CN114134059A - 生产毛喉素的重组酿酒酵母菌及构建方法 - Google Patents

生产毛喉素的重组酿酒酵母菌及构建方法 Download PDF

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CN114134059A
CN114134059A CN202111364198.8A CN202111364198A CN114134059A CN 114134059 A CN114134059 A CN 114134059A CN 202111364198 A CN202111364198 A CN 202111364198A CN 114134059 A CN114134059 A CN 114134059A
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卢文玉
张传波
鞠海燕
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Abstract

本发明公开了一种生产毛喉素的重组酿酒酵母菌及构建方法，构建方法为：向酿酒酵母菌中导入优化的泪柏醚合酶编码基因tCfTPS2和tCfTPS3，得到重组菌1；再以重组菌1为基础依次导入相关基因制备出重组菌2、重组菌3、重组菌4、重组菌5、重组菌6和重组菌7，实验证明，本发明的生产毛喉素的重组酿酒酵母，发酵使得毛喉素摇瓶产量达到18.37mg/L以上，为人工合成毛喉素奠定了基础。

Description

生产毛喉素的重组酿酒酵母菌及构建方法
技术领域
本发明涉及生物技术领域，尤其涉及生产毛喉素的重组酿酒酵母菌及构建方法及用途。
背景技术
毛喉素(Forskolin)，一种半日花烷型的杂环二萜类天然产物，存在于毛喉鞘蕊花Coleus forskohlii的根软木组织中。由于毛喉素作为腺苷酸环化酶的激活剂，提高了细胞内环磷酸腺苷(cAMP)的浓度，所以毛喉素目前已经被研究发现的医药应用价值有：缓解青光眼、治疗哮喘、抗肿瘤、抗HIV、治疗心力衰竭及降压、减肥的功效等。由于植物原材料有限，毛喉素的生产显得供不应求，而毛喉素的化学合成过程步骤繁琐，且对环境不友好。
随着合成生物学的发展，以分子生物学为手段，对基因进行改造，构建外源代谢途径或调节内源途径，以廉价的碳源作为底物，通过大规模发酵生产萜类化合物，既避免了植物提取法对资源的浪费，又避免了化学合成法复杂的工序，减少了对环境的危害等问题。因此，利用合成生物学方法来生产萜类化合物越来越重要。目前，利用合成生物学方法，通过分子生物学手段，基因改造微生物可以实现目标产物的有效积累，但从系统代谢角度进行毛喉素生物合成路径优化来生产毛喉素还未见报道。
发明内容
本发明的目的是克服现有技术的不足，提供一种生产毛喉素的重组酿酒酵母菌。
本发明的第二个目的是提供一种生产毛喉素的重组酿酒酵母菌的构建方法。
本发明的第三个目的是提供一种生产毛喉素的重组酿酒酵母菌发酵生产毛喉素的用途。
本发明的技术方案概述如下：
生产毛喉素的重组酿酒酵母菌的构建方法，包括如下步骤：
(1)向酿酒酵母中导入含有优化的泪柏醚合酶编码基因tCfTPS2和优化的泪柏醚合酶编码基因tCfTPS3的表达盒，得到重组菌1；
所述优化的泪柏醚合酶编码基因tCfTPS2的核苷酸序列如SEQ ID No.1所示；
所述优化的泪柏醚合酶编码基因tCfTPS3的核苷酸序列如SEQ ID No.2所示；
(2)向重组菌1中导入截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1、牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1和法呢基焦磷酸合酶编码突变基因ERG20F96C表达盒，得到重组菌2；
所述截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1的核苷酸序列如SEQ ID No.3所示；
所述牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1的核苷酸序列如SEQ ID No.4所示；
所述法呢基焦磷酸合酶编码突变基因ERG20F96C的核苷酸序列如SEQ ID No.5所示；
(3)向重组菌2中导入含有优化的细胞色素P450酶编码基因CfCYP76AH15、优化的细胞色素P450酶编码基因CfCYP76AH11、优化的细胞色素P450酶编码基因CfCYP76AH16，优化的细胞色素P450还原酶编码基因CfCPR和优化的乙酰基转移酶编码基因CfACT1-8的表达盒，得到重组菌3；
所述优化的细胞色素P450酶编码基因CfCYP76AH15的核苷酸序列如SEQ ID No.6所示；所述优化的细胞色素P450酶编码基因CfCYP76AH11的核苷酸序列如SEQ ID No.7所示；所述优化的细胞色素P450酶编码基因CfCYP76AH16的核苷酸序列如SEQ ID No.8所示；所述优化的细胞色素P450还原酶编码基因CfCPR的核苷酸序列如SEQ ID No.9所示；所述优化的乙酰基转移酶编码基因CfACT1-8的核苷酸序列如SEQ ID No.10所示；
(4)向重组菌3中导入含优化的泪柏醚合酶编码基因tCfTPS2、优化的泪柏醚合酶编码基因tCfTPS3、截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1、牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1和法呢基焦磷酸合酶编码突变基因ERG20F96C的表达盒得到重组菌4；
(5)向重组菌4中导入含有CfCYP76AH15～t66CPR～t30CYB5、fCYP76AH11～t66CPR～t30CYB5、CfCYP76AH16～t66CPR～t30CYB5和CfACT1-8表达盒，得到重组菌5；
所述t66CPR是CfCPR从N端截短了60个氨基酸后获得；
所述t30CYB5是细胞色素CYB5从C端截短了30个氨基酸后获得，细胞色素CYB5的核苷酸序列如SEQ ID No.11所示；
(6)向重组菌5中导入内质网调控因子INO2表达盒，得到重组菌6；
所述内质网调控因子INO2的核苷酸序列如SEQ ID No.12所示；
(7)向重组菌6中导入含有6-磷酸葡萄糖脱氢酶编码基因ZWF1，磷酸葡萄糖酸脱氢酶编码基因GND1，乙醛脱氢酶编码基因ALD6和乙酰辅酶A合酶编码基因ACS的表达盒，得到重组菌7；
所述6-磷酸葡萄糖脱氢酶编码基因ZWF1的核苷酸序列如SEQ ID No.13所示；所述磷酸葡萄糖酸脱氢酶编码基因GND1的核苷酸序列如SEQ ID No.14所示；所述乙醛脱氢酶编码基因ALD6的核苷酸序列如SEQ ID No.15所示；所述乙酰辅酶A合酶编码基因ACS的核苷酸序列如SEQ ID No.16所示。
酿酒酵母优选酿酒酵母Saccharomyces cerevisiae W303-1a。
上述方法构建的生产毛喉素的重组酿酒酵母菌。
上述重组酿酒酵母菌发酵生产毛喉素的用途。
实验证明，本发明的生产毛喉素的重组酿酒酵母，发酵使得毛喉素摇瓶产量达到18.37mg/L以上，为人工合成毛喉素奠定了基础。
附图说明
图1为毛喉素HPLC-MS检测图，其中a是液相图，b为质谱图。
具体实施方式
下面通过具体实施例对本发明作进一步的说明。
下面实施例中所使用的实验方法如无特殊说明，均为常规方法。
本发明所用的酿酒酵母Saccharomyces cerevisiae W303-1a(ATCC：208352)(购买时间，2016.6，网址：https://www.atcc.org/)为出发菌株。
酿酒酵母的公开是为了使本领域技术人员能够更好地理解本发明，但并不对本发明作任何限定。其它酿酒酵母也可以用于本发明。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、重组酿酒酵母菌1(重组菌1)的构建
向酿酒酵母W303-1a中导入含有优化的泪柏醚合酶编码基因tCfTPS2和优化的泪柏醚合酶编码基因tCfTPS3的表达盒，得到重组菌1；
(1)模块构建
泪柏醚合酶编码基因tCfTPS2和泪柏醚合酶编码基因tCfTPS3均来源于毛喉鞘蕊花(Coleus forskohlii)，针对酿酒酵母进行密码子优化，由武汉金开瑞生物工程有限公司通过化学合成的方法合成。得到优化的泪柏醚合酶编码基因tCfTPS2(SEQ ID No.1)和优化的泪柏醚合酶编码基因tCfTPS3(SEQ ID No.2)连接在大肠杆菌质粒pUC18(商业化质粒)上。
利用表1引物对及模板分别PCR扩增HOL，PTEF1，tCfTPS2，TADH1，PTDH3，tCfTPS3，TTDH2，URA3，HOR片段；将片段HOL，tCfTPS2，PTEF1，TADH1，PTDH3片段融合，得到模块HOL-PTEF1-tCfTPS2-TADH1-PTDH3；将片段tCfTPS3，TTDH2，URA3，HOR片段融合，得到模块tCfTPS3-TTDH2-URA3-HOR。
