CN112625994B - 一种重组需钠弧菌及其应用 - Google Patents

一种重组需钠弧菌及其应用 Download PDF

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CN112625994B
CN112625994B CN202110008125.9A CN202110008125A CN112625994B CN 112625994 B CN112625994 B CN 112625994B CN 202110008125 A CN202110008125 A CN 202110008125A CN 112625994 B CN112625994 B CN 112625994B
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陈振
张冶
刘德华
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Guangdong Tsinghua Smart Biotech Co ltd
Tsinghua University
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Abstract

本发明涉及生物技术领域，具体涉及一种重组需钠弧菌及其应用。本发明提供一种重组需钠弧菌，所述重组需钠弧菌表达甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶，且所述重组需钠弧菌与野生型需钠弧菌相比，具有降低的转录调控因子的表达和/或酶活性，所述转录调控因子为arcA和/或glpR。本发明通过改造需钠弧菌，使其能够高效利用甘油发酵生产1,3‑丙二醇，1,3‑丙二醇的产量高，副产物少，提取分离过程简单；而且，使用需钠弧菌解决了传统1,3‑丙二醇生产菌株潜在的生物安全性问题以及菌株对粗甘油中盐的耐受性较低的问题，具有重要的工业应用价值。

Description

一种重组需钠弧菌及其应用
技术领域
本发明涉及生物技术领域，具体涉及一种重组需钠弧菌及其应用。
背景技术
1,3-丙二醇是一种重要的化工原料，可作为有机溶剂应用于油墨、印染、涂料、润滑剂、抗冻剂等行业，其最主要的用途是作为聚酯和聚氨酯合成的单体，特别是与对苯二甲酸聚合生成聚对苯二甲酸丙二酯(PTT)。PTT与PET(聚对苯二甲酸乙二醇酯)、PBT(聚对苯二甲酸丁二醇酯)相比具有更优良的性能，例如：更好的耐污染性、韧性和回弹性以及抗紫外性能等，此外，还具有耐磨、吸水性低、低静电等优点。因此PTT被认为是PET的升级产品，具有广阔的市场前景。
目前，1,3-丙二醇的生产方法主要是利用微生物转化甘油或者葡萄糖进行生产。近年来，由于生物柴油产业的快速发展，作为生物柴油副产物的甘油的价格急剧下降，使得利用粗甘油发酵生产1,3-丙二醇的方法具有重要的工业应用价值。目前利用甘油生产1,3-丙二醇主要是利用一些天然的微生物如克雷伯氏肺炎杆菌(Klebsiella pneumoniae)、丁酸梭状芽胞杆菌(Clostridium butyricum)及弗氏柠檬酸菌(Citrobacter freundii)等在厌氧或者微氧的条件下进行。目前甘油法生产1,3-丙二醇工艺路线的主要缺点是：(1)常用的1,3-丙二醇生产菌株如克雷伯氏肺炎杆菌等为条件致病菌，其生产过程中生物安全性需要严格控制；(2)大量副产物如乙酸、乳酸，丁二酸，2,3-丁二醇的合成，使得整个后提取过程变得十分复杂；(3)粗甘油中含有高浓度的NaCl，会抑制菌体的生产性能，从而降低利用粗甘油生产1,3-丙二醇的产量。
发明内容
本发明的目的是提供一种重组需钠弧菌，该菌株能够利用甘油高效生产1,3-丙二醇。本发明的另一目的是提供该重组需钠弧菌的应用以及制备1,3-丙二醇的方法。
为实现上述目的，本发明开发能够以甘油(尤其是粗甘油)为底物，具有较高1,3-丙二醇产量和较低副产物的重组微生物。在众多的微生物中，本发明选择需钠弧菌(Vibrionatriegens)作为出发菌株构建能够产生1,3-丙二醇的重组需钠弧菌，需钠弧菌是目前已知的生长最快的微生物，且能耐受高浓度盐，但是其本身并不能合成1,3-丙二醇。本发明首先将1,3-丙二醇合成模块导入需钠弧菌中，但发现仅导入合成模块的重组菌的1,3-丙二醇产量很低，在进一步对菌株进行代谢工程改进的过程中，本发明创造性地发现了能够有效促进1,3-丙二醇积累的改造靶点，并通过多个改造靶点的组合和适配性优化，显著提高了需钠弧菌利用甘油发酵生产1,3-丙二醇的性能。
具体地，本发明提供以下技术方案：
第一方面，本发明提供一种重组需钠弧菌，该重组需钠弧菌表达甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶，且与野生型需钠弧菌相比，该需钠弧菌具有降低的转录调控因子的表达和/或酶活性，所述转录调控因子为arcA和/或glpR。
本发明发现，单独降低需钠弧菌的转录调控因子arcA、glpR的表达量或活性，均能够显著促进需钠弧菌合成1,3-丙二醇，而且，将两者组合改造，同时降低转录调控因子arcA和glpR的表达量或活性，促进需钠弧菌合成1,3-丙二醇的效果更优。
以上所述的转录调控因子arcA的氨基酸序列如SEQ ID NO.11所示，转录调控因子glpR的氨基酸序列如SEQ ID NO.12所示。
因此，具体地，本发明提供以下任一种重组需钠弧菌：
(1)该重组需钠弧菌表达甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶，且与野生型需钠弧菌相比，该需钠弧菌具有降低的转录调控因子arcA的表达和/或酶活性；
(2)该重组需钠弧菌表达甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶，且与野生型需钠弧菌相比，该需钠弧菌具有降低的转录调控因子glpR的表达和/或酶活性；
(3)该重组需钠弧菌表达甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶，且与野生型需钠弧菌相比，该需钠弧菌具有降低的转录调控因子arcA和glpR的表达和/或酶活性。
为配合arcA、glpR的靶点改造，本发明进一步对1,3-丙二醇合成模块(PDO)的酶的选择进行了优化。
具体地，所述甘油脱水酶和所述甘油脱水酶激活因子来源于克雷伯氏肺炎杆菌。所述醇脱氢酶来源于大肠杆菌。
优选地，所述甘油脱水酶DhaBCE的氨基酸序列如SEQ ID NO.1-3所示，所述甘油脱水酶激活因子OrfXY的氨基酸序列如SEQ ID NO.4-5所示，所述醇脱氢酶YqhD的氨基酸序列如SEQ ID NO.6所示。
以上所述的甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的表达可通过如下(1)、(2)中的一种或多种方式实现：
(1)导入携带甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因的表达质粒；
(2)在基因组上整合一个或多个拷贝的甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因。
对于导入携带上述酶的编码基因的质粒的方式，这些编码基因可存在于同一质粒上，或者分别存在于不同的质粒上。
对于表达质粒，本发明没有特别的限制，只要能够在需钠弧菌中克隆、进行基因表达的质粒均可使用。对于质粒的拷贝数，本发明没有特别的限制，可为高拷贝质粒、中拷贝质粒或低拷贝质粒。优选采用拷贝数为5～100的质粒，例如：pXMJ19、pTrc99a等。
作为本发明的优选方案，将甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因置于一个质粒上进行表达，质粒的拷贝数为20～50。
具体地，将甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因置于pTrc99a上，从距离pTrc99a多克隆位点区启动子由近至远的方向，依次为甘油脱水酶编码基因、甘油脱水酶激活因子编码基因和醇脱氢酶编码基因。
对于在基因组上整合上述酶的编码基因的方式，整合编码基因的拷贝数至少为1个，对于整合拷贝数的具体数量，本发明没有特别的限制，优选的整合拷贝数为1～10个。
本发明所述的表达和/或酶活性的降低可通过如下(1)、(2)中的一种或多种方式实现：
(1)对所述转录因子的编码基因进行一个或多个碱基的插入、缺失或替换以使得转录因子失活、表达量或活性降低；
(2)将所述转录因子的编码基因的转录或翻译调控元件替换为活性更低的调控元件。
上述重组需钠弧菌能够利用甘油发酵生产1,3-丙二醇，具有较高的产量，但是，由于携带表达质粒，在发酵时需要添加抗生素维持质粒的稳定性。本发明进一步提供了一种重组需钠弧菌，该菌株在发酵时不需要添加抗生素维持质粒的稳定性。本发明对有利于维持表达质粒稳定性的改造靶点进行了大量筛选，最终发现将甘油-3-磷酸脱氢酶失活并将其在质粒在表达质粒上表达，能够显著提高该表达质粒的稳定性，使得在不添加抗生素进行发酵的情况下，该质粒也能够在发酵过程中稳定存在。
基于此，以上所述的重组需钠弧菌的基因组甘油-3-磷酸脱氢酶编码基因被失活，且携带含有甘油-3-磷酸脱氢酶编码基因的表达质粒。
优选地，所述表达质粒上还含有甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因。
以上所述的甘油-3-磷酸脱氢酶的氨基酸序列如SEQ ID NO.7所示。
在上述重组需钠弧菌的基础上，可进一步引入分子伴侣phaP、转氢酶pntAB的改造，提高重组需钠弧菌中分子伴侣phaP、转氢酶pntAB的表达能够进一步提高1,3-丙二醇的产量并降低发酵副产物。
具体地，所述重组需钠弧菌与野生型需钠弧菌相比，还具有提高的分子伴侣phaP和/或转氢酶pntAB的表达量和/或酶活性。
分子伴侣phaP、转氢酶pntAB的表达量的提高可通过如下(1)、(2)中的一种或多种方式实现：
(1)导入携带甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因的表达质粒；
(2)在基因组上整合一个或多个拷贝的甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因。
分子伴侣phaP、转氢酶pntAB的酶活性的提高可利用目前已报道的以上蛋白的突变体，或者利用本领域常规技术手段对以上蛋白进行突变，获得活性提高的突变体。
以上所述的分子伴侣PhaP的氨基酸序列如SEQ ID NO.8所示，所述转氢酶PntAB的氨基酸序列如SEQ ID NO.9-10所示。
作为本发明的一种实施方式，本发明提供一种重组需钠弧菌，该重组需钠弧菌中，转录调控因子arcA和glpR均失活，基因组甘油-3-磷酸脱氢酶编码基因glpD失活，且携带表达甘油脱水酶、甘油脱水酶激活因子、醇脱氢酶以及分子伴侣phaP、转氢酶pntAB和甘油-3-磷酸脱氢酶编码基因glpD的质粒。
作为本发明的另一种实施方式，本发明提供一种重组需钠弧菌，该重组需钠弧菌中，转录调控因子arcA和glpR均失活，且携带表达甘油脱水酶、甘油脱水酶激活因子、醇脱氢酶以及分子伴侣phaP、转氢酶pntAB的质粒。
作为本发明的另一种实施方式，本发明提供一种重组需钠弧菌，该重组需钠弧菌中，转录调控因子arcA和glpR均失活，基因组甘油-3-磷酸脱氢酶编码基因glpD失活，且携带表达甘油脱水酶、甘油脱水酶激活因子、醇脱氢酶和甘油-3-磷酸脱氢酶编码基因glpD的质粒。
本发明所述的重组需钠弧菌可采用本领域常用的分子生物学等技术手段(包括基因的克隆、表达、敲除等)构建得到。
第二方面，本发明提供所述重组需钠弧菌的应用，具体为以下任一种应用：
(1)在制备1,3-丙二醇或其衍生物中的应用；
(2)在用于生产1,3-丙二醇或其衍生物的微生物的遗传育种中的应用。
本发明所述的1,3-丙二醇的衍生物包括但不限于3-羟基丙酸等。
以上所述的应用具体为：利用所述重组需钠弧菌转化甘油制备1,3-丙二醇或其衍生物。
第三方面，本发明提供一种制备1,3-丙二醇的方法，该方法为利用所述重组需钠弧菌转化甘油生成1,3-丙二醇。
具体地，所述方法包括：在包含甘油的培养基中，对所述重组需钠弧菌进行培养，收集培养液并分离得到1,3-丙二醇。
优选地，所述培养为在28-37℃(更优选35-37℃)、pH 5-8(更优选pH 6-7)、溶氧10％以上的条件下培养。
发酵过程的pH可通过流加NaOH控制，溶氧可通过改变转数控制。
优选地，所述培养所使用的培养基包括如下组分：甘油10-100g/L，(NH4)2SO4 2-10g/L，KH2PO4 0.5-5.0g/L，MgSO4 0.5-3g/L，NaCl 2-8g/L，MnCl2 0.005-0.02g/L，CaCl20.005-0.02g/L，FeSO4 0.005-0.02g/L,酵母粉2-8g/L，维生素B12 0.005-0.02g/L。
为降低1,3-丙二醇的生产成本，所述甘油可以为未经精制处理、含高浓度NaCl的粗甘油。
本发明所述的粗甘油是指生物柴油生产过程中所获得的副产的甘油，一般含甘油含量在50％-80％，同时含有脂肪酸、NaCl等杂质。
本发明的有益效果在于：本发明通过改造需钠弧菌，使其能够高效利用甘油(尤其是粗甘油)发酵生产1，3-丙二醇，1，3-丙二醇的产量高，副产物少，提取分离过程简单；而且，使用需钠弧菌解决了传统1,3-丙二醇生产菌株潜在的生物安全性问题以及菌株对粗甘油中盐的耐受性较低的问题，具有重要的工业应用价值。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例使用的粗甘油中甘油的含量为80％，氯化钠的含量为3％。
以下实施例中，为构建自然感受态的需钠弧菌，便于质粒的转化，使用携带质粒pXMJ19-tfoX的需钠弧菌进行遗传改造。本领域技术人员可以理解，pXMJ19-tfoX的导入仅为了方便需钠弧菌的遗传改造，对于1,3-丙二醇的生产并无作用。若采用其他转化方法对需钠弧菌进行改造，也可使用不含有质粒pXMJ19-tfoX的需钠弧菌。
tfoX基因编码蛋白氨基酸序列如SEQ ID NO.13所示，核苷酸序列如SEQ ID NO.14所示。
质粒pXMJ19-tfoX的构建方法如下：以tfoX-F(5’-agcttgcatgcctgcaggtcgactagaaaggtgtgttgatgattaaaggatcaatggatatgaatgagcaaca-3’)和tfoX-R(5’-gatattatcgtgaggatgcgattaacgctgctgacaactttctaa cag-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到0.7kb的PCR片段。以PFL-F(5’-aagttgtcagcagcgttaatcgcatcctcacgataatatccgg-3’)和PFL-R(5’-atccgccaaaacagccaagctgttaatctttctgcgaattgagatgacgcc-3’)为引物，以质粒pSIJ8(购自Addgene)为模板，扩增得到3.7kb的PCR片段。利用Gibson组装的方法将以上两个基因片段插入到质粒pXMJ19(购自addgene)的EcoRI/XbaI酶切位点处，获得质粒pXMJ19-tfoX。将pXMJ19-tfoX转化至需钠弧菌ATCC 14048中，获得含有pXMJ19-tfoX的需钠弧菌ATCC 14048。
实施例1表达质粒的构建
1、1,3-丙二醇合成模块表达质粒的构建
将甘油脱水酶基因dhaBCE(氨基酸序列如SEQ ID NO.1-3所示，核苷酸序列如SEQID NO.15所示)、甘油脱水酶激活因子基因orfXY(氨基酸序列如SEQ ID NO.4-5所示，核苷酸序列如SEQ ID NO.16所示)和醇脱氢酶基因yqhD(氨基酸序列如SEQ ID NO.6所示，核苷酸序列如SEQ ID NO.17所示)连接至表达质粒中，构建含有甘油转化1,3-丙二醇合成模块的质粒pTrc99a-PDO，具体方法如下：
分别以dhaBCE-F(5’-aagcggcatgcatttacgttttgacggctagctcagtcctag gtacagtgctagcttcaccttttgagccgatgaacaatgaa-3’)和dhaBCE-R(5’-cgcaccaggataacgctagcactgtacctaggactgagctagccgtcaattaattcgcctgaccggccag-3’)，orfXY-F(5’-cagtcctaggtacagtgctagcgttatcctggtgcgggagagaatgatgaacaagagccaacaagttca-3’)和orfXY-R(5’-ctccttgctagcactgtacctaggactgagctagccgtcaatcaagcgcaagcatcaggcc-3’)为引物，以克雷伯氏肺炎杆菌(Klebsiella Pneumoniae)ACR30为模板扩增得到2.7kb的甘油脱水酶基因的PCR片段和3.2kb的甘油脱水酶激活因子基因的PCR片段。以大肠杆菌W3110(购自中国工业微生物菌种保藏中心)为模板，以yqhd-F(5’-ctcagtcctaggtacagtgctagcaaggagatataccatgaacaactttaatctgcacacccca-3’)和yqhd-R(5’-gtacctaggactgagctagccgtcaatgcctgacgccagaagcatt-3’)为引物扩增得到1.3kb的醇脱氢酶基因PCR片段。
以vector-F(5’-gcgtctggtaaaaaaaccgcgtaatgcaggcatgcaagcttggctgttttg-3’)和vector-R(5’-ggactgagctagccgtcaaaacgtaaatgcatgccgcttcg-3’)为引物，以质粒pTrc99a(购自addgene)为模板，扩增得到2.6kb的片段。用Gibson组装的方法将以上片段组装，获得携带甘油脱水酶基因、甘油脱水酶激活因子基因和醇脱氢酶基因的表达质粒pTrc99a-PDO，该质粒上，甘油脱水酶基因、甘油脱水酶激活因子基因和醇脱氢酶基因的连接顺序为：启动子-甘油脱水酶基因-甘油脱水酶激活因子基因-醇脱氢酶基因。
2、1,3-丙二醇合成模块以及phaP和pntAB共表达质粒的构建
将甘油脱水酶基因、甘油脱水酶激活因子基因、醇脱氢酶基因、分子伴侣基因phaP和转氢酶基因pntAB连接至表达质粒中，构建含有甘油转化1,3-丙二醇合成模块以及分子伴侣基因phaP(氨基酸序列如SEQ ID NO.8所示，核苷酸序列如SEQ ID NO.18所示)和转氢酶基因pntAB(氨基酸序列如SEQ ID NO.9-10所示，核苷酸序列如SEQ ID NO.19所示)的表达质粒pTrc99a-PDO-phaP-pntAB，具体方法如下：
