatataatgg ttcggggaaa 1800
ttgtttccca aaacacctat acctgaaaat gctttttctc tttctattat tccatggact 1860
tcatttactg ggtttaactt aaatatcaat aataatagta attaccttct acccattatt 1920
acagcaggaa aattcattaa taaaggtaat tcaatatatt taccgctatc tttacaggta 1980
catcattctg tttgtgatgg ttatcatgca ggattgttta tgaactctat tcaggaattg 2040
tcagataggc ctaatgactg gcttttataa tatgagataa tgccgactgt actttttaca 2100
gtcggttttc taaaacgata cattaatagg tacgaaaaag caactttttt tgcgcttaaa 2160
accagtcata ccaataaata acttcgtata gcatacatta tacgaacggt attcagaacg 2220
ctcggttgcc gccgggcgtt ttttatgcag caatggcaag aacgtcccgg ggagctccta 2280
acttataggg gtaacactta aaaaagaatc aataacgata gaaaccgctc ctaaagcagg 2340
tgcatttttt cctaacgaag aaggcaatag ttcacattta ttgtctaaat gagaatggac 2400
tctagaagaa acttcgtttt taatcgtatt taaaacaatg ggatgagatt caattatatg 2460
atttctcaag ataacagctt ctatatcaaa tgtattaagg atattggtta atccaattcc 2520
gatataaaag ccaaagtttt gaagtgcatt taacatttct acatcatttt tatttgcgcg 2580
ttccacaatc tcttttcgag aaatattctt ttcttcttta gagagcgaag ccagtaacgc 2640
tttttcagaa gcatataatt cccaacagcc tcgatttcca cagctgcatt tgggtccatt 2700
aaaatctatc gtcatatgac ccatttcccc agaaaaaccc tgaacacctt tatacaattc 2760
gttgttaata acaagtccag ttccaattcc gatattaata ctgatgtaaa cgatgttttc 2820
atagtttttt gtcataccaa atactttttc accgtatgct cctgcattag cttcattttc 2880
aacaaaaacc ggaacattaa actcactctc aattaaaaac tgcaaatctt tgatattcca 2940
atttaagtta ggcatgaaaa taatttgctg atgacgatct acaaggcctg gaacacaaat 3000
tcctattccg actagaccat aaggggactc aggcatatgg gttacaaaac catgaataag 3060
tgcaaataaa atctctttta cttcactagc ggaagaacta gacaagtcag aagtcttctc 3120
gagaataata tttccttcta agtcggttag aattccgtta agatagtcga ctcctatatc 3180
aataccaatc gagtagcctg cattcttatt aaaaacaagc attacaggtc ttctgccgcc 3240
tctagattgc cctgccccaa tttcaaaaat aaaatctttt tcaagcagtg tatttacttg 3300
agaggagaca gtagacttgt ttaatcctgt aatctcagag agagttgccc tggagacagg 3360
ggagttcttc aaaatttcat ctaatattaa tttttgattc atttttttta ctaaagcttg 3420
atctgcaatt tgaataataa ccactccttt gtttatccac cgaactaagt tggtgttttt 3480
tgaagcttga attagatatt taaaagtatc atatctaata ttataactaa attttctaaa 3540
aaaaacattg aaataaacat ttattttgta tatgatgaga taaagttagt ttattggata 3600
aacaaactaa ctcaattaag atagttgatg gataaacttg ttcacttaaa tcaaaggggg 3660
aaatgacaaa tggtccaaac tagtgatatc taaaaatcaa agggggaaat gggatccatg 3720
agtcagaaaa cagacgcacc tttagaatcg tatgaagtga acggcgcaac aattgccgtg 3780
ctgccagaag aaatagacgg caaaatctgt tccaaaatta ttgaaaaaga ttgcgtgttt 3840
tatgtaaaca tgaagccgct gcaaattgtc gacagaagct gccgattttt tggatcaagc 3900
tatgcgggaa gaaaagcagg aacttatgaa gtgacaaaaa tttcacacaa gccgccgatc 3960
atggtggacc cttcgaacca aatcttttta ttccctacac tttcttcgac aagaccccaa 4020
tgcggctgga tttcccatgt gcatgtaaaa gaattcaaag cgactgaatt cgacgatacg 4080
gaagtgacgt tttccaatgg gaaaacgatg gagctgccga tctcttataa ttcgttcgag 4140
aaccaggtat accgaacagc gtggctcaga accaaattcc aagacagaat cgaccaccgc 4200
gtgccgaaaa gacaggaatt tatgctgtac ccgaaagaag agcggacgaa gatgatttat 4260
gattttattt tgcgtgagct cggggaacgg tattagaaaa ataggaagga gctgaccgaa 4320
cagggcagct cctttcataa agctatgccg tcggaattcc tccggtgatg ccatatacct 4380
gagatgtcac atagcttgaa ttctctgaag ctagaaatac ataaacatcg gccaattcca 4440
ctggctgccc agcgcgattt aatggagctg gcggcgtgcc ttgtccgaat ttcgggatgt 4500
tttccgtcgg ctgtccgccg gaaatttgca gcggtgtcca aattggtccg ggcgcaactg 4560
aatttact 4568
<210> 23
<211> 25
<212> DNA
<213> 人工序列(未知)
<400> 23
gggacaagga atagtaagcc ggcaa 25
<210> 24
<211> 56
<212> DNA
<213> 人工序列(未知)
<400> 24
tcctgtgtga aattgttatc cgctcctaca tactctctgt agcagaggta gcttga 56
<210> 25
<211> 59
<212> DNA
<213> 人工序列(未知)
<400> 25
tgaagcccgc ctaatgagcg ggcttttttc tgataagaac tgcaaaagct gcggattat 59
<210> 26
<211> 33
<212> DNA
<213> 人工序列(未知)
<400> 26
ccaccctata gataaatttt tcggctgcca tat 33
<210> 27
<211> 36
<212> DNA
<213> 人工序列(未知)
<400> 27
agagtatgta ggagcggata acaatttcac acagga 36
<210> 28
<211> 57
<212> DNA
<213> 人工序列(未知)
<400> 28
aaagaaatga catgattttg catgtgtaca ttcctctctt acctataatg gtaccgc 57
<210> 29