表1重组菌PCR扩增片段
Figure BDA0003360019060000031
Figure BDA0003360019060000041
(2)酿酒酵母感受态制备
将酿酒酵母Saccharomyces cerevisiae W303-1a的单菌落接种于3ml YPD液体培养基中，在30℃，220rpm的摇床中培养12h；将培养的酵母种子液转接到新的3ml YPD液体培养基中，30℃，在220rpm的转速下培养5h；取1ml菌液于提前灭过菌的2ml离心管中，4000rpm离心机离心3min，弃上清，收集菌体；1ml无菌水洗涤一次，4000rpm离心机离心3min，弃上清，收集菌体。然后将1ml 100mM LiAc水溶液加入菌体中混匀，室温静置5min。将静置结束后的细胞4000rpm离心3min，用移液枪除去LiAc液体。将鲑鱼精DNA(市售，Solarbio，10mg/ml)进行开水煮沸5min，然后快速放入准备好的冰上冷却。
(3)酿酒酵母转化
a依次向菌体沉淀离心管中加入120μl PEG3350(50g PEG3350/100ml水)、18μL1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、17μL HOL-PTEF1-tCfTPS2-TADH1-PTDH3片段和20μL tCfTPS3-TTDH2-URA3-HOR片段，体系共180μl。
b然后温和地用移液枪吹打1min，使其混匀；将上述混匀的离心管放置42℃水浴锅热激30min后，4000rpm离心机离心3min，除去上清；继续加入干净的1ml YPD液体培养基，30℃，220rpm摇床进行复苏培养2h；将复苏好的离心管在4000rpm转速下离心3min，弃去上层培养基，再用1ml无菌水洗涤两次；最后向含有菌体的离心管中加入200μl的无菌水并混匀，涂布于缺尿嘧啶的SC选择性固体培养基平板上，放置于30℃恒温培养箱中培养2天，待单菌落长出。挑取转化子过夜培养，提基因组，并PCR验证，得到正确目的片段的为正确克隆，命名为重组酿酒酵母菌1，简称重组菌1。
实施例2、重组酿酒酵母菌2(重组菌2)的构建
向重组菌1中导入截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1(SEQID No.3)、牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1(SEQ ID No.4)和法呢基焦磷酸合酶编码突变基因ERG20F96C(SEQ ID No.5)表达盒，得到重组菌2，
3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1(酿酒酵母Saccharomycescerevisiae)；
牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1来源(酿酒酵母Saccharomycescerevisiae)；
法呢基焦磷酸合酶编码突变基因ERG20F96C的原始基因来源(酿酒酵母Saccharomyces cerevisiae))。
(1)模块构建
利用表2引物对及模板分别PCR扩增LPP1up，PPGK1，tHMG1，TPGK1，PTDH3，BTS1，ERG20(F96C)up，ERG20(F96C)-TERG20，HIS3，LPP1down；
将片段LPP1up、PPGK1、tHMG1、TPGK1、PTDH3融合获得LPP1up-PPGK1-tHMG1-TPGK1-PTDH3，将片段BTS1、ERG20(F96C)up、ERG20(F96C)-TERG20融合获得BTS1～ERG20F96C-TERG20，将片段HIS3，LPP1down融合获得HIS3-LPP1down。ERG20(F96C)up与ERG20(F96C)融合后变成ERG20F96C)
表2重组菌PCR扩增片段
Figure BDA0003360019060000051
(2)重组菌1酿酒酵母感受态制备
按照实施例1中步骤(2)的方法制备重组菌1酿酒酵母感受态细胞。
(3)酿酒酵母转化
a依次向重组菌1感受态细胞中加入120μl PEG3350(50g PEG3350/100ml水)、18μl1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、18μlLPP1up-PPGK1-tHMG1-TPGK1-PTDH3片段和10μl BTS1～ERG20F96C-TERG20，9μlHIS3-LPP1down片段，体系共180μl。
b同实施例1中步骤(3)中的b相同，但是重组菌涂布于缺组氨酸的SC选择性固体培养基平板上，筛选正确的克隆命名为重组酿酒酵母菌2(重组菌2)。
实施例3、重组酿酒酵母菌3(重组菌3)的构建
向重组菌2中导入含有优化的细胞色素P450酶编码基因CfCYP76AH15(SEQ IDNo.6)、优化的细胞色素P450酶编码基因CfCYP76AH11(SEQ ID No.7)、优化的细胞色素P450酶编码基因CfCYP76AH16(SEQ ID No.8)，优化的细胞色素P450还原酶编码基因CfCPR(SEQID No.9)和优化的乙酰基转移酶编码基因CfACT1-8(SEQ ID No.10)的表达盒，得到重组菌3；
细胞色素P450酶编码基因CfCYP76AH15(毛喉鞘蕊花(Coleus forskohlii))
细胞色素P450酶编码基因CfCYP76AH11(毛喉鞘蕊花(Coleus forskohlii))
细胞色素P450酶编码基因CfCYP76AH16(毛喉鞘蕊花(Coleus forskohlii))
细胞色素P450还原酶编码基因CfCPR(毛喉鞘蕊花(Coleus forskohlii))
乙酰基转移酶编码基因CfACT1-8(毛喉鞘蕊花(Coleus forskohlii))
(1)模块构建
优化的毛喉素合成相关的P450酶编码基因CfCYP76AH15、CfCYP76AH11、CfCYP76AH16、细胞色素P450还原酶编码基因CfCPR和乙酰基转移酶CfACT1-8均来源于毛喉鞘蕊花(Coleus forskohlii)，均由武汉金开瑞生物工程有限公司通过化学合成的方法合成，针对酿酒酵母进行密码子优化，并连接在大肠杆菌质粒pUC18上，保存在大肠杆菌中。
利用表3引物对及模板分别PCR扩增DPPA，PTEF2，CfCPR，TADH2，PTEF1，CfCYP76AH15，TPGK1，PPGK1，CfCYP76AH11，TADH1，PTDH3，CfCYP76AH16，TCYC1，PFBA1，CfACT1-8，TTDH2，TRP1，DPPdown片段，将DPPA，PTEF2，CfCPR，TADH2片段融合，得到模块DPPA-PTEF2-CfCPR-TADH2；将PTEF1，CfCYP76AH15，TPGK1，PPGK1片段融合，得到模块PTEF1-CfCYP76AH15-TPGK1-PPGK1；将CfCYP76AH11，TADH1，PTDH3片段融合，得到模块CfCYP76AH11-TADH1-PTDH3；将CfCYP76AH16，TCYC1，PFBA1，CfACT1-8片段融合，得到模块CfCYP76AH16-TCYC1-PFBA1-CfACT1-8；将TTDH2，TRP1，DPPdown片段融合，得到模块TTDH2-TRP1-DPPdown。
表3重组菌PCR扩增片段
Figure BDA0003360019060000061
Figure BDA0003360019060000071
(2)重组菌2酿酒酵母感受态制备
按照实施例1中步骤(2)的方法制备重组菌2酿酒酵母感受态细胞。
(3)酿酒酵母转化
a依次向重组菌2感受态细胞中加入120μl PEG3350(50g PEG3350/100ml水)、18μl1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、8μl DPPA-PTEF2-CfCPR-TADH2片段、8μl PTEF1-CfCYP76AH15-TPGK1-PPGK1片段、8μl CfCYP76AH11-TADH1-PTDH3片段、6μl CfCYP76AH16-TCYC1-PFBA1-CfACT1-8片段和7μl TTDH2-TRP1-DPPdown片段，体系共180μl。
b同实施例1中步骤(3)中的b，但是重组菌涂布于缺色氨酸的SC选择性固体培养基平板上，筛选正确的克隆命名为重组酿酒酵母菌3(重组菌3)。
实施例4、重组酿酒酵母菌4(重组菌4)的构建