分别以dhaBCE-F(5’-aagcggcatgcatttacgttttgacggctagctcagtcctag gtacagtgctagcttcaccttttgagccgatgaacaatgaa-3’)和dhaBCE-R(5’-cgcaccag gataacgctagcactgtacctaggactgagctagccgtcaattaattcgcctgaccggccag-3’)，orfXY-F(5’-cagtcctaggtacagtgctagcgttatcctggtgcgggagagaatgatgaacaagagccaacaagttca-3’)和orfXY-R(5’-ctccttgctagcactgtacctaggactgagctagccgtca atcaagcgcaagcatcaggcc-3’)为引物，以克雷伯氏肺炎杆菌(Klebsiella Pneumoniae)ACR30为模板扩增得到2.7kb的甘油脱水酶基因的PCR片段和3.2kb的甘油脱水酶激活因子基因的PCR片段。
分别以yqhd-F(5’-ctcagtcctaggtacagtgctagcaaggagatataccatgaacaactttaatctgcacacccca-3’)和yqhd-R(5’-gtacctaggactgagctagccgtcaatgcctgacgccagaagcatt-3’)，pntAB-F(5’-gcttctggcgtcaggcattgacggctagctcagtcctaggt acagtgctagcaaccgatggaagggaatatcatgc-3’)和pntAB-R(5’-atatctccttt gcaggtcgactcttacagagctttcaggattgcatccac-3’)为引物，以大肠杆菌W3110(购自中国工业微生物菌种保藏中心)为模板扩增得到1.3kb的醇脱氢酶基因PCR片段和3.0kb的NAD(P)+转氢酶(transhydrogenase)基因PCR片段。
人工合成分子伴侣phaP基因，其基因序列如SEQ ID NO.18所示。
以vector-F(5’-gcgtctggtaaaaaaaccgcgtaatgcaggcatgcaagcttggctgttttg-3’)和vector-R(5’-ggactgagctagccgtcaaaacgtaaatgcatgccgcttcg-3’)为引物，以质粒pTrc99a(购自addgene)为模板，扩增得到2.6kb的片段。用Gibson组装的方法将以上片段组装，获得携带甘油脱水酶基因、甘油脱水酶激活因子基因、醇脱氢酶基因、分子伴侣基因和转氢酶基因的表达质粒pTrc99a-PDO-phaP-pntAB，该质粒上，甘油脱水酶基因、甘油脱水酶激活因子基因、醇脱氢酶基因、分子伴侣基因和转氢酶基因的连接顺序为：启动子-甘油脱水酶基因-甘油脱水酶激活因子基因-醇脱氢酶基因-分子伴侣基因-转氢酶基因。
实施例2敲除arcA基因以提高需钠弧菌生产1,3-丙二醇的产量与得率
1、敲除arcA基因
在需钠弧菌ATCC 14048中敲除转录调控因子基因arcA(氨基酸序列如SEQ IDNO.11所示，核苷酸序列如SEQ ID NO.20所示)，arcA基因敲除方法如下：
分别以arcA-up-F(5’-ccgaatagtctgaaccgttacgacc-3’)，arcA-up-R(5’-gacagcttatcactgatcagggcggtacctaaatttgtgacaaaatt-3’)和arcA-down-F(5’-aagcagctccagcctacacgtgcgctactgcacttctgtga-3’)与arcA-down-R(5’-acgctttgtgacctcttgct-3’)为引物，以需钠弧菌ATCC 14048为模板，分别扩增得到3.0kb的PCR片段。以arcA-kana-F(5’-gtcacaaatttaggtaccgccctgatcagtgataagctgtcaaacatgag-3’)和arcA-kana-R(5’-cacagaagtgcagtagcgcacgtgtaggctggagctgcttc-3’)为引物，以质粒pSIJ8(购自Addgene)为模板扩增得到1.3kb的PCR片段。通过overlap的方法将以上三个片段连接，并将该片段通过自然转化的方法转入含有pXMJ19-tfoX的需钠弧菌ATCC 14048中。在包含100mg/L的卡那霉素LBv2平板上筛选得到抗性的菌株。挑取单克隆菌株在包含50mM鼠李糖的LBv2培养基中过夜培养，获得抗性消失的菌株，即为arcA基因敲除的需钠弧菌ATCC 14048，命名为VN△arcA。
2、菌株VN△arcA/pTrc99a-PDO的构建和发酵验证
将实施例1构建的表达质粒pTrc99a-PDO电转入VN△arcA中，在包含100mg/L氨苄青霉素的LBv2平板上获得抗性菌株，获得菌株VN△arcA/pTrc99a-PDO。
同时，将质粒pTrc99a-PDO电转入需钠弧菌ATCC 14048中，在包含100mg/L氨苄青霉素的LBv2平板上获得抗性菌株，命名为VN/pTrc99a-PDO，获得对照菌株VN/pTrc99a-PDO。
将需钠弧菌VN△arcA/pTrc99a-PDO和对照菌株VN/pTrc99a-PDO，在500ml的摇瓶中进行培养，培养基为改良M9培养基(Glycerol 20g/L，Na2HPO4·12H2O 15.11g/L,KH2PO43g/L,NH4Cl 1g/L,NaCl 15g/L,MgSO4·7H2O 2g/L,CaCl2 0.015g/L,CaCO3 30g/L,vitaminB12 0.005g/L,Ampicillin 0.100g/L,FeCl3·6H2O 0.0024g/L,ZnCl2 0.003g/L,Na2MO4·2H2O 0.003g/L,MnCl2·4H2O 0.005g/L,CoCl2·6H2O 0.003g/L,CuCl2·2H2O 0.00015g/Land H3BO30.00008g/L)，培养温度为37℃，转速200rpm，发酵周期24小时。
利用高效液相色谱检测菌株的底物代谢情况与生产1,3-丙二醇的情况。需钠弧菌VN△arcA/pTrc99a-PDO的1,3-丙二醇产量与得率分别达到6.54g/L和0.40mol/mol，而对照菌株1,3-丙二醇产量与得率分别为5.82g/L和0.36mol/mol。以上结果证实，敲除需钠弧菌的转录调节因子ArcA可以提高1,3-丙二醇的产量和得率。
3、菌株VN△arcA/pTrc99a-PDO-phaP-pntAB的构建和发酵验证
将实施例1构建的表达质粒pTrc99a-PDO-phaP-pntAB电转入VN△arcA中，在包含100mg/L氨苄青霉素的LBv2平板上获得抗性菌株，命名为VN△arcA/pTrc99a-PDO-phaP-pntAB。
同时，将质粒pTrc99a-PDO-phaP-pntAB电转入需钠弧菌ATCC 14048中，在包含100mg/L氨苄青霉素的LBv2平板上获得抗性菌株，命名为VN/pTrc99a-PDO-phaP-pntAB，获得对照菌株VN/pTrc99a-PDO-phaP-pntAB。
将需钠弧菌VN△arcA/pTrc99a-PDO-phaP-pntAB和对照菌株VN/pTrc99a-PDO-phaP-pntAB，在500ml的摇瓶中进行培养，培养基为改良M9培养基(Glycerol 20g/L，Na2HPO4·12H2O 15.11g/L,KH2PO4 3g/L,NH4Cl 1g/L,NaCl15g/L,MgSO4·7H2O 2g/L,CaCl20.015g/L,CaCO3 30g/L,vitamin B12 0.005g/L,Ampicillin 0.100g/L,FeCl3·6H2O0.0024g/L,ZnCl2 0.003g/L,Na2MO4·2H2O 0.003g/L,MnCl2·4H2O 0.005g/L,CoCl2·6H2O 0.003g/L,CuCl2·2H2O0.00015g/L and H3BO3 0.00008g/L)，培养温度为37℃，转速200rpm，发酵周期24小时。
利用高效液相色谱检测菌株的底物代谢情况与生产1,3-丙二醇的情况。需钠弧菌VN△arcA/pTrc99a-PDO-phaP-pntAB的1,3-丙二醇产量与得率分别达到7.93g/L和0.48mol/mol，而对照菌株1,3-丙二醇产量与得率分别为7.34g/L和0.44mol/mol。以上结果证实，敲除需钠弧菌的转录调节因子ArcA可以提高1,3-丙二醇的产量和得率。
实施例3敲除GlpR基因以提高需钠弧菌生产1,3-丙二醇的产量与得率
1、在需钠弧菌ATCC 14048和VN△arcA中分别敲除转录调控因子基因glpR
glpR基因敲除方法如下：
分别以glpR-up-F(5’-cggtcatccaggataaagctacagc-3’)，glpR-up-R(5’-cgaagcagctccagcctacactttcggtattctcggaatcagtgg-3’)和glpR-down-F(5’-tttgacagcttatcactgatcagttaggactccattgtgcacggg-3’)与glpR-down-R(5’-cttactccccccctttaatagagccc-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到长为3.0kb的glpR基因(氨基酸序列如SEQ ID NO.12所示，核苷酸序列如SEQ ID NO.21所示)上下游片段。以glpR-kana-F(5’-ccactgattccgagaataccgaaagtgtaggctggagctgcttc-3’)和glpR-kana-R(5’-ctacccgtgcacaatggagtcctaactgatcagtgataagctgtcaaacatga ga-3’)为引物，以质粒pSIJ8(购自Addgene)为模板扩增获得1.3kb的PCR片段。通过overlap的方法将三个片段连接，并将该片段通过自然转化的方法分别转入含有pXMJ19-tfoX的需钠弧菌ATCC 14048和VN△arcA中。在包含100mg/L的卡那霉素LBv2平板上筛选得到抗性的菌株。挑取单克隆菌株在包含50mM鼠李糖的LBv2培养基中过夜培养，获得抗性消失的菌株，分别命名为VN△glpR、VN△arcA△glpR。
2、菌株VN△glpR/pTrc99a-PDO和VN△arcA△glpR/pTrc99a-PDO的构建和发酵验证
将实施例1构建的表达质粒pTrc99a-PDO分别电转入VN△glpR和VN△arcA△glpR中，在包含100mg/L氨苄青霉素的LBv2平板上获得抗性菌株，获得菌株VN△glpR/pTrc99a-PDO和VN△arcA△glpR/pTrc99a-PDO。
将需钠弧菌VN△glpR/pTrc99a-PDO和VN△arcA△glpR/pTrc99a-PDO和对照菌株VN/pTrc99a-PDO，在500ml的摇瓶中进行培养，培养基为改良M9培养基(Glycerol 20g/L，Na2HPO4·12H2O 15.11g/L,KH2PO4 3g/L,NH4Cl 1g/L,NaCl 15g/L,MgSO4·7H2O 2g/L,CaCl20.015g/L,CaCO3 30g/L,vitamin B12 0.005g/L,Ampicillin 0.100g/L,FeCl3·6H2O0.0024g/L,ZnCl2 0.003g/L,Na2MO4·2H2O 0.003g/L,MnCl2·4H2O 0.005g/L,CoCl2·6H2O0.003g/L,CuCl2·2H2O 0.00015g/L and H3BO3 0.00008g/L)，培养温度为37度，转速200rpm，发酵周期24小时。
利用高效液相色谱检测菌株的底物代谢情况与生产1,3-丙二醇的情况。需钠弧菌VN△glpR/pTrc99a-PDO和VN△arcA△glpR/pTrc99a-PDO的1,3-丙二醇产量分别为6.28g/L和7.21g/L，得率分别为0.39mol/mol和0.44mol/mol，而对照菌株的1,3-丙二醇产量与得率分别为5.82g/L和0.36mol/mol。以上结果证实，敲除需钠弧菌的转录调节因子GlpR可以提高1,3-丙二醇的产量和得率，且若同时敲除转录调节因子GlpR与ArcA，1,3-丙二醇的产量和得率提升效果更为显著。
3、菌株VN△glpR/pTrc99a-PDO-phaP-pntAB和VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB的构建
将实施例1构建的表达质粒pTrc99a-PDO-phaP-pntAB分别电转入VN△glpR和VN△arcA△glpR中，在包含100mg/L氨苄青霉素的LBv2平板上获得抗性菌株，获得菌株VN△glpR/pTrc99a-PDO-phaP-pntAB和VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB。
将需钠弧菌VN△glpR/pTrc99a-PDO-phaP-pntAB、VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB和对照菌株VN/pTrc99a-PDO-phaP-pntAB，在500ml的摇瓶中进行培养，培养基为改良M9培养基(Glycerol 20g/L，Na2HPO4·12H2O 15.11g/L,KH2PO4 3g/L,NH4Cl 1g/L,NaCl 15g/L,MgSO4·7H2O 2g/L,CaCl2 0.015g/L,CaCO3 30g/L,vitamin B12 0.005g/L,Ampicillin 0.100g/L,FeCl3·6H2O 0.0024g/L,ZnCl2 0.003g/L,Na2MO4·2H2O 0.003g/L,MnCl2·4H2O 0.005g/L,CoCl2·6H2O 0.003g/L,CuCl2·2H2O 0.00015g/L and H3BO30.00008g/L)，培养温度为37度，转速200rpm，发酵周期24小时，利用高效液相色谱检测菌株的底物代谢情况与生产1,3-丙二醇的情况。需钠弧菌VN△glpR/pTrc99a-PDO-phaP-pntAB与VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB的1,3-丙二醇产量分别为7.45g/L和8.32g/L，得率分别为0.45mol/mol和0.50mol/mol而对照菌株1,3-丙二醇产量与得率分别为7.34g/L和0.44mol/mol。本发明证实敲除需钠弧菌的转录调节因子GlpR可以提高1,3-丙二醇的产量和得率，且若同时敲除转录调节因子GlpR与ArcA，效果更为显著。
实施例4敲除glpD基因并在质粒上互补表达glpD基因以提高质粒的稳定性和减少抗生素的使用
在1,3-丙二醇的发酵罐放大实验中发现，即使在培养基中添加抗生素，所用菌株也很快出现质粒丢失的情况。本发明通过敲除需钠弧菌中的甘油代谢的关键酶甘油-3-磷酸脱氢酶基因glpD(氨基酸序列如SEQ ID NO.7所示，核苷酸序列如SEQ ID NO.22所示)，并在质粒上互补表达glpD基因，使其在保持甘油代谢能力的同时，提高质粒稳定性，避免抗生素的使用。同时，对比了glpD基因表达强度的变化对1,3-丙二醇产量和得率的影响。
1、glpD基因敲除
glpD基因敲除方法如下：
分别以glpD-up-F(5’-attggcgatctttgctaactttgcc-3’)，glpD-up-R(5’-cgaagcagctccagcctacaccgttctcgcagcacgtgaaaac-3’)和glpD-down-F(5’-gacagcttatcactgatcagaaatttgacctctttggtgagcgaac-3’)与glpD-down-R(5’-tcgtgactttggaccaaaactgttcg-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到长为3kb的glpD基因上下游片段。以glpD-kana-F(5’-ttttcacgtgctgcgagaacggtgtaggctggagctgcttcg-3’)和glpD-kana-R(5’-tcaccaaagaggtcaaatttctgatcagtgataagctgtcaaacatgag-3’)为引物，以质粒pSIJ8(购自Addgene)为模板扩增获得1.3kb的PCR片段。通过overlap的方法将三个片段连接，并将该片段通过自然转化的方法转入含有pXMJ19-tfoX的需钠弧菌VN△arcA△glpR中。在包含100mg/L的卡那霉素LBv2平板上筛选得到抗性的菌株。挑取单克隆菌株在包含50mM鼠李糖的LBv2培养基中过夜培养，获得抗性消失的菌株，命名为VN△arcA△glpR△glpD。
2、glpD的互补
(1)构建质粒pTrc99a-PDO-promoter2-glpD并转入菌株VN△arcA△glpR△glpD中
glpD编码蛋白的氨基酸序列如SEQ ID NO.7所示，核苷酸序列如SEQ ID NO.22所示。
质粒pTrc99a-PDO-promoter2-glpD的构建方法如下：以Terminator2-F(5’-ggtaaaaaaaccgcgtaatgcaggcatgcacaaataaaacgaaaggctcagtcgaaag-3’)和Terminator2-R(5’-acaaaatttgtttgcagcacaaaaggcca tccgtcaggat-3’)为引物，以pTrc99a-PDO-phaP-pntAB为模板，扩增得到0.3kb的PCR片段。以Promoter2-F(5’-atcctgacggatggccttttgtgctgcaaacaaattttgtacagacaataat-3’)和Promoter2-R(5’-gcatttttttgtatactcataaaacctaacaaaaaagaaaaatcatgattg gtg-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到1.0kb的PCR片段。以glpD2-F(5’-tttcttttttgttaggttttatgagtatacaaaaaaatgcttccacaactg-3’)和glpD2-R(5’-aaaatcttctctcatccgccaaaacagccattagcccacttgtgagaggttcac-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到1.6kb的PCR片段。利用Gibson组装的方法将以上三个基因片段插入到质粒pTrc99a-PDO的HindIII酶切位点处，获得质粒pTrc99a-PDO-promoter2-glpD。