<211> 55
<212> DNA
<213> 人工序列(未知)
<400> 29
aggtaagaga ggaatgtaca catgcaaaat catgtcattt ctttagcatc agcag 55
<210> 30
<211> 55
<212> DNA
<213> 人工序列(未知)
<400> 30
cccgctcatt aggcgggctt catcattact gaaacggaac gataaactgt tcgcg 55
<210> 31
<211> 24
<212> DNA
<213> 人工序列(未知)
<400> 31
cgtatccgat ttgggtgagt gtga 24
<210> 32
<211> 28
<212> DNA
<213> 人工序列(未知)
<400> 32
tgatgtcaat ctaatgcctc cttactgg 28
<210> 33
<211> 4313
<212> DNA
<213> 人工序列(未知)
<400> 33
cgtatccgat ttgggtgagt gtgacaatca atccagcacc gcttggagaa agtccaattg 60
ccttactaat taatccgacc aagggttgtg catagtaaag attagcaaca atgataccgc 120
atgcagttgc gagaagaagg gttaaactgg gtgaaatcct ctgacctgct tgtttgtttc 180
cattagccat atgaatgaac tcctttttaa gtggtttttt gagtgtaatc atgacttctc 240
acaagtgatt ttataaactg aacgttcagt ttcgtcgatt aataaaaata atatactaaa 300
cgttcagttt tgtaaatgga tttctgtttt ctttttttat tcaatttaag tatactgaac 360
gtatagttta tattgcggga aggaatgatt tatgtgcaga gtaaacgagg gcggccgcgt 420
gatgaaggaa cgcataaggc gattctctct gcagcctatg acctattgct ggaaaaaggc 480
ttcgatgcgg tgacagtcga taaaattgcc gagcgtgcga aagtgagtaa agcaacgatt 540
tataaatggt ggtctaacaa ggctgccgtt atcatggaca gctttctttc gaccgcgacg 600
gacaggctgc ctgtgcctga tacagggtcg tcagtacaag atatagtaac ccacgccacg 660
aatttagcta ggtttttgac aagccgggaa ggaaccgtta ttaaggaatt aataggtgca 720
gggcagttgg atgaaaaatt ggcagaagaa tatcgcacgc gatttttcca gcctcgccgc 780
ctccaagcga aaggccttct agaaaaggga attcagaaag gtgaattgag agagaatctt 840
gatattgaag taagcataga tctcatttac ggaccaattt tctatcgtct gcttataaca 900
ggggatgagg tgaatgattc ctatgtgcgt gatttggtga tgaatgcgtt taagggagtt 960
caagctacct ctgctacaga gagtatgtag gagcggataa caatttcaca caggaaacag 1020
ctatgaccat gattacgaat tcgagctcgg tacccgggga tcctctagag attgtaccgt 1080
tcgtatagca tacattatac gaagttatcg attttcgttc gtgaatacat gttataataa 1140
ctataactaa taacgtaacg tgactggcaa gagatatttt taaaacaatg aataggttta 1200
cacttacttt agttttatgg aaatgaaaga tcatatcata tataatctag aataaaatta 1260
actaaaataa ttattatcta gataaaaaat ttagaagcca atgaaatcta taaataaact 1320
aaattaagtt tatttaatta acaactatgg atataaaata ggtactaatc aaaatagtga 1380
ggaggatata tttgaataca tacgaacaag ttaataaagt gaaaaaaata cttcggaaac 1440
atttaaaaaa taaccttatt ggtacttaca tgtttggatc aggagttgag agtggactaa 1500
aaccaaatag tgatcttgac tttttagtcg tcgtatctga accattgaca gatcaaagta 1560
aagaaatact tatacaaaaa attagaccta tttcaaaaaa aataggagat aaaagcaact 1620
tacgatatat tgaattaaca attattattc agcaagaaat ggtaccgtgg aatcatcctc 1680
ccaaacaaga atttatttat ggagaatggt tacaagagct ttatgaacaa ggatacattc 1740
ctcagaagga attaaattca gatttaacca taatgcttta ccaagcaaaa cgaaaaaata 1800
aaagaatata cggaaattat gacttagagg aattactacc tgatattcca ttttctgatg 1860
tgagaagagc cattatggat tcgtcagagg aattaataga taattatcag gatgatgaaa 1920
ccaactctat attaacttta tgccgtatga ttttaactat ggacacgggt aaaatcatac 1980
caaaagatat tgcgggaaat gcagtggctg aatcttctcc attagaacat agggagagaa 2040
ttttgttagc agttcgtagt tatcttggag agaatattga atggactaat gaaaatgtaa 2100
atttaactat aaactattta aataacagat taaaaaaatt ataaataact tcgtatagca 2160
tacattatac gaacggtaga atcgtcgact gataggtggt atgttttcgc ttgaactttt 2220
aaatacagcc attgaacata cggttgattt aataactgac aaacatcacc ctcttgctaa 2280
agcggccaag gacgccgccg ccggggctgt ttgcgttctt gccgtgattt cgtgtaccat 2340
tggtttactt atttttttgc caaggctgta atggctgaaa attcttacat ttattttaca 2400
tttttagaaa tgggcgtgaa aaaaagcgcg cgattatgta aaatataaag tgatagcggt 2460
accattatag gtaagagagg aatgtacaca tgcaaaatca tgtcatttct ttagcatcag 2520
cagcggaaag acgcgctcat attgccgata catttggcag acatggaatc ccgtttcaat 2580
ttttcgatgc gcttatgccg tcagaacgct tagaacaggc aatggcggaa ttagttccgg 2640
gcctgtcagc tcatccgtat cttagcggag tggaaaaagc atgctttatg agccatgcgg 2700
tcttatggaa acaagctctt gatgaaggcc tgccgtacat cacagttttt gaagatgatg 2760
tgctgcttgg cgaaggagcc gaaaaatttc tggcagaaga tgcgtggctt caggaaagat 2820
ttgatccgga tacagcattt atcgtgcgct tagaaacaat gtttatgcat gtcctgacat 2880
caccgagcgg cgttgccgat tattgtggaa gagcatttcc gttactggaa tctgaacatt 2940