向重组菌3中导入含优化的泪柏醚合酶编码基因tCfTPS2、优化的泪柏醚合酶编码基因tCfTPS3、截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1、牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1和法呢基焦磷酸合酶编码突变基因ERG20F96C表达盒得到重组菌4；
(1)模块构建
除左右同源臂，其他部分均参照实施例1和实施例2，本实施例使用的同源臂δ2，δ1均来自酿酒酵母ATCC208352基因组,筛选标记基因HYGR(SEQ ID NO.17)由武汉金开瑞生物工程有限公司通过化学合成的方法合成，针对酿酒酵母酵母进行密码子优化，并连接在大肠杆菌质粒pUC18上，保存在大肠杆菌中。
利用表4中引物对及模板分别PCR扩增δ2，PTEF1-tCfTPS2-TADH1-PTDH3，tCfTPS3，TCYC1，PPGK1-tHMG1-TPGK1-PTDH3，BTS1～ERG20(F96C)-TERG20，HYG，δ1片段；
将δ2，PTEF1-tCfTPS2-TADH1-PTDH3片段融合，得到模块δ2-PTEF1-tCfTPS2-TADH1-PTDH3；将tCfTPS3，TCYC1，PPGK1-tHMG1-TPGK1-PTDH3片段融合，得到模块tCfTPS3-TCYC1-PPGK1-tHMG1-TPGK1-PTDH3；将HYG，δ1片段融合，得到模块HYG-δ1。
表4重组菌PCR扩增片段
Figure BDA0003360019060000081
(2)重组菌3酿酒酵母感受态制备
按照实施例1中步骤(2)的方法制备重组菌3酿酒酵母感受态细胞。
(3)酿酒酵母转化
a依次向重组菌3感受态细胞中加入120μl PEG3350(50g PEG3350/100ml水)、18μl1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、10μLδ2-PTEF1-tCfTPS2-TADH1-PTDH3片段、10μL tCfTPS3-TCYC1-PPGK1-tHMG1-TPGK1-PTDH3片段、10μL BTS1～ERG20(F96C)-TERG20片段和7μL HYG-δ1片段，体系共180μL。
b同实施例1中步骤(3)中的b，但是重组菌涂布于含有潮霉素的YPD培养基平板上，筛选正确的克隆命名为重组酿酒酵母菌4(重组菌4)。
实施例5、重组酿酒酵母菌5(重组菌5)的构建
向重组菌4中导入含有CfCYP76AH15～t66CPR～t30CYB5、CfCYP76AH11～t66CPR～t30CYB5、CfCYP76AH16～t66CPR～t30CYB5和CfACT1-8表达盒，得到重组菌5；
所述t66CPR是CfCPR从N端截短了60个氨基酸后获得；
所述t30CYB5是细胞色素CYB5从C端截短了30个氨基酸后获得，细胞色素CYB5的核苷酸序列如SEQ ID No.11所示；
CfCYP76AH15人工合成、CfCYP76AH11人工合成、CfCYP76AH16人工合成、CfCPR人工合成、CfACT1-8版段，人工合成。
细胞色素CYB5基因的来源(原始来源，黄花蒿，Artemisia annua)
(1)模块构建
利用表5中的引物对及模板分别PCR扩增片段rDNA2，PPGK1，CfCYP76AH15，t66CPR，t30CYB5，融合片段获得模块rDNA2-PPGK1-CfCYP76AH15～t66CPR～t30CYB5；PCR扩增片段TADH1，PTEF1，CfCYP76AH11，t66CPR，融合片段获得模块TADH1-PTEF1-CfCYP76AH11～t66CPR；PCR扩增片段t30CYB5，TADH2，PTDH3，CfCYP76AH16，融合片段获得模块t30CYB5-TADH2-PTDH3-CfCYP76AH16；PCR扩增片段t66CPR～t30CYB5，TPGK1，PTEF2，融合片段获得模块t66CPR～t30CYB5-TPGK1-PTEF2；PCR扩增片段CfACT1-8，TCYC1，G418(人工合成SEQ ID No.18)，rDNA1，融合片段获得模块CfACT1-8-TCYC1-G418-rDNA1。
表5重组菌PCR扩增片段
Figure BDA0003360019060000091
Figure BDA0003360019060000101
(2)重组菌4酿酒酵母感受态制备
按照实施例1中步骤(2)的方法制备重组菌4酿酒酵母感受态细胞。
(3)酿酒酵母转化
a依次向重组菌4感受态细胞中加入120μl PEG3350(50g PEG3350/100ml水)、18μl1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、8μl rDNA2-PPGK1-CfCYP76AH15～t66CPR～t30CYB5片段、8μl TADH1-PTEF1-CfCYP76AH11～t66CPR片段、7μl t30CYB5-TADH2-PTDH3-CfCYP76AH16片段、6μl t66CPR～t30CYB5-TPGK1-PTEF2片段和8μl CfACT1-8-TCYC1-G418-rDNA1片段，体系共180μl。
b同实施例1中步骤(3)中的b，但是重组菌涂布于含有G418的YPD培养基平板上，筛选正确的克隆命名为重组酿酒酵母菌5(重组菌5)。
实施例6、重组酿酒酵母菌6(重组菌6)的构建
向重组菌5中导入内质网调控因子INO2表达盒，得到重组菌6；所述内质网调控因子INO2的核苷酸序列如SEQ ID No.12所示。(内质网调控因子INO2来源：酿酒酵母Saccharomyces cerevisiae)
一、模块构建
以表6引物对及模板分别PCR扩增MET17up，PPGK1，INO2，TCYC1，LEU2，MET17down片段，融合MET17up，PPGK1，INO2片段，得到模块MET17up-PPGK1-INO2；融合TCYC1，LEU2，MET17down片段，得到模块TCYC1-LEU2-MET17down。
表6重组菌PCR扩增片段
Figure BDA0003360019060000111
(2)重组菌5酿酒酵母感受态制备
按照实施例1中步骤(2)的方法制备重组菌1酿酒酵母感受态细胞。
(3)酿酒酵母转化
a依次向重组菌5酿酒酵母感受态细胞中加入120μl PEG3350(50g PEG3350/100ml水)、18μl 1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、20μl MET17up-PPGK1-INO2片段和17μl TCYC1-LEU2-MET17down片段，体系共180μl。
b同实施例1中步骤(3)中的b，但是重组菌涂布于缺亮氨酸的SC选择性固体培养基平板上，筛选正确的克隆命名为重组酿酒酵母菌6(重组菌6)。
实施例7、重组酿酒酵母菌7(重组菌7)的构建
向重组菌6中导入含有6-磷酸葡萄糖脱氢酶编码基因ZWF1(SEQ ID No.13)，磷酸葡萄糖酸脱氢酶编码基因GND1(SEQ ID No.14)，乙醛脱氢酶编码基因ALD6(SEQ ID No.15)和乙酰辅酶A合酶编码基因ACS(SEQ ID No.16)的表达盒，得到重组菌7；
6-磷酸葡萄糖脱氢酶编码基因ZWF1、磷酸葡萄糖酸脱氢酶编码基因GND1、乙醛脱氢酶编码基因ALD6和乙酰辅酶A合酶编码基因ACS来源于：酿酒酵母Saccharomycescerevisiae。
(1)模块构建
利用表7中的引物对及模板PCR扩增TRP1up，PTEF1，ACS，TCYC1片段，融合片段获得模块TRP1up-PTEF1-ACS-TCYC1；PCR扩增PADH2，ALD6，TTDH2，PPGK1片段，融合片段获得模块PADH2-ALD6-TTDH2-PPGK1；PCR扩增ZWF1，TADH2，PTDH3，GND1片段，融合片段获得模块ZWF1-TADH2-PTDH3-GND1；PCR扩增TADH1，MET17，TRP1down片段，融合片段获得模块TADH1-MET17-TRP1down。
表7重组菌PCR扩增片段
Figure BDA0003360019060000121
Figure BDA0003360019060000131
(2)重组菌6酿酒酵母感受态制备
按照实施例1中步骤(2)的方法制备重组菌6酿酒酵母感受态细胞。
(3)酿酒酵母转化
a依次向重组菌6感受态细胞中加入120μl PEG3350(50g PEG3350/100ml水)、18μl1.0M LiAc水溶液、将开水煮沸后冷却的5μl鲑鱼精DNA、10μl TRP1up-PTEF1-ACS-TCYC1片段、9μl PADH2-ALD6-TTDH2-PPGK1片段、9μl ZWF1-TADH2-PTDH3-GND1片段和9μl TADH1-MET17-TRP1down片段，体系共180μl。
b同实施例1中步骤(3)中的b，但是重组菌涂布于缺甲硫氨酸的SC选择性固体培养基平板上，筛选正确的克隆命名为重组酿酒酵母菌7。
实施例8、重组酿酒酵母菌发酵生成毛喉素