将质粒pTrc99a-PDO-promoter2-glpD电转入需钠弧菌VN△arcA△glpR△glpD，在包含100mg/L氨苄青霉素的LBv2平板上筛选抗性菌株，获得菌株VN△arcA△glpR△glpD/pTrc99a-PDO-promoter2-glpD。
(2)构建质粒pTrc99a-PDO-phaP-pntAB-promoter2-glpD并转入菌株VN△arcA△glpR△glpD中
质粒pTrc99a-PDO-phaP-pntAB-promoter2-glpD的构建方法如下：以Terminator2-F(5’-ggtaaaaaaaccgcgtaatgcaggcatgcacaaataaaacgaaaggctcagtcgaaag-3’)和Terminator2-R(5’-acaaaatttgtttgcagcacaaaaggccatccgtcaggat-3’)为引物，以pTrc99a-PDO-phaP-pntAB为模板，扩增得到0.3kb的PCR片段。以Promoter2-F(5’-atcctgacggatggccttttgtgctgcaaacaaattttgtacagacaataat-3’)和Promoter2-R(5’-gcatttttttgtatactcataaaacctaacaaaaaagaaaaatcatgattggtg-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到1.0kb的PCR片段。以glpD2-F(5’-tttcttttttgttaggttttatgagtatacaaaaaaatgcttccacaactg-3’)和glpD2-R(5’-aaaatcttctctcatccgccaaaacagccattagcccacttgtgagaggttcac-3’)为引物，以需钠弧菌ATCC 14048为模板，扩增得到1.6kb的PCR片段。利用Gibson组装的方法将以上三个基因片段插入到质粒pTrc99a-PDO-phaP-pntAB的HindIII酶切位点处，获得质粒pTrc99a-PDO-phaP-pntAB-promoter2-glpD。
将质粒pTrc99a-PDO-phaP-pntAB-promoter2-glpD电转入需钠弧菌VN△arcA△glpR△glpD，在包含100mg/L氨苄青霉素的LBv2平板上筛选抗性菌株，获得菌株VN△arcA△glpR△glpD/pTrc99a-PDO-phaP-pntAB-promoter2-glpD。
将需钠弧菌VN△arcA△glpR△glpD/pTrc99a-PDO-promoter2-glpD、VN△arcA△glpR△glpD/pTrc99a-PDO-phaP-pntAB-promoter2-glpD以及菌株VN△arcA△glpR/pTrc99a-PDO和VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB，在发酵罐中培养，培养基为改良VN培养基(粗甘油50g/L，KH2PO41g/L,(NH4)2SO4 5g/L,酵母粉5g/L,NaCl 5g/L,MgSO4·7H2O 2g/L,CaCl2 0.01g/L CoCl2·6H2O 0.01g/L,MnCl2·4H2O 0.01g/L,FeSO4·7H2O0.01g/L,vitamin B12 0.005g/L.)，实验组不添加抗生素，对照组添加100mg/L的氨苄青霉素。种子培养基为LBv2培养基，接种量10％，培养温度为37℃，使用5M氢氧化钠溶液调节pH在6.5，溶氧控制在10％。当甘油浓度低于5g/L时，脉冲式流加600g/L的甘油以维持甘油浓度在5g/L以上，利用高效液相色谱检测菌株的底物代谢情况与生产1,3-丙二醇的情况。
结果显示，发酵24h，菌株VN△arcA△glpR△glpD/pTrc99a-PDO-promoter2-glpD的1,3-丙二醇产量为42.8g/L，得率为0.56mol/mol，而菌株VN△arcA△glpR/pTrc99a-PDO的1,3-丙二醇产量为38.8g/L，得率为0.52mol/mol，两菌株在发酵过程中均没有副产物乙酸、乳酸，丁二酸，2，3-丁二醇等的积累。
发酵24h，菌株VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB的1,3-丙二醇产量为47.1g/L，得率为0.59mol/mol，而菌株VN△arcA△glpR△glpD/pTrc99a-PDO-phaP-pntAB-promoter2-glpD的1,3-丙二醇产量为62.4g/L,得率为0.61mol/mol。两菌株在发酵过程中均没有副产物乙酸、乳酸，丁二酸，2，3-丁二醇等的积累。菌株VN△arcA△glpR/pTrc99a-PDO-phaP-pntAB的质粒保存率仅为55％，而菌株VN△arcA△glpR△glpD/pTrc99a-PDO-phaP-pntAB-promoter2–glpD的质粒保存率为100％。
以上实验结果证实在质粒上互补表达甘油-3-磷酸脱氢酶可以提高需钠弧菌在以甘油为唯一碳源的培养基中的质粒保存率，且可以提高1,3-丙二醇产量和得率；同时，不依赖抗生素的1,3-丙二醇发酵体系不仅避免了抗生素的滥用，也降低了生产成本，具有重要的工业应用潜力。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 清华大学
广东清大智兴生物技术有限公司
<120> 一种重组需钠弧菌及其应用
<130> KHP201120113.1
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Met Lys Arg Ser Lys Arg Phe Ala Val Leu Ala Gln Arg Pro Val Asn
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Gln Asp Gly Leu Ile Gly Glu Trp Pro Glu Glu Gly Leu Ile Ala Met
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Asp Ser Pro Phe Asp Pro Val Ser Ser Val Lys Val Asp Asn Gly Leu
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Ile Val Glu Leu Asp Gly Lys Arg Arg Asp Gln Phe Asp Met Ile Asp
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Met Arg Leu Glu Ala Val Glu Ile Ala Arg Met Leu Val Asp Ile His
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Val Ser Arg Glu Glu Ile Ile Ala Ile Thr Thr Ala Ile Thr Pro Ala
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Ala Leu Gln Lys Met Arg Ala Arg Arg Thr Pro Ser Asn Gln Cys His
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Val Thr Asn Leu Lys Asp Asn Pro Val Gln Ile Ala Ala Asp Ala Ala
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Ala Arg Tyr Ala Pro Phe Asn Ala Leu Ala Leu Leu Val Gly Ser Gln
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Ser Val Tyr Gly Thr Glu Ala Val Phe Thr Asp Gly Asp Asp Thr Pro
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Trp Ser Lys Ala Phe Leu Ala Ser Ala Tyr Ala Ser Arg Gly Leu Lys
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Met Arg Tyr Thr Ser Gly Thr Gly Ser Glu Ala Leu Met Gly Tyr Ser
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Glu Ser Lys Ser Met Leu Tyr Leu Glu Ser Arg Cys Ile Phe Ile Thr
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Gly Met Thr Gly Ala Val Pro Ser Gly Ile Arg Ala Val Leu Ala Glu
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Asn Leu Ile Ala Ser Met Leu Asp Leu Glu Val Ala Ser Ala Asn Asp
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Gln Thr Phe Ser His Ser Asp Ile Arg Arg Thr Ala Arg Thr Leu Met
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Gln Met Leu Pro Gly Thr Asp Phe Ile Phe Ser Gly Tyr Ser Ala Val
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Pro Asn Tyr Asp Asn Met Phe Ala Gly Ser Asn Phe Asp Ala Glu Asp
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Phe Asp Asp Tyr Asn Ile Leu Gln Arg Asp Leu Met Val Asp Gly Gly
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Met Gln Gln Thr Thr Gln Ile Gln Pro Ser Phe Thr Leu Lys Thr Arg
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Glu Gly Gly Val Ala Ser Ala Asp Glu Arg Ala Asp Glu Val Val Ile
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Glu Gly Leu His Ala Arg Val Val Arg Ile Leu Arg Thr Ser Asp Val
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Ser Phe Met Ala Trp Asp Ala Ala Asn Leu Ser Gly Ser Gly Ile Gly
85 90 95
Ile Gly Ile Gln Ser Lys Gly Thr Thr Val Ile His Gln Arg Asp Leu
100 105 110
Leu Pro Leu Ser Asn Leu Glu Leu Phe Ser Gln Ala Pro Leu Leu Thr
115 120 125
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Lys Glu Ser Pro Ser Pro Val Pro Val Val Asn Asp Gln Met Val Arg
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His Val Val Gln Asp Ala Glu Pro Val Thr Leu His Val Asp Leu Val
180 185 190
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Met Ser Glu Lys Thr Met Arg Val Gln Asp Tyr Pro Leu Ala Thr Arg
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20 25 30
Thr Leu Glu Lys Val Leu Ser Gly Glu Val Gly Pro Gln Asp Val Arg
35 40 45
Ile Ser Arg Gln Thr Leu Glu Tyr Gln Ala Gln Ile Ala Glu Gln Met
50 55 60
Gln Arg His Ala Val Ala Arg Asn Phe Arg Arg Ala Ala Glu Leu Ile
65 70 75 80
Ala Ile Pro Asp Glu Arg Ile Leu Ala Ile Tyr Asn Ala Leu Arg Pro
85 90 95
Phe Arg Ser Ser Gln Ala Glu Leu Leu Val Ile Ala Asp Glu Leu Glu
100 105 110
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115 120 125
Glu Val Tyr Gln Gln Arg His Lys Leu Arg Lys Gly Ser
130 135 140
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Met Pro Leu Ile Ala Gly Ile Asp Ile Gly Asn Ala Thr Thr Glu Val
1 5 10 15
Ala Leu Ala Ser Asp Asp Pro Gln Ala Arg Ala Phe Val Ala Ser Gly
20 25 30
Ile Val Ala Thr Thr Gly Met Lys Gly Thr Arg Asp Asn Ile Ala Gly
35 40 45
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Ser Asp Val Ser Arg Ile Tyr Leu Asn Glu Ala Ala Pro Val Ile Gly
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Asp Val Ala Met Glu Thr Ile Thr Glu Thr Ile Ile Thr Glu Ser Thr
85 90 95
Met Ile Gly His Asn Pro Gln Thr Pro Gly Gly Val Gly Val Gly Val
100 105 110
Gly Thr Thr Ile Ala Leu Gly Arg Leu Ala Thr Leu Pro Ala Ala Gln
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Tyr Ala Glu Gly Trp Ile Val Leu Ile Asp Asp Ala Val Asp Phe Leu
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Asp Ala Val Trp Trp Leu Asn Glu Ala Leu Asp Arg Gly Ile Asn Val
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Val Ala Ala Ile Leu Lys Lys Asp Asp Gly Val Leu Val Asn Asn Arg
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180 185 190
Val Pro Glu Gly Val Met Ala Ala Val Glu Val Ala Ala Pro Gly Gln
195 200 205
Val Val Arg Ile Leu Ser Asn Pro Tyr Gly Ile Ala Thr Phe Phe Gly
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Leu Ser Pro Glu Glu Thr Gln Ala Ile Val Pro Ile Ala Arg Ala Leu
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Ile Gly Asn Arg Ser Ala Val Val Leu Lys Thr Pro Gln Gly Asp Val
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Gln Ser Arg Val Ile Pro Ala Gly Asn Leu Tyr Ile Ser Gly Glu Lys
260 265 270
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Thr His Ala Gly Gly Met Leu Glu Arg Val Arg Lys Val Met Ala Ser
305 310 315 320
Leu Thr Gly His Glu Met Ser Ala Ile Tyr Ile Gln Asp Leu Leu Ala