ggggcacagc gggatacatc atctcaagaa aagctatgag atttttcctg gatagatttg 3000
ctgcccttcc gccggaaggc ttacatccgg ttgatctgat gatgttttct gatttctttg 3060
atcgcgaagg aatgccggtg tgccaactga atccggctct ttgtgcccag gaacttcatt 3120
acgccaaatt tcatgatcaa aacagcgcac tgggatctct tatcgaacat gatagacttc 3180
tgaaccgcaa acaacagaga cgcgatagcc cggcgaacac atttaaacat agattaattc 3240
gcgctctgac aaaaatctct agagaacgcg aaaaaagacg ccaaagacgc gaacagttta 3300
tcgttccgtt tcagtaatga tgaagcccgc ctaatgagcg ggcttttttc tgataagaac 3360
tgcaaaagct gcggattata tgacaaattg gttccatcgt acagtgaaat ataagatttt 3420
tgttgctgag cggaatcatc gagggaaaag ttctcttatc ggatgtcgaa aaagaagtca 3480
ttcaatcgtg aatacataac atcatattat tacattcctt ttcatcgatt ggaacaaggg 3540
ttctatactt caatggaata ccgcaaagtt ggagtattta aacaacaggg gaaaatcgat 3600
ggccattatg taggtgtcat ggcaatggaa aaaatacttg caatgtaaac aggcctctaa 3660
agagacctgt tttttaatat ccagatgatt gtctagtttc agcttgggct ctatgaccac 3720
tcccataact gaaatagtct aatatttata ccagaaggga ttgtttatat aaataaaagt 3780
ttatataatg acgggggcgc taaggaaacg cggggagcga ctcaggtttt cgtttcaaag 3840
ttttgatcct taagatatac ggagcgctac atatggaaac gtaaagggga atgtataatc 3900
atttggcgtg tatataacga tttgcttata tattgatttg ggaaggagga gctcatatga 3960
cgattgatgt tgcggcgatg actcgttgtt tgaaaacact tagtgatcaa actaggctta 4020
tcatgatgag attatttctt gaacaagaat attgtgtctg tcaattggtt gatatgttcg 4080
aaatgagcca gcccgccata agccagcatt tgcggaaatt aaaaaacgca ggttttgtga 4140
atgaggacag aagaggccaa tggcgttatt attcaataaa tggttcctgt cctgagtttg 4200
atacattgca attgatctta catcaaattg atcaagagga tgaattgtta aaccatatca 4260
aacaaaagaa aactcaagcg tgttgccagt aaggaggcat tagattgaca tca 4313
<210> 34
<211> 59
<212> DNA
<213> 人工序列(未知)
<400> 34
tgaagcccgc ctaatgagcg ggctttttta agcttggcgt aatcatggtc atagctgtt 59
<210> 35
<211> 56
<212> DNA
<213> 人工序列(未知)
<400> 35
ctgtgtgaaa ttgttatccg ctcccagcct ttcttattaa aaccactttg tcagcc 56
<210> 36
<211> 7781
<212> DNA
<213> 人工序列(未知)
<400> 36
tgaagcccgc ctaatgagcg ggctttttta agcttggcgt aatcatggtc atagctgttt 60
cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 120
tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 180
cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 240
gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 300
tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 360
acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 420
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 480
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 540
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 600
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 660
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 720
cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 780
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 840
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 900
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 960
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 1020
agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 1080
aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 1140
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 1200
tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 1260
tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 1320
tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 1380
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 1440
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 1500
ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 1560
gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 1620
aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 1680
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 1740
tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 1800
ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 1860
aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 1920
ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 1980
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 2040
agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 2100