(1)重组酿酒酵母菌培养及产物提取
将实施例1-7分别得到的重组菌1-7，连同酿酒酵母Saccharomyces cerevisiaeW303-1a，在SC选择性固体培养基上活化；然后将各个重组菌接种到试管YPD液体培养基中，过夜培养，待OD600长到约为4.0，接种到含30mLYPD液体培养基的摇瓶中，使初始OD600为0.1，30℃，220rpm发酵4d后，测毛喉素产量。
毛喉素的提取方法为:
细胞外的毛喉素提取，5mL发酵液和500μL正己烷混合，用涡旋振荡器振荡1h,然后12000g离心5min，收集正己烷层，烘干然后用甲醇复溶。
胞内的毛喉素按照如下方式提取：将细胞与0.5mL正己烷及0.25mL直径为0.5mm的玻璃珠混合，涡旋震荡1h破碎细胞，然后12000g离心5min，收集正己烷，烘干然后用甲醇复溶。
(2)毛喉素的HPLC-MS检测
毛喉素检测液质联用进行检测(HPLC-MS)。在Hypersil C18色谱柱(4.6mm×250mm，5μm；Elite Analytical Instruments Co.，Ltd.，中国大连)
毛喉素的HPLC-MS检测方法为：乙腈-水(50：50，v/v)用作流动相。检测波长为210nm。MS操作条件如下：信号源类型，ESI；离子极性，正；所有光谱都是在50/1300的m/z范围内获得的；干气流，8.0L/min；干燥温度250℃；雾化器压力为1.2bar；探头电压+4.5kV。
(3)检测结果(各重组菌为胞内胞外总产物)
A.酿酒酵母Saccharomyces cerevisiae W303-1a没有检测到毛喉素；
B.重组菌1：提取重组菌1的发酵产物，没有检测到毛喉素；
C.重组菌2：提取重组菌2发酵产物，没有检测到毛喉素；
D.重组菌3：提取重组菌3发酵产物，检测到毛喉素的含量为0.09mg/L；
E.重组菌4：提取重组菌4发酵产物，检测到毛喉素的含量为1.73mg/L；
F.重组菌5：提取重组菌5发酵产物，检测到毛喉素的含量为4.28mg/L；
G.重组菌6：提取重组菌6发酵产物，检测到毛喉素的含量为11.12mg/L；
H.重组菌7：提取重组菌7发酵产物，检测到毛喉素的含量为18.37mg/L。
(4)实施例所用培养基
YPD液体培养基：葡萄糖终浓度为20g/L，酵母浸粉终浓度为10g/L，蛋白胨终浓度为20g/L，以蒸馏水配制。YPD固体培养基则在YPD液体培养基中加20g/L的琼脂粉。
SC选择性固体培养基：葡萄糖终浓度为2g/L，无氨基酵母氮源(YNB)终浓度为6.7g/L，氨基酸混合物终浓度为0.2g/L，20g/L的琼脂粉,以蒸馏水配制，缺失氨基酸混合物指去掉氨基酸混合物中的对应成份。
氨基酸混合物：甘氨酸，2.0g；丙氨酸，2.0g；甲硫氨酸，2.0g；赖氨酸，2.0g；精氨酸，2.0g；丝氨酸，2.0g；天冬酰胺，2.0g；天冬氨酸，2.0g；苯丙氨酸，2.0g；半胱氨酸，2.0g；脯氨酸，2.0g；酪氨酸，2.0g；谷氨酸，2.0g；缬氨酸，2.0g；苏氨酸，2.0g；丝氨酸，2.0g；异亮氨酸，2.0g；肌醇，2.0g；谷酰胺，2.0g；对氨基苯甲酸0.2g；腺嘌呤，0.5g；亮氨酸10g；甲硫氨酸，2g；色氨酸，2g；组氨酸，2g；尿嘧啶，2g。
序列表
<110> 天津大学
<120> 生产毛喉素的重组酿酒酵母菌及构建方法
<160> 182
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2190
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggttgcat ctttagatgc tttgaacggt attcaaaagg ttggtccagc tactattggt 60
acaccagaag aagaaaataa gaaaattgaa gattctatcg aatatgttaa agaattgtta 120
aaaacaatgg gtgacggtag aatttctgtt tcaccatacg atactgcaat cgttgctttg 180
attaaagatt tggaaggtgg tgacggtcca gaatttccat catgtttgga atggattgca 240
caaaatcaat tagctgatgg ttcttggggt gaccatttct tttgtatcta tgatagagtt 300
gttaatacag ctgcatgtgt tgttgcattg aagtcttgga acgttcatgc agataagatc 360
gaaaagggtg ctgtttactt gaaggaaaac gttcataagt tgaaagatgg taaaattgaa 420
catatgccag caggttttga atttgttgtt ccagctactt tagaaagagc aaaagctttg 480
ggtattaaag gtttgccata cgatgatcct tttattagag aaatctattc agctaagcaa 540
actagattga caaagatccc aaagggtatg atctatgaat ctccaacatc attgttgtac 600
tctttagatg gtttggaagg tttagaatgg gataagatct tgaagttgca atctgcagat 660
ggttctttta ttacttctgt ttcttcaaca gctttcgttt ttatgcatac taacgatttg 720
aaatgtcatg cttttattaa aaatgctttg acaaactgta atggtggtgt tccacatact 780
tacccagttg atatcttcgc aagattatgg gctgttgata gattgcaaag attgggtatt 840
tcaagatttt tcgaaccaga aattaaatac ttgatggatc atatcaacaa cgtttggaga 900
gaaaagggtg ttttctcttc aagacattca caattcgctg atatcgatga tacatctatg 960
ggtattagat tgttgaagat gcatggttac aatgttaatc caaacgcatt ggaacatttc 1020
aagcaaaagg atggtaaatt cacttgttac gctgatcaac atatcgaatc tccatcacca 1080
atgtacaatt tgtacagagc tgcacaattg agattcccag gtgaagaaat cttgcaacaa 1140
gcattgcaat tcgcttacaa tttcttgcat gaaaatttgg cttcaaacca tttccaagaa 1200
aagtgggtta tttctgatca tttgatcgat gaagttagaa tcggtttgaa gatgccatgg 1260
tatgcaactt tgccaagagt tgaagcttca tactacttac aacattacgg tggttcttca 1320
gatgtttgga tcggtaaaac attgtacaga atgccagaaa tctctaacga tacttacaag 1380
atcttggcac aattggattt caataagtgt caagctcaac atcaattaga atggatgtca 1440
atgaaggaat ggtatcaatc taacaacgtt aaggaatttg gtatctctaa gaaagaattg 1500
ttgttggcat actttttggc tgcagctact atgtttgaac cagaaagaac acaagaaaga 1560
attatgtggg ctaagactca agttgtttca agaatgatca catcattttt gaataaggaa 1620
aatactatgt catttgattt gaaaattgca ttgttgacac aaccacaaca tcaaattaat 1680
ggttctgaaa tgaagaatgg tttggctcaa actttaccag cagcttttag acaattgttg 1740
aaggaatttg ataagtacac tagacatcaa ttgagaaaca catggaataa gtggttgatg 1800
aagttgaagc aaggtgacga taacggtggt gcagatgctg aattattggc aaacacattg 1860
aacatctgtg ctggtcataa cgaagatatc ttgtctcatt acgaatacac tgccttgtct 1920
tcattgacaa ataagatctg tcaaagatta tcacaaattc aagataagaa aatgttggaa 1980