325 330 335
Val Asp Thr Phe Ile Pro Arg Lys Val Gln Gly Gly Met Ala Gly Glu
340 345 350
Cys Ala Met Glu Asn Ala Val Gly Met Ala Ala Met Val Lys Ala Asp
355 360 365
Arg Leu Gln Met Gln Val Ile Ala Arg Glu Leu Ser Ala Arg Leu Gln
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Thr Glu Val Val Val Gly Gly Val Glu Ala Asn Met Ala Ile Ala Gly
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Ala Leu Thr Thr Pro Gly Cys Ala Ala Pro Leu Ala Ile Leu Asp Leu
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Gly Ala Gly Ser Thr Asp Ala Ala Ile Val Asn Ala Glu Gly Gln Ile
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Thr Ala Val His Leu Ala Gly Ala Gly Asn Met Val Ser Leu Leu Ile
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Lys Thr Glu Leu Gly Leu Glu Asp Leu Ser Leu Ala Glu Ala Ile Lys
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Lys Tyr Pro Leu Ala Lys Val Glu Ser Leu Phe Ser Ile Arg His Glu
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Asn Gly Ala Val Glu Phe Phe Arg Glu Ala Leu Ser Pro Ala Val Phe
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Ala Lys Val Val Tyr Ile Lys Glu Gly Glu Leu Val Pro Ile Asp Asn
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Ala Ser Pro Leu Glu Lys Ile Arg Leu Val Arg Arg Gln Ala Lys Glu
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Lys Val Phe Val Thr Asn Cys Leu Arg Ala Leu Arg Gln Val Ser Pro
530 535 540
Gly Gly Ser Ile Arg Asp Ile Ala Phe Val Val Leu Val Gly Gly Ser
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Ser Leu Asp Phe Glu Ile Pro Gln Leu Ile Thr Glu Ala Leu Ser His
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580 585 590
Arg Asn Ala Val Ala Thr Gly Leu Leu Leu Ala Gly Gln Ala Asn
595 600 605
<210> 5
<211> 143
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Met Asn Lys Ser Gln Gln Val Gln Thr Ile Thr Leu Ala Ala Ala
1 5 10 15
Gln Gln Met Ala Ala Ala Val Glu Lys Lys Ala Thr Glu Ile Asn Val
20 25 30
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35 40 45
Gln Arg Met Asp Glu Ala Phe Val Ser Ser Cys Asp Ile Ser Leu Asn
50 55 60
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65 70 75 80
Ser Ala Val Gln Pro Gly Gln Ser Leu Tyr Gly Leu Gln Leu Thr Asn
85 90 95
Gln Gln Arg Ile Ile Ile Phe Gly Gly Gly Leu Pro Val Ile Phe Asn
100 105 110
Glu Gln Val Ile Gly Ala Val Gly Val Ser Gly Gly Thr Val Glu Gln
115 120 125
Asp Gln Leu Leu Ala Gln Cys Ala Leu Asp Cys Phe Ser Ala Leu
130 135 140
<210> 6
<211> 387
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys
1 5 10 15
Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val
20 25 30
Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp
35 40 45
Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly
50 55 60
Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu
65 70 75 80
Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser
85 90 95
Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110
Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys
115 120 125
Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser
130 135 140
Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys
145 150 155 160
Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp
165 170 175
Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val
180 185 190
Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val
195 200 205
Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu
210 215 220
Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val
225 230 235 240
Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile
245 250 255
Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu
260 265 270
Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val
275 280 285
Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu
290 295 300
Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp
305 310 315 320
Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln
325 330 335
Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350
Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu
355 360 365
Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu
370 375 380
Ala Ala Arg
385
<210> 7
<211> 519
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ser Ile Gln Lys Asn Ala Ser Thr Thr Asp Ser Ser Thr Pro Leu
1 5 10 15
Asp Leu Ile Ile Ile Gly Gly Gly Ile Asn Gly Ala Gly Ile Ala Ala
20 25 30
Asp Ala Ala Gly Arg Gly Leu Ser Val Gly Leu Tyr Glu Ala Asn Asp
35 40 45
Phe Ala Ser Ala Thr Ser Ser Ala Ser Ser Lys Leu Ile His Gly Gly
50 55 60
Leu Arg Tyr Leu Glu His Tyr Glu Phe Arg Leu Val Ser Glu Ala Leu
65 70 75 80
Ala Glu Arg Glu Val Ile Leu Arg Lys Ala Pro His Val Ala Leu Pro
85 90 95
Met Arg Phe Arg Leu Pro His Arg Pro Phe Leu Arg Pro Ala Trp Met
100 105 110
Ile Arg Cys Gly Leu Phe Leu Tyr Asp Asn Leu Gly Lys Arg Thr Thr
115 120 125
Leu Pro Gly Ser Lys Thr Val Asn Leu Ala Lys Ser Gly Leu Leu Lys
130 135 140
Pro Glu Ile Lys Thr Gly Phe Glu Tyr Ser Asp Cys Trp Val Asp Asp
145 150 155 160
Ala Arg Leu Val Leu Leu Asn Val Leu Ala Ala Arg Glu Asn His Ala
165 170 175
Glu Val Arg Asn Tyr Cys Arg Val Glu Lys Ala His Arg Glu Ser Gly
180 185 190
Ile Trp His Val Thr Ile His Asp Thr Met Thr Asp Gln Arg Phe Glu
195 200 205
Arg Lys Ala Lys Ala Leu Val Asn Ala Ala Gly Pro Trp Val Lys Gln
210 215 220
Phe Phe Asp Glu Gly Leu Glu Gln Ala Ser Pro Arg Asn Ile Arg Leu
225 230 235 240
Ile Lys Gly Ser His Ile Val Val Pro Arg Ile His Asn Glu Pro Gln
245 250 255
Ala Tyr Ile Leu Gln Asn Lys Asp Asn Arg Ile Val Phe Met Ile Pro
260 265 270
Tyr Leu Asp Lys Phe Ser Ile Val Gly Thr Thr Asp Val Glu Tyr Lys
275 280 285
Gly Asp Pro Arg Glu Val Ala Ile Ser Asp Asp Glu Val Asp Tyr Leu
290 295 300
Ile Asp Ile Val Asn Gln His Phe Val His Gln Leu Ser Arg Glu Asp
305 310 315 320
Val Val Trp Thr Tyr Ser Gly Val Arg Pro Leu Cys Asp Asp Glu Ser
325 330 335
Asp Ser Pro Gln Ala Ile Thr Arg Asp Tyr Thr Leu Glu Leu Asp Ala
340 345 350
Glu Phe Asp Gln Ala Pro Leu Leu Ser Val Phe Gly Gly Lys Leu Thr
355 360 365
Thr Tyr Arg Lys Leu Gly Glu Ala Ala Met Lys Lys Leu Ala Pro Phe
370 375 380
Leu Pro Gln Met Gly Gly Asn Trp Thr Ala Asn Gln Ala Leu Pro Gly
385 390 395 400
Gly Asn Phe Ser Cys Ser Arg Glu Gln Leu Ala Lys Gln Ile His Ala
405 410 415
Lys Tyr Ala Trp Ala Pro Lys Ala Leu Ile Leu Arg Tyr Val Thr Gln
420 425 430
Phe Gly Thr Gln Thr Trp Glu Leu Met Lys Gly Ala Thr Ser Glu Ala
435 440 445
Asp Leu Gly Gln Ala Phe Ser Thr Gln Ala Gly Gly Val Tyr Gln Arg
450 455 460
Glu Ile Asp Tyr Leu Met Asn His Glu Met Ala Leu Thr Asp Glu Asp
465 470 475 480
Ile Leu Trp Arg Arg Thr Lys Ile Gly Leu Tyr Met Ser Asp Glu Glu
485 490 495
Lys Leu Ser Leu Ala Glu Tyr Leu Lys Glu Lys Leu Gln Gln Lys Val
500 505 510
Val Asn Leu Ser Gln Val Gly
515
<210> 8
<211> 187
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Phe Phe Asp Leu Glu Lys Met Glu Ser Ala Ser Lys Ser Asn
1 5 10 15
Leu Asp Ala Val Gln Gln Leu Asn Ser Lys Ile Phe Glu Ser Ala Glu
20 25 30
Glu Leu Tyr Lys Leu Gln Phe Lys Thr Leu Arg Ala Ala Ala Asp Asp
35 40 45
Asn Phe Glu Ser Leu Arg Lys Leu Leu Ser Val Arg Asp Pro Gln Ala
50 55 60
Phe Leu Glu Leu Gln Ala Ser Phe Phe Lys Pro Asn Glu Gln Ala Glu
65 70 75 80
Arg Leu Val Glu Phe Ser Arg Gln Thr Tyr Asp Leu Ile Ser Arg Phe
85 90 95
Gln Ala Glu Val Thr Lys Leu Thr Glu Arg Gln Ile Glu Ile Gly Thr
100 105 110
Gln Gln Ala Gln Glu Ile Val Glu Glu Ile Cys Lys Asn Ala Pro Ala
115 120 125
Ser Ala Glu Pro Val Val Ser Val Phe Lys Ser Ala Val Glu Gly Ala
130 135 140
Gly Asn Val Tyr Glu Ser Ala Gln Lys Ala Ala Lys Gln Ala Ser Glu
145 150 155 160
Ile Thr Ala Ser Gly Ile Glu Ala Ala Ala Asn Ala Ala Gly Gln Ala
165 170 175
Ala Ala Gln Ala Ala Ser Gly Lys Lys Thr Ala
180 185
<210> 9
<211> 510
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Arg Ile Gly Ile Pro Arg Glu Arg Leu Thr Asn Glu Thr Arg Val
1 5 10 15
Ala Ala Thr Pro Lys Thr Val Glu Gln Leu Leu Lys Leu Gly Phe Thr
20 25 30
Val Ala Val Glu Ser Gly Ala Gly Gln Leu Ala Ser Phe Asp Asp Lys
35 40 45
Ala Phe Val Gln Ala Gly Ala Glu Ile Val Glu Gly Asn Ser Val Trp
50 55 60
Gln Ser Glu Ile Ile Leu Lys Val Asn Ala Pro Leu Asp Asp Glu Ile
65 70 75 80
Ala Leu Leu Asn Pro Gly Thr Thr Leu Val Ser Phe Ile Trp Pro Ala