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 2160
ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 2220
atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc 2280
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 2340
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 2400
cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg 2460
tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc 2520
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 2580
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 2640
cgacgttgta aaacgacggc cagtgaattc cttaaggaac gtacagacgg cttaaaagcc 2700
tttaaaaacg tttttaaggg gtttgtagac aaggtaaagg ataaaacagc acaattccaa 2760
gaaaaacacg atttagaacc taaaaagaac gaatttgaac taactcataa ccgagaggta 2820
aaaaaagaac gaagtcgaga tcagggaatg agtttataaa ataaaaaaag cacctgaaaa 2880
ggtgtctttt tttgatggtt ttgaacttgt tctttcttat cttgatacat atagaaataa 2940
cgtcattttt attttagttg ctgaaaggtg cgttgaagtg ttggtatgta tgtgttttaa 3000
agtattgaaa acccttaaaa ttggttgcac agaaaaaccc catctgttaa agttataagt 3060
gactaaacaa ataactaaat agatgggggt ttcttttaat attatgtgtc ctaatagtag 3120
catttattca gatgaaaaat caagggtttt agtggacaag acaaaaagtg gaaaagtgag 3180
accatggaga gaaaagaaaa tcgctaatgt tgattacttt gaacttctgc atattcttga 3240
atttaaaaag gctgaaagag taaaagattg tgctgaaata ttagagtata aacaaaatcg 3300
tgaaacaggc gaaagaaagt tgtatcgagt gtggttttgt aaatccaggc tttgtccaat 3360
gtgcaactgg aggagagcaa tgaaacatgg cattcagtca caaaaggttg ttgctgaagt 3420
tattaaacaa aagccaacag ttcgttggtt gtttctcaca ttaacagtta aaaatgttta 3480
tgatggcgaa gaattaaata agagtttgtc agatatggct caaggatttc gccgaatgat 3540
gcaatataaa aaaattaata aaaatcttgt tggttttatg cgtgcaacgg aagtgacaat 3600
aaataataaa gataattctt ataatcagca catgcatgta ttggtatgtg tggaaccaac 3660
ttattttaag aatacagaaa actacgtgaa tcaaaaacaa tggattcaat tttggaaaaa 3720
ggcaatgaaa ttagactatg atccaaatgt aaaagttcaa atgattcgac cgaaaaataa 3780
atataaatcg gatatacaat cggcaattga cgaaactgca aaatatcctg taaaggatac 3840
ggattttatg accgatgatg aagaaaagaa tttgaaacgt ttgtctgatt tggaggaagg 3900
tttacaccgt aaaaggttaa tctcctatgg tggtttgtta aaagaaatac ataaaaaatt 3960
aaaccttgat gacacagaag aaggcgattt gattcataca gatgatgacg aaaaagccga 4020
tgaagatgga ttttctatta ttgcaatgtg gaattgggaa cggaaaaatt attttattaa 4080
agagtagttc aacaaacggg ccagtttgtt gaagattaga tgctataatt gttattaaaa 4140
ggattgaagg atgcttagga agacgagtta ttaatagctg aataagaacg gtgctctcca 4200
aatattctta tttagaaaag caaatctaaa attatctgaa aagggaatga gaatagtgaa 4260
tggaccaata ataatgacta gagaagaaag aatgaagatt gttcatgaaa ttaaggaacg 4320
aatattggat aaatatgggg atgatgttaa ggctattggt gtttatggct ctcttggtcg 4380
tcagactgat gggccctatt cggatattga gatgatgtgt gtcatgtcaa cagaggaagc 4440
agagttcagc catgaatgga caaccggtga gtggaaggtg gaagtgaatt ttgatagcga 4500
agagattcta ctagattatg catctcaggt ggaatcagat tggccgctta cacatggtca 4560
atttttctct attttgccga tttatgattc aggtggatac ttagagaaag tgtatcaaac 4620
tgctaaatcg gtagaagccc aaacgttcca cgatgcgatt tgtgccctta tcgtagaaga 4680
gctgtttgaa tatgcaggca aatggcgtaa tattcgtgtg caaggaccga caacatttct 4740
accatccttg actgtacagg tagcaatggc aggtgccatg ttgattggtc tgcatcatcg 4800
catctgttat acgacgagcg cttcggtctt aactgaagca gttaagcaat cagatcttcc 4860
ttcaggttat gaccatctgt gccagttcgt aatgtctggt caactttccg actctgagaa 4920
acttctggaa tcgctagaga atttctggaa tgggattcag gagtggacag aacgacacgg 4980
atatatagtg gatgtgtcaa aacgcatacc attttgaacg atgacctcta ataattgtta 5040
atcatgttgg ttacgtattt attaacttct cctagtatta gtaattatca tggctgtcat 5100
ggcgcattaa cggaataaag ggtgtgctta aatcgggcca ttttgcgtaa taagaaaaag 5160
gattaattat gagcgaattg aattaataat aaggtaatag atttacatta gaaaatgaaa 5220
ggggatttta tgcgtgagaa tgttacagtc tatcccggca ttgccagtcg gggatattaa 5280
aaagagtata ggtttttatt gggataaagt aggtttcact ttggttcacc atgaagatgg 5340
attcgcagtt ctaatgtgta atgaggttcg gattcatcta tgggaggcaa gtgatgaagg 5400
ctggcgcctc gtagtaatga ttcaccggtt tgtacaggtg cggagtcgtt tattgctggt 5460
actgctagtt gccgcattga agtagaggga attgatgaat tatatcaaca tattaagcct 5520