atcgaagaag gttctattaa agataaggaa atggaattgg aaattcaaac attagttaaa 2040
ttagttttgc aagaaacttc aggtggtatc gatagaaaca tcaagcaaac atttttgtct 2100
gtttttaaaa cattctacta cagagcatac catgatgcta agactatcga tgctcatatc 2160
ttccaagttt tgtttgaacc agttgtttaa 2190
<210> 2
<211> 1689
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtcagctg cagttaaatg ttcattgact acaccaactg atttgatggg taaaattaaa 60
gaagttttta atagagaagt tgatacttct ccagctgcaa tgactacaca ttctacagat 120
atcccatcaa atttgtgtat catcgatact ttgcaaagat tgggtatcga tcaatacttc 180
caatctgaaa tcgatgctgt tttgcatgat acatacagat tgtggcaatt gaaaaagaaa 240
gatattttct ctgatatcac tacacatgct atggctttta gattgttgag agttaagggt 300
tacgaagttg catcagatga attggctcca tacgcagatc aagaaagaat taatttgcaa 360
actattgatg ttccaacagt tgttgaattg tatagagctg cacaagaaag attgactgaa 420
gaagattcta cattggaaaa gttgtacgtt tggacttcag catttttgaa gcaacaattg 480
ttgacagatg caatcccaga taagaaattg cataagcaag ttgaatacta cttgaaaaat 540
tatcatggta ttttagatag aatgggtgtt agaagaaatt tggatttgta cgatatctct 600
cattacaaat cattgaaggc tgcacataga ttctacaatt tgtctaacga agatatcttg 660
gctttcgcaa gacaagattt caacatctca caagctcaac atcaaaagga attgcaacaa 720
ttgcaaagat ggtatgcaga ttgtagattg gatactttga agttcggtag agatgttgtt 780
agaatcggta atttcttgac atctgctatg attggtgacc cagaattatc agatttgaga 840
ttggctttcg caaagcatat cgttttggtt actagaatcg atgatttctt tgatcatggt 900
ggtccaaagg aagaatctta cgaaattttg gaattagtta aggaatggaa ggaaaagcca 960
gctggtgaat acgtttcaga agaagttgaa atcttgttta ctgcagttta caacacagtt 1020
aacgaattag ctgaaatggc acatatcgaa caaggtagat ctgttaagga tttgttggtt 1080
aagttgtggg ttgaaatctt gtcagttttt agaatcgaat tggatacttg gacaaacgat 1140
actgctttga cattagaaga atatttgtct caatcatggg tttctattgg ttgtagaatc 1200
tgtatcttga tctcaatgca attccaaggt gttaagttgt ctgatgaaat gttgcaatca 1260
gaagaatgta ctgatttgtg tagatacgtt tctatggttg atagattgtt gaacgatgtt 1320
caaactttcg aaaaggaaag aaaggaaaac actggtaatt ctgtttcatt gttacaagct 1380
gcacataagg atgaaagagt tattaatgaa gaagaagctt gtatcaaagt taaagaattg 1440
gcagaataca acagaagaaa gttgatgcaa attgtttaca agactggtac aatcttccca 1500
agaaagtgta aggatttgtt tttaaaagct tgtagaatcg gttgttattt gtactcttca 1560
ggtgacgagt ttacttctcc acaacaaatg atggaagata tgaagtcatt ggtttacgaa 1620
ccattgccaa tctctccacc agaagctaat aatgcatcag gtgaaaagat gtcttgtgtt 1680
tcaaattaa 1689
<210> 3
<211> 1512
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgccagttt taaccaataa aacagtcatt tctggatcga aagtcaaaag tttatcatct 60
gcgcaatcga gctcatcagg accttcatca tctagtgagg aagatgattc ccgcgatatt 120
gaaagcttgg ataagaaaat acgtccttta gaagaattag aagcattatt aagtagtgga 180
aatacaaaac aattgaagaa caaagaggtc gctgccttgg ttattcacgg taagttacct 240
ttgtacgctt tggagaaaaa attaggtgat actacgagag cggttgcggt acgtaggaag 300
gctctttcaa ttttggcaga agctcctgta ttagcatctg atcgtttacc atataaaaat 360
tatgactacg accgcgtatt tggcgcttgt tgtgaaaatg ttataggtta catgcctttg 420
cccgttggtg ttataggccc cttggttatc gatggtacat cttatcatat accaatggca 480
actacagagg gttgtttggt agcttctgcc atgcgtggct gtaaggcaat caatgctggc 540
ggtggtgcaa caactgtttt aactaaggat ggtatgacaa gaggcccagt agtccgtttc 600
ccaactttga aaagatctgg tgcctgtaag atatggttag actcagaaga gggacaaaac 660
gcaattaaaa aagcttttaa ctctacatca agatttgcac gtctgcaaca tattcaaact 720
tgtctagcag gagatttact cttcatgaga tttagaacaa ctactggtga cgcaatgggt 780
atgaatatga tttctaaagg tgtcgaatac tcattaaagc aaatggtaga agagtatggc 840
tgggaagata tggaggttgt ctccgtttct ggtaactact gtaccgacaa aaaaccagct 900
gccatcaact ggatcgaagg tcgtggtaag agtgtcgtcg cagaagctac tattcctggt 960
gatgttgtca gaaaagtgtt aaaaagtgat gtttccgcat tggttgagtt gaacattgct 1020
aagaatttgg ttggatctgc aatggctggg tctgttggtg gatttaacgc acatgcagct 1080
aatttagtga cagctgtttt cttggcatta ggacaagatc ctgcacaaaa tgttgaaagt 1140
tccaactgta taacattgat gaaagaagtg gacggtgatt tgagaatttc cgtatccatg 1200
ccatccatcg aagtaggtac catcggtggt ggtactgttc tagaaccaca aggtgccatg 1260