85 90 95
Gln Asn Pro Glu Leu Met Gln Lys Leu Ala Glu Arg Asn Val Thr Val
100 105 110
Met Ala Met Asp Ser Val Pro Arg Ile Ser Arg Ala Gln Ser Leu Asp
115 120 125
Ala Leu Ser Ser Met Ala Asn Ile Ala Gly Tyr Arg Ala Ile Val Glu
130 135 140
Ala Ala His Glu Phe Gly Arg Phe Phe Thr Gly Gln Ile Thr Ala Ala
145 150 155 160
Gly Lys Val Pro Pro Ala Lys Val Met Val Ile Gly Ala Gly Val Ala
165 170 175
Gly Leu Ala Ala Ile Gly Ala Ala Asn Ser Leu Gly Ala Ile Val Arg
180 185 190
Ala Phe Asp Thr Arg Pro Glu Val Lys Glu Gln Val Gln Ser Met Gly
195 200 205
Ala Glu Phe Leu Glu Leu Asp Phe Lys Glu Glu Ala Gly Ser Gly Asp
210 215 220
Gly Tyr Ala Lys Val Met Ser Asp Ala Phe Ile Lys Ala Glu Met Glu
225 230 235 240
Leu Phe Ala Ala Gln Ala Lys Glu Val Asp Ile Ile Val Thr Thr Ala
245 250 255
Leu Ile Pro Gly Lys Pro Ala Pro Lys Leu Ile Thr Arg Glu Met Val
260 265 270
Asp Ser Met Lys Ala Gly Ser Val Ile Val Asp Leu Ala Ala Gln Asn
275 280 285
Gly Gly Asn Cys Glu Tyr Thr Val Pro Gly Glu Ile Phe Thr Thr Glu
290 295 300
Asn Gly Val Lys Val Ile Gly Tyr Thr Asp Leu Pro Gly Arg Leu Pro
305 310 315 320
Thr Gln Ser Ser Gln Leu Tyr Gly Thr Asn Leu Val Asn Leu Leu Lys
325 330 335
Leu Leu Cys Lys Glu Lys Asp Gly Asn Ile Thr Val Asp Phe Asp Asp
340 345 350
Val Val Ile Arg Gly Val Thr Val Ile Arg Ala Gly Glu Ile Thr Trp
355 360 365
Pro Ala Pro Pro Ile Gln Val Ser Ala Gln Pro Gln Ala Ala Gln Lys
370 375 380
Ala Ala Pro Glu Val Lys Thr Glu Glu Lys Cys Thr Cys Ser Pro Trp
385 390 395 400
Arg Lys Tyr Ala Leu Met Ala Leu Ala Ile Ile Leu Phe Gly Trp Met
405 410 415
Ala Ser Val Ala Pro Lys Glu Phe Leu Gly His Phe Thr Val Phe Ala
420 425 430
Leu Ala Cys Val Val Gly Tyr Tyr Val Val Trp Asn Val Ser His Ala
435 440 445
Leu His Thr Pro Leu Met Ser Val Thr Asn Ala Ile Ser Gly Ile Ile
450 455 460
Val Val Gly Ala Leu Leu Gln Ile Gly Gln Gly Gly Trp Val Ser Phe
465 470 475 480
Leu Ser Phe Ile Ala Val Leu Ile Ala Ser Ile Asn Ile Phe Gly Gly
485 490 495
Phe Thr Val Thr Gln Arg Met Leu Lys Met Phe Arg Lys Asn
500 505 510
<210> 10
<211> 462
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ser Gly Gly Leu Val Thr Ala Ala Tyr Ile Val Ala Ala Ile Leu
1 5 10 15
Phe Ile Phe Ser Leu Ala Gly Leu Ser Lys His Glu Thr Ser Arg Gln
20 25 30
Gly Asn Asn Phe Gly Ile Ala Gly Met Ala Ile Ala Leu Ile Ala Thr
35 40 45
Ile Phe Gly Pro Asp Thr Gly Asn Val Gly Trp Ile Leu Leu Ala Met
50 55 60
Val Ile Gly Gly Ala Ile Gly Ile Arg Leu Ala Lys Lys Val Glu Met
65 70 75 80
Thr Glu Met Pro Glu Leu Val Ala Ile Leu His Ser Phe Val Gly Leu
85 90 95
Ala Ala Val Leu Val Gly Phe Asn Ser Tyr Leu His His Asp Ala Gly
100 105 110
Met Ala Pro Ile Leu Val Asn Ile His Leu Thr Glu Val Phe Leu Gly
115 120 125
Ile Phe Ile Gly Ala Val Thr Phe Thr Gly Ser Val Val Ala Phe Gly
130 135 140
Lys Leu Cys Gly Lys Ile Ser Ser Lys Pro Leu Met Leu Pro Asn Arg
145 150 155 160
His Lys Met Asn Leu Ala Ala Leu Val Val Ser Phe Leu Leu Leu Ile
165 170 175
Val Phe Val Arg Thr Asp Ser Val Gly Leu Gln Val Leu Ala Leu Leu
180 185 190
Ile Met Thr Ala Ile Ala Leu Val Phe Gly Trp His Leu Val Ala Ser
195 200 205
Ile Gly Gly Ala Asp Met Pro Val Val Val Ser Met Leu Asn Ser Tyr
210 215 220
Ser Gly Trp Ala Ala Ala Ala Ala Gly Phe Met Leu Ser Asn Asp Leu
225 230 235 240
Leu Ile Val Thr Gly Ala Leu Val Gly Ser Ser Gly Ala Ile Leu Ser
245 250 255
Tyr Ile Met Cys Lys Ala Met Asn Arg Ser Phe Ile Ser Val Ile Ala
260 265 270
Gly Gly Phe Gly Thr Asp Gly Ser Ser Thr Gly Asp Asp Gln Glu Val
275 280 285
Gly Glu His Arg Glu Ile Thr Ala Glu Glu Thr Ala Glu Leu Leu Lys
290 295 300
Asn Ser His Ser Val Ile Ile Thr Pro Gly Tyr Gly Met Ala Val Ala
305 310 315 320
Gln Ala Gln Tyr Pro Val Ala Glu Ile Thr Glu Lys Leu Arg Ala Arg
325 330 335
Gly Ile Asn Val Arg Phe Gly Ile His Pro Val Ala Gly Arg Leu Pro
340 345 350
Gly His Met Asn Val Leu Leu Ala Glu Ala Lys Val Pro Tyr Asp Ile
355 360 365
Val Leu Glu Met Asp Glu Ile Asn Asp Asp Phe Ala Asp Thr Asp Thr
370 375 380
Val Leu Val Ile Gly Ala Asn Asp Thr Val Asn Pro Ala Ala Gln Asp
385 390 395 400
Asp Pro Lys Ser Pro Ile Ala Gly Met Pro Val Leu Glu Val Trp Lys
405 410 415
Ala Gln Asn Val Ile Val Phe Lys Arg Ser Met Asn Thr Gly Tyr Ala
420 425 430
Gly Val Gln Asn Pro Leu Phe Phe Lys Glu Asn Thr His Met Leu Phe
435 440 445
Gly Asp Ala Lys Ala Ser Val Asp Ala Ile Leu Lys Ala Leu
450 455 460
<210> 11
<211> 238
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Gln Thr Pro Gln Ile Leu Ile Val Glu Asp Glu Gln Val Thr Arg
1 5 10 15
Asn Thr Leu Lys Ser Ile Phe Glu Ala Glu Gly Tyr Ala Val Phe Glu
20 25 30
Ala Ser Asp Gly Glu Glu Met His Gln Val Leu Ser Asp Asn Ser Val
35 40 45
Asn Leu Val Ile Met Asp Ile Asn Leu Pro Gly Lys Asn Gly Leu Leu
50 55 60
Leu Ala Arg Glu Leu Arg Glu Gln Ala Asn Ile Ala Leu Met Phe Leu
65 70 75 80
Thr Gly Arg Asp Asn Glu Val Asp Lys Ile Leu Gly Leu Glu Ile Gly
85 90 95
Ala Asp Asp Tyr Ile Thr Lys Pro Phe Asn Pro Arg Glu Leu Thr Ile
100 105 110
Arg Ala Arg Asn Leu Leu Ser Arg Ser Met Ser Ala Ser Ala Val Gln
115 120 125
Glu Glu Lys Arg Ser Val Glu Lys Tyr Glu Phe Asn Gly Trp Val Leu
130 135 140
Asp Ile Asn Ser Arg Ser Leu Val Ser Pro Ser Gly Asp Ser Tyr Lys
145 150 155 160
Leu Pro Arg Ser Glu Phe Arg Ala Leu Leu His Phe Cys Glu Asn Pro
165 170 175
Gly Lys Ile Gln Thr Arg Ala Asp Leu Leu Lys Lys Met Thr Gly Arg
180 185 190
Glu Leu Lys Pro His Asp Arg Thr Val Asp Val Thr Ile Arg Arg Ile
195 200 205
Arg Lys His Phe Glu Ser Val Ser Gly Thr Pro Glu Ile Ile Ala Thr
210 215 220
Ile His Gly Glu Gly Tyr Arg Phe Cys Gly Asp Leu Glu Asp
225 230 235
<210> 12
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Val Lys Gln Ile Pro Arg His Gln Gln Ile Val Glu Leu Val Lys Lys
1 5 10 15
Gln Gly Tyr Val Ser Thr Asp Glu Leu Val Glu Arg Phe Asn Val Ser
20 25 30
Pro Gln Thr Ile Arg Arg Asp Leu Asn Glu Leu Ala Asp Asp Asn Arg
35 40 45
Ile Arg Arg Tyr His Gly Gly Ala Thr Ile Pro Leu Ser Ser Glu Asn
50 55 60
Thr Ser Tyr Asn Thr Arg Lys Ala Leu Asn Phe Asn Glu Lys Asp Val
65 70 75 80
Ile Ala Glu Glu Val Val Lys His Ile Pro Asp Gly Ala Thr Leu Phe
85 90 95
Ile Asp Ile Gly Thr Thr Pro Glu Ala Val Ala Arg Ala Leu Asn Lys
100 105 110
Asn His Lys Gln Leu Arg Val Val Thr Asn Asn Ile Asn Val Ala Thr
115 120 125
Ile Leu Tyr Pro Asn Pro Glu Ile Lys Val Ile Leu Ala Gly Gly Glu
130 135 140
Val Arg Asn Arg Asp Gly Gly Ile Val Gly Glu Ala Thr Leu Asp Phe
145 150 155 160
Val Lys Gln Phe Arg Leu Asp Phe Gly Ile Leu Gly Ile Ser Gly Ile
165 170 175
Asp Phe Asp Gly Ser Leu Leu Asp Phe Asp Tyr His Glu Val Arg Val
180 185 190
Lys Gln Val Ile Ile Asp Asn Ser Arg Ser Val Phe Leu Ala Val Asp
195 200 205
His Thr Lys Phe Gly Arg Asn Ala Met Val Lys Leu Gly Asn Ile Ala
210 215 220
Gln Leu Asn Met Ile Phe Thr Asn Lys Gln Pro Pro Glu Glu Ile Leu
225 230 235 240
Ser Ile Leu Lys Glu Ala Ser Ile Pro Leu Glu Val Val Asp Ala Asn
245 250 255
Gly Ala Thr Asp Glu Glu Asn Lys
260
<210> 13
<211> 202
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ile Lys Gly Ser Met Asp Met Asn Glu Gln Gln Phe Phe Asp Tyr
1 5 10 15
Val Thr Lys Phe Gly Ala Tyr Gln Lys Arg Ser Met Phe Gly Gly Ile
20 25 30
Gly Leu Phe Gln His Asp Ala Met Tyr Val Leu Val Ser Glu Asp Arg
35 40 45
Ile Phe Val Arg Gly Gly Glu Glu Leu Asp Pro Glu Leu Leu Ala Leu
50 55 60
Gly Cys Glu Lys Tyr Arg His Val Lys Lys Gln Thr Thr Ala Thr Val
65 70 75 80
Asn Tyr Tyr Asp Ile Thr Glu Leu Tyr Glu Gln His His Pro Glu Leu
85 90 95
Asp Ser Ile Ile Glu Arg Ser Ile Gln Phe Ser Val Asn Gln Arg Glu
100 105 110
Phe Gln Arg Ser Ala Ala Ser Arg Arg Leu Arg Asp Leu Pro Asn Met
115 120 125
Gln Leu Thr Leu Glu Arg Met Val Lys Lys Ala Gly Ile Asp Asp Val
130 135 140
Glu Thr Phe Met Ser Leu Gly Ala Pro Glu Val Phe Asn Lys Val Arg
145 150 155 160
Gln Ala Tyr Gly Ser Asp Val Asp Val Lys Leu Leu Trp Lys Phe Ala
165 170 175
Gly Ala Ile Glu Gly Ile His Trp Lys Leu Leu Gln Glu Pro Arg Lys
180 185 190
Arg Gln Leu Leu Glu Ser Cys Gln Gln Arg
195 200
<210> 14
<211> 609
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgattaaag gatcaatgga tatgaatgag caacagtttt tcgactacgt aactaagttt 60
ggcgcctacc aaaaacgctc gatgtttggt ggtattggct tgtttcaaca cgacgctatg 120
tatgtgttgg tcagtgaaga ccgcattttt gtgcgtggtg gagaagagct cgaccctgag 180