ttgggcattt tgcaccccaa tacatcatta aaagatcagt ggtgggatga acgagacttt 5580
gcagtaattg atcccgacaa caatttgatt agcttttttc aacaaataaa aagctaaaat 5640
ctattattaa tctgttcagc aatcgggcgc gattgctgaa taaaagatac gagagacctc 5700
tcttgtatct tttttatttt gagtggtttt gtccgttaca ctagaaaacc gaaagacaat 5760
aaaaatttta ttcttgctga gtctggcttt cggtaagcta gacaaaacgg acaaaataaa 5820
aattggcaag ggtttaaagg tggagatttt ttgagtgatc ttctcaaaaa atactacctg 5880
tcccttgctg atttttaaac gagcacgaga gcaaaacccc cctttgctga ggtggcagag 5940
ggcaggtttt tttgtttctt ttttctcgta aaaaaaagaa aggtcttaaa ggttttatgg 6000
ttttggtcgg cactgccgcg cctcgcagag cacacacttt atgaatataa agtatagtgt 6060
gttatacttt acttggaagt ggttgccgga aagagcgaaa atgcctcaca tttgtgccac 6120
ctaaaaagga gcgatttaca tatgagttat gcagtttgta gaatgcaaaa agtgaaatca 6180
gctggactaa aaggcatgca atttcataat caaagagagc gaaaaagtag aacgaatgat 6240
gatattgacc atgagcgaac acgtgaaaat tatgatttga aaaatgataa aaatattgat 6300
tacaacgaac gtgtcaaaga aattattgaa tcacaaaaaa caggtacaag aaaaacgagg 6360
aaagatgctg ttcttgtaaa tgagttgcta gtaacatctg accgagattt ttttgagcaa 6420
ctggatcctg ataggtggta tgttttcgct tgaactttta aatacagcca ttgaacatac 6480
ggttgattta ataactgaca aacatcaccc tcttgctaaa gcggccaagg acgccgccgc 6540
cggggctgtt tgcgttcttg ccgtgatttc gtgtaccatt ggtttactta tttttttgcc 6600
aaggctgtaa tggctgaaaa ttcttacatt tattttacat ttttagaaat gggcgtgaaa 6660
aaaagcgcgc gattatgtaa aatataaagt gatagcggta ccattatagg taagagagga 6720
atgtacacat gccgtctgaa gcttttagac gccatagagc ctatcgcgaa aataaacttc 6780
aaccgttagt ctctgtttta atctgcgctt ataacgtgga aaaatatttt gcccaatcac 6840
tggcagcggt tgtgaatcag acatggagaa acctggatat tcttatcgtc gatgatggct 6900
caacagatgg aacacttgct attgcccaaa gatttcaaga acaggatggc agaattcgca 6960
tcttagcaca gccgcgcaat tctggcctta ttccgtcatt aaacatcgga ttagatgaac 7020
tggcgaaaag cggcggaggc ggagaatata tcgctagaac agatgccgat gatattgctg 7080
ccccggattg gattgaaaaa atcgttggcg aaatggaaaa agatagatca atcatcgcaa 7140
tgggagcgtg gcttgaagtg ttaagcgaag aaaaagatgg caatagactg gctcgccatc 7200
atgaacatgg aaaaatctgg aaaaaaccga caagacatga agatattgcc gatttctttc 7260
cgtttggcaa tccgattcat aataacacaa tgatcatgag acgcagcgtc attgatggcg 7320
gacttagata taacacagaa cgcgattggg cggaagatta ccagttttgg tacgatgttt 7380
ctaaactggg aagacttgca tattatccgg aagcgctggt gaaatatcgc cttcatgcta 7440
atcaagtctc aagcaaatat agcatcagac agcatgaaat tgcacaaggc atccagaaaa 7500
cagcgcgcaa cgattttctt caaagcatgg gatttaaaac aagatttgat tctctggaat 7560
accgccagat caaagcagtt gcgtatgaac tgcttgaaaa acatctgccg gaagaagatt 7620
ttgaaagagc aagacgcttt ctttaccaat gttttaaacg cacagataca ttaccggctg 7680
gcgcctggct ggattttgca gcggatggaa gaatgagacg cttatttaca ctgcgccagt 7740
actttggaat cctgcataga ctgctgaaaa accgctaatg a 7781
<210> 37
<211> 33
<212> DNA
<213> 人工序列(未知)
<400> 37
ggctttcgtt gtcttgtgtt caagaaattt cca 33
<210> 38
<211> 56
<212> DNA
<213> 人工序列(未知)
<400> 38
ctgtgtgaaa ttgttatccg ctcccagcct ttcttattaa aaccactttg tcagcc 56
<210> 39
<211> 54
<212> DNA
<213> 人工序列(未知)
<400> 39
aagcccgcct aatgagcggg cttttttatc ataaagatcc agcctttttg cgct 54
<210> 40
<211> 32
<212> DNA
<213> 人工序列(未知)
<400> 40
ctaaagccca agtcacaata tattgatcgc ct 32
<210> 41
<211> 51
<212> DNA
<213> 人工序列(未知)
<400> 41
gttttaataa gaaaggctgg gagcggataa caatttcaca caggaaacag c 51
<210> 42
<211> 57
<212> DNA
<213> 人工序列(未知)
<400> 42
aaagaaatga catgattttg catgtgtaca ttcctctctt acctataatg gtaccgc 57
<210> 43
<211> 55
<212> DNA
<213> 人工序列(未知)
<400> 43
aggtaagaga ggaatgtaca catgcaaaat catgtcattt ctttagcatc agcag 55
<210> 44
<211> 55
<212> DNA
<213> 人工序列(未知)
<400> 44
cccgctcatt aggcgggctt catcattact gaaacggaac gataaactgt tcgcg 55
<210> 45
<211> 31
<212> DNA
<213> 人工序列(未知)
<400> 45
aacctccttg tcttctttcc agtcttatct c 31
<210> 46
<211> 29
<212> DNA
<213> 人工序列(未知)
<400> 46
atcacttctc tttctttcac gctcatcct 29
<210> 47
<211> 3627
<212> DNA
<213> 人工序列(未知)
<400> 47
aacctccttg tcttctttcc agtcttatct ctaggatgaa attggaggaa gaataacgtg 60
catgtcatta caacacaagt actttttatt ttttgttttt tattgctgat tcactcgata 120