ttggacttat taggtgtaag aggcccgcat gctaccgctc ctggtaccaa cgcacgtcaa 1320
ttagcaagaa tagttgcctg tgccgtcttg gcaggtgaat tatccttatg tgctgcccta 1380
gcagccggcc atttggttca aagtcatatg acccacaaca ggaaacctgc tgaaccaaca 1440
aaacctaaca atttggacgc cactgatata aatcgtttga aagatgggtc cgtcacctgc 1500
attaaatcct aa 1512
<210> 4
<211> 1008
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 4
atggaggcca agatagatga gctgatcaat aatgatcctg tttggtccag ccaaaatgaa 60
agcttgattt caaaacctta taatcacatc cttttgaaac ctggcaagaa ctttagacta 120
aatttaatag ttcaaattaa cagagttatg aatttgccca aagaccagct ggccatagtt 180
tcgcaaattg ttgagctctt gcataattcc agccttttaa tcgacgatat agaagataat 240
gctcccttga gaaggggaca gaccacttct cacttaatct tcggtgtacc ctccactata 300
aacaccgcaa attatatgta tttcagagcc atgcaacttg tatcgcagct aaccacaaaa 360
gagcctttgt atcataattt gattacgatt ttcaacgaag aattgatcaa tctacatagg 420
ggacaaggct tggatatata ctggagagac tttctgcctg aaatcatacc tactcaggag 480
atgtatttga atatggttat gaataaaaca ggcggccttt tcagattaac gttgagactc 540
atggaagcgc tgtctccttc ctcacaccac ggccattcgt tggttccttt cataaatctt 600
ctgggtatta tttatcagat tagagatgat tacttgaatt tgaaagattt ccaaatgtcc 660
agcgaaaaag gctttgctga ggacattaca gaggggaagt tatcttttcc catcgtccac 720
gcccttaact tcactaaaac gaaaggtcaa actgagcaac acaatgaaat tctaagaatt 780
ctcctgttga ggacaagtga taaagatata aaactaaagc tgattcaaat actggaattc 840
gacaccaatt cattggccta caccaaaaat tttattaatc aattagtgaa tatgataaaa 900
aatgataatg aaaataagta tttacctgat ttggcttcgc attccgacac cgccaccaat 960
ttacatgacg aattgttata tataatagac cacttatccg aattgtga 1008
<210> 5
<211> 1059
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60
gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120
gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180
gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240
aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttactgctt ggtcgccgat 300
gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360
gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420
aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480
accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540
gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600
tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660
gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720
gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780
gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840
aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900
attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960
gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020
actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059
<210> 6
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggaaacca tgactttgtt gttgcccttg ttctttattg ccttgaccta ctttttgtcc 60
tggcgtagaa gaagaaattt gccaccaggt ccatttccat tgccaattat tggtaacttg 120
ttgcagattg gttccaagcc acatcaatct tttgctcagt tgtctaaaaa gtacggccca 180
ttgatgtctg ttcaattggg ttctgtctac accgttattg cttcatctcc agaaatggcc 240
aaagaaatct tgcaaaaaca cggtcaagtt ttctccggta gaactattgc tcaagcagct 300
caagcttgtg gtcatgatca aatttctatt ggtttcttgc cagttgctac cacttggaga 360
gatatgagaa agatctgcaa agaacagatg ttctcccatc actctctgga atcttctaaa 420
gaattgaggc acgaaaagct gcaaaagttg ttggattacg ctcaaaagtg ttgtgaagct 480
ggtagagctg ttgatattag agaagctgct ttcattacca ccttgaactt aatgtctgct 540
accttgtttt ctacccaagc tactgaattt gattccgaag ctaccaaaga attcaaagaa 600
gttattgaag gcgttgccgt tatagttggt gaacctaatt ttgctgatta cttcccaatc 660
ttgaagccat ttgacttgca aggtattaag agaagggcta actcttattt cggtaggttg 720
ttgaagttga tggaaaggta cttgaacgaa aggttggaat ctagaagatt gaatccagat 780
gctccaaaga agaacgattt cttggaaacc ttggttgaca ttattcaagc cgatgagtac 840
aagttgacta ccgatcatgt tacccatttg atgttggatt tgttcgttgg tggttctgaa 900
acttctgcta catctttgga atggatcatg tccgaattgg tttccaatcc atctaagttg 960