ctcttggcct tagggtgcga gaaatatcgc catgttaaaa aacagacaac ggcaaccgta 240
aactattacg acatcactga actgtatgag caacatcacc ctgagctaga ctcgattatt 300
gaacgctcga ttcagttttc tgtgaatcag cgggaatttc aacgctcagc agccagtcgt 360
cgtctacggg atttacccaa tatgcaactg actttggagc gtatggtaaa aaaagcgggc 420
attgacgatg tggaaacttt tatgagcctc ggtgcgccag aagtgtttaa taaggtgcgt 480
caagcttatg gaagtgatgt cgatgtaaaa ctactgtgga agtttgcggg tgcgattgaa 540
ggcattcact ggaaactgct gcaagagccg cgtaagcgcc aactgttaga aagttgtcag 600
cagcgttaa 609
<210> 15
<211> 2693
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atgaaaagat caaaacgatt tgcagtactg gcccagcgcc ccgtcaatca ggacgggctg 60
attggcgagt ggcctgaaga gggactgatc gccatggaca gcccctttga cccggtctct 120
tcagtaaaag tggacaacgg tctgatcgtc gaactggacg gcaagcgccg ggaccagttt 180
gacatgatcg accggtttat cgccgattac gcgatcaacg ttgagcgcac agagcaggca 240
atgcgcctgg aggcggtgga aatagcccgc atgctggtgg atattcacgt cagccgggag 300
gagatcattg ccatcactac cgccatcacc ccggccaaag cggtcgaggt gatggcgcag 360
atgaacgtgg tggagatgat gatggcgctg cagaagatgc gtgcccgccg gaccccctcc 420
aaccagtgcc acgtcaccaa tctcaaagat aatccggtgc agattgccgc tgacgccgcc 480
gaggccggga tccgcggctt ctcagaacag gagaccacgg tcggtatcgc gcgctacgcg 540
ccgtttaacg ccctggcgct gttggtcggt tcgcagtgcg gccgccccgg cgtgttgacg 600
cagtgctcgg tggaagaggc caccgagctg gagctgggca tgcgtggctt aaccagctac 660
gccgagacgg tgtcggtcta cggcactgaa gcggtattta ccgacggcga tgatactccg 720
tggtcaaagg cgttcctcgc ctcggcctac gcctcccgcg ggttgaaaat gcgctacacc 780
tccggcaccg gatccgaagc gctgatgggc tattcggaga gcaagtcgat gctctacctc 840
gaatcgcgct gcatcttcat taccaaaggc gccggggttc agggactgca aaacggcgcg 900
gtgagctgta tcggcatgac cggcgctgtg ccgtcgggca ttcgggcggt gctggcggaa 960
aacctgatcg cctctatgct cgacctcgaa gtggcgtccg ccaacgacca gactttctcc 1020
cactcggata ttcgccgcac cgcgcgcacc ctgatgcaga tgctgccggg caccgacttt 1080
attttctccg gctacagcgc ggtgccgaac tacgacaaca tgttcgccgg ctcgaacttc 1140
gatgcggaag attttgatga ttacaacatc ctgcagcgtg acctgatggt tgacggcggc 1200
ctgcgtccgg tgaccgaggc ggaaaccatt gccattcgcc agaaagcggc gcgggcgatc 1260
caggcggttt tccgcgagct ggggctgccg ccaatcgccg acgaggaggt ggaggccgcc 1320
acctacgcgc acggcagcaa cgagatgccg ccgcgtaacg tggtggagga tctgagtgcg 1380
gtggaagaga tgatgaagcg caacatcacc ggcctcgata ttgtcggcgc gctgagccgc 1440
agcggctttg aggatatcgc cagcaatatt ctcaatatgc tgcgccagcg ggtcaccggc 1500
gattacctgc agacctcggc cattctcgat cggcagttcg aggtggtgag tgcggtcaac 1560
gacatcaatg actatcaggg gccgggcacc ggctatcgca tctctgccga acgctgggcg 1620
gagatcaaaa atattccggg cgtggttcag cctgacacca ttgaataagg cggtattcct 1680
gtgcaacaga caacccaaat tcagccctct tttaccctga aaacccgcga gggcggggta 1740
gcttctgccg atgaacgtgc cgatgaagtg gtgatcggcg tcggccctgc cttcgataaa 1800
caccagcatc acactctgat cgatatgccc catggcgcga tcctcaaaga gctgattgcc 1860
ggggtggaag aagaggggct tcacgcccgg gtggtgcgca ttctgcgcac gtccgacgtc 1920
tcctttatgg cctgggatgc ggccaacctg agcggctcgg ggatcggcat cggtatccag 1980
tcgaagggga ccacggtcat ccatcagcgc gatctgctgc cgctcagcaa cctggagctg 2040
ttctcccagg cgccgctgct gacgctggag acctaccggc agattggcaa aaacgccgcg 2100
cgctatgcgc gcaaagagtc gccttcgccg gtgccagtgg tgaacgatca gatggtgcgg 2160
ccgaaattta tggccaaagc cgcgctattt catatcaaag agaccaaaca tgtggtgcag 2220
gacgccgagc ccgtcaccct gcacgtcgac ttagtaaggg agtgaccatg agcgagaaaa 2280
ccatgcgcgt gcaggattat ccgttagcca cccgctgccc ggagcatatc ctgacgccta 2340
ccggcaaacc attgaccgat attaccctcg agaaggtgct ctctggcgag gtgggcccgc 2400
aggatgtgcg gatctcccgc cagacccttg agtaccaggc gcagattgcc gagcagatgc 2460
agcgccatgc ggtggcgcgc aatttccgcc gcgcggcgga gcttatcgcc attcctgacg 2520
agcgcattct ggctatctat aacgcgctgc gcccgttccg ctcctcgcag gcggagctgc 2580
tggtgatcgc cgacgagctg gagcacacct ggcatgcgac agtgaatgcc gcctttgtcc 2640
gggagtcggc ggaagtgtat cagcagcggc ataagctgcg taaaggaagc taa 2693
<210> 16
<211> 3271
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgccgttaa tagccgggat tgatatcggc aacgccacca ccgaggtggc gctggcgtcc 60
gacgacccgc aggcgagggc gtttgttgcc agcgggatcg ttgcgacgac gggcatgaaa 120
gggacgcggg acaatatcgc cgggaccctc gccgcgctgg agcaggccct ggcgaaaaca 180
ccgtggtcga tgagcgatgt ctctcgcatc tatcttaacg aagccgcgcc ggtgattggc 240
gatgtggcga tggagaccat caccgagacc attatcaccg aatcgaccat gatcggtcat 300
aacccgcaga cgccgggcgg ggtgggcgtt ggcgtgggga cgactatcgc tctcgggcgg 360
ctggcgacgc tgccggcggc gcagtatgcc gaggggtgga tcgtactgat tgacgacgcc 420
gtcgatttcc ttgacgccgt gtggtggctc aatgaggcgc tcgaccgggg gatcaacgtg 480
gtggcggcga tcctcaaaaa ggacgacggc gtgctggtga acaaccgcct gcgtaaaacc 540
ctgccggtgg tggatgaagt gacgctgctg gagcaggtcc ccgagggggt gatggcggcg 600
gtggaagtgg ccgcgccggg ccaggtggtg cggatcctgt cgaatcccta cgggatcgcc 660
accttcttcg ggctaagccc ggaagagacc caggctatcg tccccatcgc ccgcgccctg 720
attggcaacc gttccgcggt ggtgctcaag accccgcagg gggacgtgca gtcgcgggtg 780
atcccggcgg gcaacctcta cattagcggc gaaaagcgcc gcggagaggc cgatgtcgcc 840
gagggcgcgg aagccatcat gcaggcgatg agcgcctgcg ctccggtacg cgacatccgc 900
ggcgaaccgg gcacccacgc cggcggcatg cttgagcggg tgcgcaaggt aatggcgtcc 960
ctgaccggcc atgagatgag cgcgatatac atccaggatc tgctggcggt ggatacgttt 1020
attccgcgca aggtgcaggg cgggatggcc ggcgagtgcg ccatggagaa tgccgtcggg 1080
atggcggcga tggtgaaagc ggatcgtctg caaatgcagg ttatcgcccg cgaactgagc 1140
gcccgactgc agaccgaggt ggtggtgggc ggcgtggagg ccaacatggc catcgccggg 1200
gcgttaacca ctcccggctg tgcggcgccg ctggcgatcc tcgacctcgg cgccggctcg 1260
acggatgcgg cgatcgtcaa cgcggagggg cagataacgg cggtccatct cgccggggcg 1320
gggaatatgg tcagcctgtt gattaaaacc gagctgggcc tcgaggatct ttcgctggcg 1380
gaagcgataa aaaagtaccc gctggccaaa gtggaaagcc tgttcagtat tcgtcacgag 1440
aatggcgcgg tggagttctt tcgggaagcc ctcagcccgg cggtgttcgc caaagtggtg 1500
tacatcaagg agggcgaact ggtgccgatc gataacgcca gcccgctgga aaaaattcgt 1560
ctcgtgcgcc ggcaggcgaa agagaaagtg tttgtcacca actgcctgcg cgcgctgcgc 1620
caggtctcac ccggcggttc cattcgcgat atcgcctttg tggtgctggt gggcggctca 1680
tcgctggact ttgagatccc gcagcttatc acggaagcct tgtcgcacta tggcgtggtc 1740
gccgggcagg gcaatattcg gggaacagaa gggccgcgca atgcggtcgc caccgggctg 1800
ctactggccg gtcaggcgaa ttaattgacg gctagctcag tcctaggtac agtgctagcg 1860
ttatcctggt gcgggagaga atgatgaaca agagccaaca agttcagaca atcaccctgg 1920
ccgccgccca gcaaatggcg gcggcggtgg aaaaaaaagc cactgagatc aacgtggcgg 1980
tggtgttttc cgtggttgac cgcggaggca acacgctgct tatccagcgg atggacgagg 2040
ccttcgtctc cagctgcgat atttctctga ataaagcctg gagcgcctgc agcctgaagc 2100
aaggtaccca tgaaattacg tcagcggtcc agccaggaca atctctgtac ggtctgcagc 2160
taaccaacca acagcgaatt attatttttg gcggtggcct gccagttatt tttaatgagc 2220
aggtaattgg cgccgtcggc gttagcggcg gtacggtcga gcaggatcaa ttattagccc 2280
agtgcgccct ggattgtttt tccgcattat aacctgaagc gagaaggtat attatgagct 2340
atcgtatgtt ccgccaggca ttctgagtgt taacgagggg accgtcatgt cgctttcacc 2400
gccaggcgta cgcctgtttt acgatccgcg cgggcaccat gccggcgcca tcaatgagct 2460
gtgctggggg ctggaggagc agggggtccc ctgccagacc ataacctatg acggaggcgg 2520
tgacgccgct gcgctgggcg ccctggcggc cagaagctcg cccctgcggg tgggtattgg 2580
gctcagcgcg tccggcgaga tagccctcac tcatgcccag ctgccggcgg acgcgccgct 2640
ggctaccgga cacgtcaccg atagcgacga tcatctgcgt acgctcggcg ccaacgccgg 2700
gcagctggtt aaagtcctgc cgttaagtga gagaaactga atgtatcgta tctatacccg 2760
caccggggat aaaggcacca ccgccctgta cggcggcagc cgcatcgaga aagaccatat 2820
tcgcgtcgag gcctacggta ccgtcgatga actgatatcc cagctgggcg tctgctacgc 2880
cacgacccgc gacgccgggc tgcgggaaag cctgcaccat attcagcaga cgctgttcgt 2940
gctgggggct gaactggcca gcgatgcgcg gggcctgacc cgcctgagcc agacgatcgg 3000
cgaagaggag atcaccgccc tggagcggct tatcgaccgc aatatggccg agagcggccc 3060
gttaaaacag ttcgtgatcc cgggaaagaa tctcgcctct gcccagctgc acgtggcgcg 3120
cacccagtcc cgtcggctcg aacgcctgct gacggccatg gaccgcgcgc atccgctgcg 3180
cgacgcgctc aaacgctaca gcaatcgcct gtcggatgcc ctgttctcca tggcgcgaat 3240
cgaagagact aggcctgatg cttgcgcttg a 3271
<210> 17
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60
ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120
gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180
gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240
gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300
accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360
caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420
gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480
caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540
tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600
gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660
ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720
cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780
ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840
cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900
cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960
gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020
acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080
gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140
cgtatatacg aagccgcccg ctaa 1164
<210> 18
<211> 564
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atggcgtttt ttgaccttga gaagatggaa tcggcttcta agagtaattt ggatgcggta 60
caacaattga actccaaaat ctttgaatcg gctgaagaat tatataaact gcagtttaaa 120
acgctgcgtg cagcggccga tgacaacttc gagtcgttac gcaagctgtt gtctgtgcgc 180
gatcctcaag cgtttctgga attacaggcg tcttttttca agccaaacga acaggccgaa 240
cgtctggtag agttttctcg tcagacttat gacttaattt cgcgcttcca agccgaagta 300
accaaactga cggagcgtca aatcgagatt gggactcagc aagcccagga gattgtagag 360
gaaatttgta aaaacgcccc ggccagtgca gagccagtag tttcagtatt caaaagtgca 420
gtggaaggtg ccggtaacgt gtatgaatcg gcccaaaagg ccgccaagca ggcgtccgag 480
attaccgcga gcggaattga ggctgccgcc aacgcggccg gtcaggccgc tgcccaagcc 540
gcgtctggta aaaaaaccgc gtaa 564
<210> 19
<211> 2932
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60
aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120
caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180
aatagcgtct ggcagtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240
gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300
ttaatgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 360
atctcacgcg cacaatcgct ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420
gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480
gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540
attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600
aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660
ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720
ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780
aaaccagcgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840
attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900
ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960
acgcaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020
gagaaagacg gcaatatcac tgttgatttt gatgatgtgg tgattcgcgg cgtgaccgtg 1080
atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140
gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200
cgtaaatacg cgttgatggc gctggcaatc attctttttg gctggatggc aagcgttgcg 1260
ccgaaagaat tccttgggca cttcaccgtt ttcgcgctgg cctgcgttgt cggttattac 1320
gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380
tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440
cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500
cagcgcatgc tgaaaatgtt ccgcaaaaat taaggggtaa catatgtctg gaggattagt 1560
tacagctgca tacattgttg ccgcgatcct gtttatcttc agtctggccg gtctttcgaa 1620
acatgaaacg tctcgccagg gtaacaactt cggtatcgcc gggatggcga ttgcgttaat 1680
cgcaaccatt tttggaccgg atacgggtaa tgttggctgg atcttgctgg cgatggtcat 1740
tggtggggca attggtatcc gtctggcgaa gaaagttgaa atgaccgaaa tgccagaact 1800
ggtggcgatc ctgcatagct tcgtgggtct ggcggcagtg ctggttggct ttaacagcta 1860
tctgcatcat gacgcgggaa tggcaccgat tctggtcaat attcacctga cggaagtgtt 1920
cctcggtatc ttcatcgggg cggtaacgtt cacgggttcg gtggtggcgt tcggcaaact 1980
gtgtggcaag atttcgtcta aaccattgat gctgccaaac cgtcacaaaa tgaacctggc 2040
ggctctggtc gtttccttcc tgctgctgat tgtatttgtt cgcacggaca gcgtcggcct 2100
gcaagtgctg gcattgctga taatgaccgc aattgcgctg gtattcggct ggcatttagt 2160
cgcctccatc ggtggtgcag atatgccagt ggtggtgtcg atgctgaact cgtactccgg 2220
ctgggcggct gcggctgcgg gctttatgct cagcaacgac ctgctgattg tgaccggtgc 2280
gctggtcggt tcttcggggg ctatcctttc ttacattatg tgtaaggcga tgaaccgttc 2340
ctttatcagc gttattgcgg gtggtttcgg caccgacggc tcttctactg gcgatgatca 2400
ggaagtgggt gagcaccgcg aaatcaccgc agaagagaca gcggaactgc tgaaaaactc 2460
ccattcagtg atcattactc cggggtacgg catggcagtc gcgcaggcgc aatatcctgt 2520
cgctgaaatt actgagaaat tgcgcgctcg tggtattaat gtgcgtttcg gtatccaccc 2580
ggtcgcgggg cgtttgcctg gacatatgaa cgtattgctg gctgaagcaa aagtaccgta 2640
tgacatcgtg ctggaaatgg acgagatcaa tgatgacttt gctgataccg ataccgtact 2700
ggtgattggt gctaacgata cggttaaccc ggcggcgcag gatgatccga agagtccgat 2760
tgctggtatg cctgtgctgg aagtgtggaa agcgcagaac gtgattgtct ttaaacgttc 2820
gatgaacact ggctatgctg gtgtgcaaaa cccgctgttc ttcaaggaaa acacccacat 2880
gctgtttggt gacgccaaag ccagcgtgga tgcaatcctg aaagctctgt aa 2932
<210> 20
<211> 717
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atgcaaaccc cgcagattct gatcgttgaa gatgagcaag taactcgtaa cactcttaag 60
agtatttttg aagcagaggg atacgctgtt tttgaagcca gtgacggtga agagatgcac 120
caagtattat cagacaactc tgtcaacttg gtcattatgg acattaacct tccaggcaag 180
aatggtcttc tacttgcacg tgaattgcgc gaacaagcaa acatcgcact gatgtttttg 240
actggccgtg acaatgaagt ggacaagatt ctaggccttg agattggtgc tgatgattac 300
atcactaaac cattcaaccc acgtgagttg actatccgtg cgcgcaactt gttgagtcgc 360
tcaatgagcg caagtgctgt tcaagaagaa aaacgctcgg tagaaaaata cgagttcaac 420
ggttgggtgc tagatatcaa tagccgttca ctggtgagcc cgtcaggtga tagctacaaa 480
ctgcctcgtt ctgaattccg tgcgctactg cacttctgtg agaacccagg caaaatccaa 540
acacgtgccg atcttctgaa gaagatgaca ggccgtgaac ttaaaccaca cgaccgtact 600
gttgacgtaa ccattcgtcg tattcgtaaa cacttcgaat ctgtatcagg tacaccagaa 660
atcatcgcaa cgattcatgg tgaaggttac cgcttctgtg gtgatttaga agactaa 717
<210> 21
<211> 795
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtgaagcaaa tacctagaca ccagcagatt gtcgagttgg tgaaaaaaca aggctatgtc 60
agcacggatg agttggttga acgattcaat gtcagcccac aaaccatccg acgcgatctc 120
aatgagctcg ccgacgataa tagaattcgt cgctatcacg gtggtgccac catccctctc 180
agctcggaaa atacctctta caacacgcgt aaagcactta acttcaacga aaaagatgtg 240
attgcagaag aggtggtcaa acacatacct gatggcgcga cgctctttat cgatatcgga 300
acgacgccgg aagccgttgc acgtgcactc aataaaaacc acaaacaact tcgagtggtc 360
actaacaaca tcaatgttgc gaccattctt taccctaacc cagaaatcaa agtgatactg 420
gctggcggcg aagttcgtaa ccgcgatggt ggtattgtcg gtgaagccac tctggacttc 480
gtaaaacaat tccgcctcga tttcggtatt ctcggaatca gtggtattga tttcgatggt 540
tcactgctgg acttcgatta ccatgaagta cgagtaaaac aggttatcat cgataacagt 600
cgcagcgtat ttctggccgt agaccacact aagtttggcc gcaatgcgat ggtgaaactg 660
ggcaatatcg ctcagttaaa catgatattt accaataagc agccacctga agaaattctg 720
tctattttaa aagaagcctc catccctctt gaagttgtcg acgcaaacgg ggcgacagac 780
gaagaaaata aataa 795
<210> 22
<211> 1560
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atgagtatac aaaaaaatgc ttccacaact gactcatcca ctcctttaga cttgatcatt 60
atcggtggcg gcattaacgg ggctggtatc gctgctgatg ctgcaggccg tggattaagt 120
gttggtttgt atgaagctaa cgactttgca tctgcaacct cttcagcaag ctccaaactt 180
attcatggtg gattacgcta ccttgaacac tacgagtttc gtttagtttc ggaagcgctc 240
gcagaacgtg aagtaatcct tcgcaaagca cctcatgttg cgttacctat gcgtttccgt 300
ttacctcatc gtccattttt acgtccagcg tggatgatcc gctgtggttt gttcctttac 360
gataacttag gtaaacgcac tacgctccca ggcagcaaga ccgttaatct ggcaaagtct 420
ggcttgttaa aaccggagat aaaaaccggt tttgaatact ccgactgctg ggtagatgat 480
gctcgcttag ttctacttaa cgttctcgca gcacgtgaaa accatgcaga agttcgtaac 540
tactgccgag tagaaaaagc gcatcgcgaa agcggcatct ggcatgtcac aattcacgac 600
acaatgacag atcaacgttt tgaacgtaaa gcgaaagcac tggttaacgc tgcgggaccg 660
tgggtaaaac agttctttga tgaaggatta gagcaagctt ctccacgtaa tattcgtttg 720
attaaaggtt cgcacattgt tgttcctcgt atccacaacg aacctcaggc ttatattctg 780
caaaacaaag ataaccgcat cgtgtttatg atcccgtact tagataagtt ctcgatcgtc 840
ggtaccacag atgtcgaata caaaggcgac ccacgagaag ttgcgatctc tgatgacgaa 900
gtcgattatc tgatcgacat tgttaaccag cacttcgtcc accagctatc gcgcgaagat 960
gtggtgtgga catacagcgg tgtgcgccca ctgtgcgatg atgagtcaga ctcgccacag 1020
gcgatcacgc gagactacac gctagagcta gatgcagaat tcgatcaagc tccattactt 1080
tcggttttcg gcggtaaact aacgacttac cgaaaactgg gtgaagcggc gatgaaaaag 1140