gaaaccttag cctatgcgac aaggctttcc ggagctcgcg ttggatttat tgcgtccgcg 180
ctttctctgt ttaatgtcat ggtcatcgta tccagaatgt cgaatatggt gcagcagccc 240
tttactgggc atttaattga tgatgctgga aaaaacgcac tggcgattgt aggggagcag 300
ttccgctttt taattttcgg atcgacagtc ggcaccattt tgggcattat cctgctcccg 360
tcttttgtcg ctcttttttc acgggcgatt attcacttgg cgggcggcgg cggctccgtt 420
tttcaagtat tccgaaaggg attctcgaaa caaggattca aaaatgccct ttcctatttg 480
cgtctgccgt ccatttcata tgtaaaagga tttcatatgc gcttgattcc gaagcgtttg 540
tttgtcatca acatgctgat cacatcgatt tatacgattg gtgtgctttc ggctttatac 600
gcaggccttt tggcgccgga gcgcagcacg acagccgtca tggcttcggg tttgatcaac 660
ggaattgcaa cgatgctgct ggctattttt gttgatccta aggtatccgt tcttgctgat 720
gatgtggcaa aaggaaaacg aagctatatc tatttaaaat ggacctctgt cacaatggtc 780
acatcaaggg tggcgggcac actcctcgcc cagctcatgt ttattcccgg ggcctactat 840
atcgcgtggc tgacaaagtg gttttaataa gaaaggctgg gagcggataa caatttcaca 900
caggaaacag ctatgaccat gattacgaat tcgagctcgg tacccgggga tcctctagag 960
ataccgttcg tatagcatac attatacgaa gttatcttga tatggctttt tatatgtgtt 1020
actctacata cagaaaggag gaactaaaca tggccaagtt gaccagtgcc gttccggtgc 1080
tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc 1140
gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca 1200
gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc 1260
tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg 1320
ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc 1380
cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgaataact tcgtatagca 1440
tacattatac gaacggtaaa tcgtcgactg ataggtggta tgttttcgct tgaactttta 1500
aatacagcca ttgaacatac ggttgattta ataactgaca aacatcaccc tcttgctaaa 1560
gcggccaagg acgccgccgc cggggctgtt tgcgttcttg ccgtgatttc gtgtaccatt 1620
ggtttactta tttttttgcc aaggctgtaa tggctgaaaa ttcttacatt tattttacat 1680
ttttagaaat gggcgtgaaa aaaagcgcgc gattatgtaa aatataaagt gatagcggta 1740
ccattatagg taagagagga atgtacacat gcaaaatcat gtcatttctt tagcatcagc 1800
agcggaaaga cgcgctcata ttgccgatac atttggcaga catggaatcc cgtttcaatt 1860
tttcgatgcg cttatgccgt cagaacgctt agaacaggca atggcggaat tagttccggg 1920
cctgtcagct catccgtatc ttagcggagt ggaaaaagca tgctttatga gccatgcggt 1980
cttatggaaa caagctcttg atgaaggcct gccgtacatc acagtttttg aagatgatgt 2040
gctgcttggc gaaggagccg aaaaatttct ggcagaagat gcgtggcttc aggaaagatt 2100
tgatccggat acagcattta tcgtgcgctt agaaacaatg tttatgcatg tcctgacatc 2160
accgagcggc gttgccgatt attgtggaag agcatttccg ttactggaat ctgaacattg 2220
gggcacagcg ggatacatca tctcaagaaa agctatgaga tttttcctgg atagatttgc 2280
tgcccttccg ccggaaggct tacatccggt tgatctgatg atgttttctg atttctttga 2340
tcgcgaagga atgccggtgt gccaactgaa tccggctctt tgtgcccagg aacttcatta 2400
cgccaaattt catgatcaaa acagcgcact gggatctctt atcgaacatg atagacttct 2460
gaaccgcaaa caacagagac gcgatagccc ggcgaacaca tttaaacata gattaattcg 2520
cgctctgaca aaaatctcta gagaacgcga aaaaagacgc caaagacgcg aacagtttat 2580
cgttccgttt cagtaatgat gaagcccgcc taatgagcgg gcttttttat cataaagatc 2640
cagccttttt gcgcttcctt atttatagtt ccttaatccg cggtcgtaca aaaaatttcc 2700
gaatgggacg aaagcggcga tgaagccggc agctgaccat ttcagcggcc atttgacaga 2760
gaaggttgca taagccaaga caagcaaata caaaatgaac aacccgccgt gaaccgaacc 2820
gacaattgtc accgcaagcg gaaggcctgc ccaatattta agcggcatag cgatgaacaa 2880
caggattaag agtgacattc cttcaataaa acccatcgtg cgaagtcttc cgatcggcgt 2940
gtgcagcata aatcgccctc cttgtggaca cgttttcatt ttatactata aacaatccgg 3000
gggggcatat gacagctttc aaaaaatgtt cggaaaacat tcatttttac atgccttttc 3060
tagggaactg tacttgtcat ttacaaaaat acccgagata atgtgtacaa aatcaaaaaa 3120
gaaggatgtt gaaatgaaac ttgaccagat tgatctgaat atcattgagg agctgaagaa 3180
ggacagccgt ttgtcgatga gggaattagg cagaaaaatt aagctgtcgc ctccatctgt 3240
aacagaacgg gtaagacagc ttgaatcgtt tggcatcatc aagcaataca cgctggaggt 3300
cgaccagaaa aaactggggc ttcccgtttc ctgcattgtg gaagcaaccg ttaaaaacgc 3360
ggattatgag cggttcaaaa gctatattca aacattgccg aatattgaat tttgctaccg 3420
gattgcgggt gcagcctgct atatgctgaa aatcaatgcc gaaagcctcg aagcggtaga 3480