gctaaggtta aggctgaatt gaagtctgtt gttggtgaaa agaaggttgt ctccgaatct 1020
gaaatggcta gattgccata cttgcaagcc gttatcaaag aggttttgag attgcatcca 1080
cctggtcctt tgttattgcc aagaaaagct ggttctgatc aagttgttaa cggttacttg 1140
attccaaagg gtactcagtt gttgtttaac gtttgggcta tgggtagaga tccatctatt 1200
tggaaaaacc cagaatcctt cgaaccagag agatttttga accagaacat cgattacaag 1260
ggtcaagact tcgaattgat tccatttggt tctggtagaa gaatctgtcc aggtatgcca 1320
ttggctgata gaattatgca tatgactacc gccactttgg ttcataattt cgattggaaa 1380
ttggaagatg gtgctggtga tgctgatcat aagggtgatg atccttttgg tttggctatt 1440
agacgtgcta ctccattgag aattatccca ttgaagcctt aa 1482
<210> 7
<211> 1487
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggaattgg ttcaagttat cgctgttgtt gccgttgttg ttgttttgtg gtcacaattg 60
aaacgtaagg gtagaaaatt gccaccaggt ccatctccat tgccaatagt tggtaacatt 120
ttccagttgt ccggtaagaa catcaacgaa tcttttgcca agctgtctaa aatctacggt 180
ccagttatgt ctttgaggtt gggttctttg ttgaccgtta ttatctcttc accagaaatg 240
gccaaagaag tcttgacttc taaggatttt gctaacagac cattgactga agctgctcat 300
gctcatggtc attctaaatt ttctgttggt ttcgttccag tctctgatcc aaaatggaaa 360
caaatgagaa gagtctgcca agaagaaatg ttcgcctcta gaattttgga aaactctcaa 420
cagagaaggc accagaagtt gcaagaattg attgatcacg tccaagaatc tagagatgct 480
ggtagagctg ttactattag agatccagtt ttcgctacca ccttgaacat tatgtccttg 540
actttgtttt ctgccgatgc tactgaattc tcttcttctg ctactgctga attgagagat 600
attatggctg gtgttgtttc tgttttgggt gctgctaatt tggctgattt cttcccaatc 660
ttgaaatact tcgatccaca gggtatgaga agaaaagctg acttgcatta cggtagattg 720
atcgaccata tcaagtccag aatggacaag agatctgaat tgaagaaggc taatcccaat 780
catccaaagc acgatgattt cttggaaaag atcatcgaca tcaccatcca aagaaactac 840
gatttgacca ttaacgagat cacccatttg ttggttgact tgtatttggc tggttccgaa 900
tctactgtta tgactattga atggaccatg gccgaattga tgttaagacc agaatctttg 960
gctaagttga agctgaacta agatctgtta tgggtgagag aaagatgatc caagagtccg 1020
atgatatttc cagattgcca tatttgaacg gtgctatcaa agaggctttg agattgcatc 1080
cacctggtcc tttgttgttt gctagaaagt ctgaaatcga tgtcgagttg tctggttact 1140
ttattccaaa gggtactcag atcttggtta acgaatgggg tatgggtaga gatccttctg 1200
tttggcctaa tccagaatgt tttcaaccag aaaggttcct ggataagaac attgattaca 1260
agggtcaaga cccacaattg attccatttg gtgcaggtag aagaatttgt ccaggtattc 1320
caattgctca cagagttgtt cattcagttg ttgctgcttt ggttcataac ttcgattggg 1380
aatttgctcc tggtggttct caatgtaaca acgaattttt cactggtgct gccttggtta 1440
gagaagttcc attgaagttg attcctttga acccaccatc catctga 1487
<210> 8
<211> 1497
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggaattgg ttgaagttat cgttgttgtt gtaggtgctg ctgctttggg tgttgttttg 60
tggtcacatt tgaaaccaga aggtagaaaa ttgccaccag gtccatctcc attgccaatt 120
tttggtaaca ttttccaatt gaccggtcca aacacttgtg aatcttttgc taacctgtcc 180
aaaaagtacg gtccagttat gtctttgaga ttgggttctt tgttcaccgt tgttatctct 240
tcaccagaaa tggccaaaga agttttgact aacaccgatt tcttggagag gccattgatg 300
caagctgttc atgctcatga tcacgctcaa ttctctattg cttttttgcc agttactacc 360
ccaaagtgga aacagttgag aagaatttgc caagaacaaa tgttcgcctc cagaatcttg 420
gaaaaatctc aaccattgag acaccagaag ttgcaagaat tgattgatca cgttcaaaag 480
tgctgtgatg ctggtagagc tgttactatt agagatgctg cttttgctac taccctgaac 540
ttgatgtctg ttaccatgtt ttctgctgat gctaccgaat tggattcttc tgttactgct 600
gaattgaggg aattgatggc tggtgttgtt actgttttgg gtactccaaa tttcgctgat 660
ttcttcccca tcttgaaata cttggatcca caaggtgtta gaagaaaggc tcattttcat 720
tacggtaaga tgttcgacca catcaagtct agaatggctg aaagagttga actgaagaag 780
gctaatccaa accatttgaa gcacgacgac tttctggaaa agatcttgga tatctccttg 840
agaagagact acgaattgac cattcaagat atcacccatt tgttggttga cttgtatgtt 900
gctggttctg aatctaccgt tatgtccatt gaatggatca tgtccgaatt gatgttgcac 960
ccacaatctt tggctaaatt gaaagccgaa ttgagatccg ttatgggtga aagaaagatg 1020
atccaagaat ccgaagatat ctccagattg ccattcttga acgccgttat caaagaaacc 1080
ttgagattgc atccacctgg tcctttgttg tttccaagac aaaacactaa cgacgtcgaa 1140