ctggcgccat tcctgccaca aatgggcggt aactggacag caaaccaagc tctgccgggc 1200
ggtaacttca gctgtagtcg cgaacaactg gcgaaacaga tccacgccaa gtacgcctgg 1260
gcaccaaaag cgcttattct acgttacgtc actcagtttg gaacacaaac gtgggagcta 1320
atgaagggag cgacaagtga agccgattta ggccaagcct tctctacaca agccggtggc 1380
gtatatcagc gtgaaattga ctacttgatg aaccatgaaa tggccctgac tgacgaagac 1440
attttatggc gtcgtaccaa gatcgggctc tacatgagcg acgaagaaaa gctatctctt 1500
gcagaatact taaaagaaaa gttacaacag aaagtcgtga acctctcaca agtgggctaa 1560
<210> 23
<211> 73
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
agcttgcatg cctgcaggtc gactagaaag gtgtgttgat gattaaagga tcaatggata 60
tgaatgagca aca 73
<210> 24
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gatattatcg tgaggatgcg attaacgctg ctgacaactt tctaacag 48
<210> 25
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
aagttgtcag cagcgttaat cgcatcctca cgataatatc cgg 43
<210> 26
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atccgccaaa acagccaagc tgttaatctt tctgcgaatt gagatgacgc c 51
<210> 27
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
aagcggcatg catttacgtt ttgacggcta gctcagtcct aggtacagtg ctagcttcac 60
cttttgagcc gatgaacaat gaa 83
<210> 28
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cgcaccagga taacgctagc actgtaccta ggactgagct agccgtcaat taattcgcct 60
gaccggccag 70
<210> 29
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
cagtcctagg tacagtgcta gcgttatcct ggtgcgggag agaatgatga acaagagcca 60
acaagttca 69
<210> 30
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ctccttgcta gcactgtacc taggactgag ctagccgtca atcaagcgca agcatcaggc 60
c 61
<210> 31
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ctcagtccta ggtacagtgc tagcaaggag atataccatg aacaacttta atctgcacac 60
ccca 64
<210> 32
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gtacctagga ctgagctagc cgtcaatgcc tgacgccaga agcatt 46
<210> 33
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gcgtctggta aaaaaaccgc gtaatgcagg catgcaagct tggctgtttt g 51
<210> 34
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ggactgagct agccgtcaaa acgtaaatgc atgccgcttc g 41
<210> 35
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
aagcggcatg catttacgtt ttgacggcta gctcagtcct aggtacagtg ctagcttcac 60
cttttgagcc gatgaacaat gaa 83
<210> 36
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
cgcaccagga taacgctagc actgtaccta ggactgagct agccgtcaat taattcgcct 60
gaccggccag 70
<210> 37
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
cagtcctagg tacagtgcta gcgttatcct ggtgcgggag agaatgatga acaagagcca 60
acaagttca 69
<210> 38
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ctccttgcta gcactgtacc taggactgag ctagccgtca atcaagcgca agcatcaggc 60
c 61
<210> 39
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ctcagtccta ggtacagtgc tagcaaggag atataccatg aacaacttta atctgcacac 60
ccca 64
<210> 40
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gtacctagga ctgagctagc cgtcaatgcc tgacgccaga agcatt 46
<210> 41
<211> 76
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gcttctggcg tcaggcattg acggctagct cagtcctagg tacagtgcta gcaaccgatg 60
gaagggaata tcatgc 76
<210> 42
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
atatctcctt tgcaggtcga ctcttacaga gctttcagga ttgcatccac 50
<210> 43
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gcgtctggta aaaaaaccgc gtaatgcagg catgcaagct tggctgtttt g 51
<210> 44
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ggactgagct agccgtcaaa acgtaaatgc atgccgcttc g 41
<210> 45
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ccgaatagtc tgaaccgtta cgacc 25
<210> 46
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gacagcttat cactgatcag ggcggtacct aaatttgtga caaaatt 47
<210> 47
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
aagcagctcc agcctacacg tgcgctactg cacttctgtg a 41
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
acgctttgtg acctcttgct 20
<210> 49
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
gtcacaaatt taggtaccgc cctgatcagt gataagctgt caaacatgag 50
<210> 50
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
cacagaagtg cagtagcgca cgtgtaggct ggagctgctt c 41
<210> 51
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
cggtcatcca ggataaagct acagc 25
<210> 52
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
cgaagcagct ccagcctaca ctttcggtat tctcggaatc agtgg 45
<210> 53
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
tttgacagct tatcactgat cagttaggac tccattgtgc acggg 45
<210> 54
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
cttactcccc ccctttaata gagccc 26
<210> 55
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ccactgattc cgagaatacc gaaagtgtag gctggagctg cttc 44
<210> 56
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
ctacccgtgc acaatggagt cctaactgat cagtgataag ctgtcaaaca tgaga 55
<210> 57
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
attggcgatc tttgctaact ttgcc 25
<210> 58
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
cgaagcagct ccagcctaca ccgttctcgc agcacgtgaa aac 43
<210> 59
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
gacagcttat cactgatcag aaatttgacc tctttggtga gcgaac 46
<210> 60
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
tcgtgacttt ggaccaaaac tgttcg 26
<210> 61
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ttttcacgtg ctgcgagaac ggtgtaggct ggagctgctt cg 42
<210> 62
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
tcaccaaaga ggtcaaattt ctgatcagtg ataagctgtc aaacatgag 49
<210> 63
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
ggtaaaaaaa ccgcgtaatg caggcatgca caaataaaac gaaaggctca gtcgaaag 58
<210> 64
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
acaaaatttg tttgcagcac aaaaggccat ccgtcaggat 40
<210> 65
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
atcctgacgg atggcctttt gtgctgcaaa caaattttgt acagacaata at 52
<210> 66
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
gcattttttt gtatactcat aaaacctaac aaaaaagaaa aatcatgatt ggtg 54
<210> 67
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
tttctttttt gttaggtttt atgagtatac aaaaaaatgc ttccacaact g 51
<210> 68
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
aaaatcttct ctcatccgcc aaaacagcca ttagcccact tgtgagaggt tcac 54
<210> 69
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
ggtaaaaaaa ccgcgtaatg caggcatgca caaataaaac gaaaggctca gtcgaaag 58
<210> 70
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
acaaaatttg tttgcagcac aaaaggccat ccgtcaggat 40
<210> 71
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
atcctgacgg atggcctttt gtgctgcaaa caaattttgt acagacaata at 52
<210> 72
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
gcattttttt gtatactcat aaaacctaac aaaaaagaaa aatcatgatt ggtg 54
<210> 73
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
tttctttttt gttaggtttt atgagtatac aaaaaaatgc ttccacaact g 51
<210> 74
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
aaaatcttct ctcatccgcc aaaacagcca ttagcccact tgtgagaggt tcac 54

Claims (10)

1.一种重组需钠弧菌，其特征在于，所述重组需钠弧菌表达甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶，且所述重组需钠弧菌与野生型需钠弧菌相比，具有降低的转录调控因子的表达和/或酶活性，所述转录调控因子为arcA和/或glpR；
所述甘油脱水酶由SEQ ID NO.15所示的序列编码，所述甘油脱水酶激活因子由SEQ IDNO.16所示的序列编码，所述醇脱氢酶由SEQ ID NO.17所示的序列编码。
2.根据权利要求1所述的重组需钠弧菌，其特征在于，所述甘油脱水酶的氨基酸序列如SEQ ID NO.1-3所示，所述甘油脱水酶激活因子的氨基酸序列如SEQ ID NO.4-5所示，所述醇脱氢酶的氨基酸序列如SEQ ID NO.6所示。
3.根据权利要求1所述的重组需钠弧菌，其特征在于，所述甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的表达通过导入携带甘油脱水酶、甘油脱水酶激活因子和醇脱氢酶的编码基因的表达质粒和/或在基因组上整合一个或多个拷贝的所述编码基因实现。
4.根据权利要求1所述的重组需钠弧菌，其特征在于，所述表达和/或酶活性的降低通过如下(1)、(2)中的一种或多种方式实现：
(1)对所述转录因子的编码基因进行一个或多个碱基的插入、缺失或替换以使得所述转录因子失活、表达量或活性降低；
(2)将所述转录因子的编码基因的转录或翻译调控元件替换为活性更低的调控元件。
5.根据权利要求1～4任一项所述的重组需钠弧菌，其特征在于，所述重组需钠弧菌的基因组甘油-3-磷酸脱氢酶编码基因被失活，且携带含有甘油-3-磷酸脱氢酶编码基因的表达质粒。
6.根据权利要求5所述的重组需钠弧菌，其特征在于，所述甘油-3-磷酸脱氢酶的氨基酸序列如SEQ ID NO.7所示。
7.根据权利要求1～4、6任一项所述的重组需钠弧菌，其特征在于，所述重组需钠弧菌与野生型需钠弧菌相比，还具有提高的分子伴侣phaP和/或转氢酶pntAB的表达量和/或酶活性。
8.根据权利要求7所述的重组需钠弧菌，其特征在于，所述分子伴侣phaP的氨基酸序列如SEQ ID NO.8所示，所述转氢酶pntAB的氨基酸序列如SEQ ID NO.9-10所示。
9.权利要求1～8任一项所述的重组需钠弧菌的如下任一种应用：
(1)在制备1,3-丙二醇或其衍生物中的应用；
(2)在用于生产1,3-丙二醇或其衍生物的微生物的遗传育种中的应用。
10.一种制备1,3-丙二醇的方法，其特征在于，利用权利要求1～8任一项所述的重组需钠弧菌转化甘油生产1,3-丙二醇。
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