agatttcatt aacaaaacat cgccctacgc gcaaaccgtc actcacgtca ttttctcaga 3540
aattgacacg aaaaacgggc gcggttagag agtgccgcgc gaagtctgtt ataataacag 3600
gatgagcgtg aaagaaagag aagtgat 3627
<210> 48
<211> 35
<212> DNA
<213> 人工序列(未知)
<400> 48
catccagcgt aaaacgttca cgggaataat ctagg 35
<210> 49
<211> 50
<212> DNA
<213> 人工序列(未知)
<400> 49
ttatccgctc tggcgcggac ttgtttgttt atatccattc taaatgaagg 50
<210> 50
<211> 59
<212> DNA
<213> 人工序列(未知)
<400> 50
gcccgcctaa tgagcgggct tttttttaag acgtggactc gttttcagcc tgaaatttt 59
<210> 51
<211> 29
<212> DNA
<213> 人工序列(未知)
<400> 51
catcccagca gctgatcagg atgaattct 29
<210> 52
<211> 49
<212> DNA
<213> 人工序列(未知)
<400> 52
acaagtccgc gccagagcgg ataacaattt cacacaggaa acagctatg 49
<210> 53
<211> 56
<212> DNA
<213> 人工序列(未知)
<400> 53
agaaatgaca tgattttgca tgtgtacatt cctctcttac ctataatggt accgct 56
<210> 54
<211> 59
<212> DNA
<213> 人工序列(未知)
<400> 54
ataggtaaga gaggaatgta cacatgcaaa atcatgtcat ttctttagca tcagcagcg 59
<210> 55
<211> 57
<212> DNA
<213> 人工序列(未知)
<400> 55
gcccgctcat taggcgggct tcatcattac tgaaacggaa cgataaactg ttcgcgt 57
<210> 56
<211> 26
<212> DNA
<213> 人工序列(未知)
<400> 56
gccaggaagc cacagcacat catagc 26
<210> 57
<211> 35
<212> DNA
<213> 人工序列(未知)
<400> 57
ttcgttcatt cgttcccgtt tacagaacaa atagc 35
<210> 58
<211> 4150
<212> DNA
<213> 人工序列(未知)
<400> 58
gccaggaagc cacagcacat catagccctg catgcgtttc attcttgtta caatgtcttg 60
cagcgttgtg tcccatgcgt ggccaaggtg aagtctccct gttacgtttg gcggcgggat 120
gacaacagag tatggctctt ttgtctggtc actgcccgct tcaaaaaatt tgccttttag 180
ccaaaaatca tatcggtctt tttcaaccgc tgccggatca tatttcgtcg gcattgtttg 240
ttcattcgtt tccatcataa ccctccagca tgtttaaaaa tggctgagaa cgaaaaaaga 300
gctcttcatc cctgtaaaag gacgaaaagc tctttcgcgg taccaccttt tttcatgaac 360
tcaatcagtt catgcactta taacggataa cgggtgtttc ccgaatttct ctactcttct 420
ttcaagaaat tatgctcccg ggcgaccttc caatcagaca acataagaag cctttcagca 480
aacggcttct ctctctgcat gcgccttttt tgtactcttc ccgttctaag caggtgtatt 540
tatcagctca ttgtatacat tatttatgtt ctttattata gtaaaagtgt ttttgattcg 600
tcaatcatct tttccaaaaa caccgctttc tatacagatg ggcaggcacc tattgcccaa 660
gtttacatat gatgcttgta aatgcgtaga ggaggatgca ccgtgaagaa acgtttcagc 720
tcttattcgc tgccgccatg ggtaaggcaa attcggcttg tatccgcaca agtgattatt 780
cccattacga tttttcaagg aatcagaacc attttctttc cgacaacctt tgatgttttg 840
ctgctcgcaa tcctaatctt tttagcttgc gcccttcatt tagaatggat ataaacaaac 900
aagtccgcgc cagagcggat aacaatttca cacaggaaac agctatgacc atgattacga 960
attcgagctc ggtacccggg gatcctctag agattgtacc gttcgtatag catacattat 1020
acgaagttat gccatagtga ctggcgatgc tgtcggaatg gacgacggca atagttaccc 1080
ttattatcaa gataagaaag aaaaggattt ttcgctacgc tcaaatcctt taaaaaaaca 1140
caaaagacca cattttttaa tgtggtcttt tattcttcaa ctaaagcacc cattagttca 1200
acaaacgaaa attggataaa gtgggatatt tttaaaatat atatttatgt tacagtaata 1260
ttgactttta aaaaaggatt gattctaatg aagaaagcag acaagtaagc ctcctaaatt 1320
cactttagat aaaaatttag gaggcatatc aaatgaactt taataaaatt gatttagaca 1380
attggaagag aaaagagata tttaatcatt atttgaacca acaaacgact tttagtataa 1440
ccacagaaat tgatattagt gttttatacc gaaacataaa acaagaagga tataaatttt 1500
accctgcatt tattttctta gtgacaaggg tgataaactc aaatacagct tttagaactg 1560
gttacaatag cgacggagag ttaggttatt gggataagtt agagccactt tatacaattt 1620
ttgatggtgt atctaaaaca ttctctggta tttggactcc tgtaaagaat gacttcaaag 1680
agttttatga tttatacctt tctgatgtag agaaatataa tggttcgggg aaattgtttc 1740
ccaaaacacc tatacctgaa aatgcttttt ctctttctat tattccatgg acttcattta 1800
ctgggtttaa cttaaatatc aataataata gtaattacct tctacccatt attacagcag 1860
gaaaattcat taataaaggt aattcaatat atttaccgct atctttacag gtacatcatt 1920
ctgtttgtga tggttatcat gcaggattgt ttatgaactc tattcaggaa ttgtcagata 1980
ggcctaatga ctggctttta taatatgaga taatgccgac tgtacttttt acagtcggtt 2040
ttctaacgat acattaatag gtacgaaaaa gcaacttttt ttgcgcttaa aaccagtcat 2100
accaataaat aacttcgtat agcatacatt atacgaacgg tatgataggt ggtatgtttt 2160
cgcttgaact tttaaataca gccattgaac atacggttga tttaataact gacaaacatc 2220
accctcttgc taaagcggcc aaggacgccg ccgccggggc tgtttgcgtt cttgccgtga 2280
tttcgtgtac cattggttta cttatttttt tgccaaggct gtaatggctg aaaattctta 2340
catttatttt acatttttag aaatgggcgt gaaaaaaagc gcgcgattat gtaaaatata 2400
aagtgatagc ggtaccatta taggtaagag aggaatgtac acatgcaaaa tcatgtcatt 2460
tctttagcat cagcagcgga aagacgcgct catattgccg atacatttgg cagacatgga 2520
atcccgtttc aatttttcga tgcgcttatg ccgtcagaac gcttagaaca ggcaatggcg 2580
gaattagttc cgggcctgtc agctcatccg tatcttagcg gagtggaaaa agcatgcttt 2640
atgagccatg cggtcttatg gaaacaagct cttgatgaag gcctgccgta catcacagtt 2700
tttgaagatg atgtgctgct tggcgaagga gccgaaaaat ttctggcaga agatgcgtgg 2760
cttcaggaaa gatttgatcc ggatacagca tttatcgtgc gcttagaaac aatgtttatg 2820
catgtcctga catcaccgag cggcgttgcc gattattgtg gaagagcatt tccgttactg 2880
gaatctgaac attggggcac agcgggatac atcatctcaa gaaaagctat gagatttttc 2940
ctggatagat ttgctgccct tccgccggaa ggcttacatc cggttgatct gatgatgttt 3000
tctgatttct ttgatcgcga aggaatgccg gtgtgccaac tgaatccggc tctttgtgcc 3060
caggaacttc attacgccaa atttcatgat caaaacagcg cactgggatc tcttatcgaa 3120
catgatagac ttctgaaccg caaacaacag agacgcgata gcccggcgaa cacatttaaa 3180
catagattaa ttcgcgctct gacaaaaatc tctagagaac gcgaaaaaag acgccaaaga 3240
cgcgaacagt ttatcgttcc gtttcagtaa tgatgaagcc cgcctaatga gcgggctttt 3300
ttttaagacg tggactcgtt ttcagcctga aattttttct cttgttcaat ctgatgaatt 3360
ttcatcacaa acggctctat gtttttcatt tgccaataag cctttaataa ttgtgtacat 3420
tcctcttttt catgctgtct tttcccagtt tgataaccgt tcagcacctt gtacatggct 3480
gccggatatg ccatatagct gagaaaaagg ctgacttccg cttcacgcaa agggaatgat 3540
ttcgtatatc cataaaacca ttcgacgcat tccggacaag ctttcggaaa gcctctgaac 3600
atttttgtat aaaagccgag caggtcgttt tgcggcggac cgacagatgc tctttcgaag 3660
tttgtaaaat atccggtccc ggcatcatta tacagaaaat ggtgtataga caggctgccg 3720
tggttcatta caacccttga aacgtccttt tcctttgcgg actcatacca gtcctccagc 3780
cgttccagcg caaagttcac cgcggaaatg gtttcagaaa aataggttat ggcctggagt 3840
tcaaagggag aaaggtacca ttccttctcc gctctatcca caaactgctc atagaaaatt 3900
ttatcttgtt cccacttttt tttcgtttgc ccgtaataac gttcaatctc atcacgtcta 3960
acctttagtt cctgcgcggt tctctcgtga agacgggctg tttctctaaa taaataagca 4020
tgtttttgat cccgttcttc ctcttggtca aattggagcc aaggcattaa atagtagata 4080
tctccgtatt gtatgcctgc tgagaaaaac tcaccgctat ttgttctgta aacgggaacg 4140
aatgaacgaa 4150
<210> 59
<211> 29
<212> DNA
<213> 人工序列(未知)
<400> 59
cgaaggaatg ccggtgtgcc aactgaatc 29
<210> 60
<211> 30
<212> DNA
<213> 人工序列(未知)
<400> 60
cgacctccag cgtgtattgc ttgatgatgc 30
<210> 61
<211> 30
<212> DNA
<213> 人工序列(未知)
<400> 61
gggcacagcg ggatacatca tctcaagaaa 30
<210> 62
<211> 29
<212> DNA
<213> 人工序列(未知)
<400> 62
tgaaccacgg cagcctgtct atacaccat 29
Claims (9)
1.一种合成乳酰-N-新四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宿主菌Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK的基因组上整合两个β-1,4-半乳糖转移酶基因,并外源表达β-1,3-N-葡糖氨基转移酶基因得到的。
2.根据权利要求1所述的合成乳酰-N-新四糖的重组枯草芽孢杆菌,其特征在于,所述宿主菌是以枯草芽孢杆菌168为出发菌株,通过以启动子PxylA调控基因comK的表达,乳糖透性酶编码基因整合到基因组amyE位点,β-1,4-半乳糖转移酶编码基因整合到基因组ydeS位点和ydzO位点中间得到的。
3.权利要求1所述的合成乳酰-N-新四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)通过融合PCR构建包含ydaH,ydzA基因同源臂、β-1,4-半乳糖转移酶基因、P43启动子、博来霉素抗性基因序列的重组片段;
(2)通过融合PCR构建包含yszA,ysxE基因同源臂、β-1,4-半乳糖转移酶基因、P43启动子、氯霉素抗性基因序列的重组片段;
(3)将步骤(1)构建的重组片段转化Bacillus subtilis 168△amyE:P43-lacY,P43-lgtB,PxylA-comK,通过验证获得重组枯草芽孢杆菌BY03a;
(4)将步骤(2)构建的重组片段转化步骤(3)的重组枯草芽孢杆菌BY03a,通过验证获得重组枯草芽孢杆菌BY03b;
(5)将pP43NMK-lgtA转化重组枯草芽孢杆菌BY03b,得到合成乳酰-N-新四糖的重组枯草芽孢杆菌BY05。
4.根据权利要求3所述的合成乳酰-N-新四糖的重组枯草芽孢杆菌的构建方法,其特征在于,步骤(1)中,使用的是ydaH基因左侧同源臂,ydzA基因右侧同源臂。
5.根据权利要求3所述的合成乳酰-N-新四糖的重组枯草芽孢杆菌的构建方法,其特征在于,步骤(2)中,使用的是yszA基因左侧同源臂,ysxE基因右侧同源臂。
6.根据权利要求3所述的合成乳酰-N-新四糖的重组枯草芽孢杆菌的构建方法,其特征在于,步骤(1)构建的重组片段序列如SEQ ID NO.47所示,步骤(2)构建的重组片段序列如SEQ ID NO.58所示。
7.根据权利要求3所述的合成乳酰-N-新四糖的重组枯草芽孢杆菌的构建方法,其特征在于,步骤(3)中,步骤(1)构建的重组片段中的β-1,4-半乳糖转移酶基因、P43启动子、博来霉素抗性基因序列通过同源重组整合到基因组ydaH位点和ydzA位点中间。
8.根据权利要求3所述的合成乳酰-N-新四糖的重组枯草芽孢杆菌的构建方法,其特征在于,步骤(4)中,步骤(2)构建的重组片段中β-1,4-半乳糖转移酶基因、P43启动子、氯霉素抗性基因序列通过同源重组整合到基因组yszA位点和ysxE位点中间。
9.权利要求1所述的重组枯草芽孢杆菌在发酵生产乳酰-N-新四糖中的应用。
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