ttgaacggtt actttattcc aaagggtact cagatcttgg ttaacgaatg ggctattggt 1200
agagatccat ctgtttggcc taatccagaa tcctttgttc ctgaaaggtt cttggataag 1260
aacatcgatt acaagggtca agatccacaa ttggttccat ttggttctgg tagaagaatc 1320
tgcttgggta ttccaattgc tcatagaatg gttcattcta ctgttgccgc tttgatccat 1380
aatttcgaat ggaaatttgc cccagatggt tctgagtata atcgtgaatt attctctggt 1440
ccagctttga gaagggaagt tcctttgaat ttgattccac tgaacccctc attctaa 1497
<210> 9
<211> 2136
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggagagta ctatcgaaaa actttctcca ttcgatctta tgactgctat ccttaaaggt 60
gttaaattgg ataactctaa cggttctgct ggagtcgagc acccagccgt tatcgccatg 120
ttgatggaga ataaagatct tgtcatgatg ttgaccacca gtgtcgccgt cttgttggga 180
ttggccgtct atcttgtctg gagaagagga gctggtagtg ccaagagagt cgtcgaacca 240
cctaagttgg tcatccctaa gggtccagtc gacgccgaag aggaagacga cggaaaaaag 300
aaggtcacca tcttcttcgg aactcaaacc ggtaccgctg agggttttgc taaagccttg 360
gctgaagaag ccaaggctag atatcctttg accaacttca aggttgtcga ccttgacgat 420
tacgccgccg atgacgaaga atacgaggaa aagatgaaga aagaaacttt tgcttttttc 480
ttcttggcta cctatggaga cggagagcca accgataacg ccgctagatt ctataagtgg 540
ttcagtgagg gtaaagaaag gggagaaatc tttaaaaatt tgaactacgg tgtttttggt 600
cttggtaata gacaatatga acattttaat aagatcgcta tcgttgtcga cgacatcttg 660
ttggagcaag gtggtaacag acttgtccca gtcggtcttg gtgacgacga ccaatgtatc 720
gaggacgact tctctgcttg gagggataac gtctggccag agttggacaa gcttcttagg 780
gatgaggatg acgctactgt cgctactcct tacaccgccg ctgtcttgga gtatagagtc 840
gtcttccatg accagagtga cgaacttcac tctgagaaca atcttgccaa cggtcatgcc 900
aacggtaatg ccagttacga tgcccagcat ccatgcaagg tcaacgtcgc tgtcaagagg 960
gagttgcaca ctccactttc tgatagatct tgtacccacc ttgagttcga catcagtgga 1020
actggtcttg agtacgagac cggtgaccac gttggtgttt attgtgaaaa tttgatcgag 1080
actgtcgaag aggccgagag acttttgggt cttagtcctc agactttctt tagtgtccat 1140
accgacaaag ctgacggaac tcctcttgga ggaagtgctt tgcctcctcc tttcccacct 1200
tgcaccttga gaaccgccct ttctaggtac gccgaccttc ttaacgcccc aaagaagtct 1260
gctcttactg ctttggccgc ctatgccagt gatccatctg aggccgatag gttgaagcac 1320
ttggcctctc cagatggaaa ggaggagtac gcccagtacg ttgtcagtgg tcagaggagt 1380
cttttggagg tcatggccga tttccctagt gccaaacctc cattgggtgt ctttttcgct 1440
gccatcgccc caagattgca gccaaggttc tacagtatct ctagttctcc taagatcgcc 1500
ccatctagga ttcacgtcac ttgtgccttg gtctatgaga agatgcctac cggtaggatc 1560
cacaaaggag tctgcagtac ttggatgaag aacgccgttc cattggagga gagtcctaac 1620
tgctctagtg ctccagtctt cgttaggacc tctaatttta gacttccagc cgacccaaag 1680
gttccagtta tcatgatcgg tcccggtacc ggtcttgccc cattcagagg tttccttcaa 1740
gaaaggcttg cccttaagga atctggtgcc gaacttggac cagctattct tttctttgga 1800
tgtagaaata gtaagatgga ctttatttat caagacgagc ttgataactt cgtcaaggcc 1860
ggtgtcgtct ctgagcttgt cttggccttc tctagggagg gaccagctaa ggagtacgtc 1920
cagcacaaaa tggcccagaa ggcctctgac gtctggaaca tgattagtga gggtggttac 1980
gtctacgtct gtggtgacgc taaaggtatg gctagagacg tccacagaac cttgcatacc 2040
atcgtccaag aacaaggaag tcttgatagt tctaaaactg agtcttttgt caaaaacttg 2100
caaatgactg gtaggtactt gagggacgtc tggtaa 2136
<210> 10
<211> 1275
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgaaggtcg aaagaatctc ccgtaagttc attaagccat atactccaac tccacagaac 60
ctgaaaaagt acaagttgtc cttgttggat aagtgcatgg gtcatatgga tttcgctgtt 120
gttttgttct acgagtctaa gccaagaaac aagaacgaat tggaagagtc cttggaaaag 180
gttttggttg atttttatcc attggccggt agatacacca tgaacgatca tatagttgat 240
tgctctgatg aaggtgccgt ttttgttgaa gctgaagctc caaatgttga attgaccgtt 300
gatcaattgg tcaagaacat ggaagctcaa accatccatg atttcttgcc agatcaatac 360
tttccagctg atgctcctaa tcctttgttg tctattcaag ttactcattt cccatgtggt 420
ggtttggcta ttggtatagt tgtttctcat gctgttttcg acggtttctc tttgggtgtt 480
tttttggctg cttggtctaa ggctactatg aatccagaaa gaaagatcga aatcacccca 540
tcttttgact tgccatcttt gttgccttac aaggatgaat ctttcggctt gaacttctcc 600
gaaattgtca aagctgaaaa catcgtcgtc aagagattga actttggtaa agaagccatc 660
accagattga ggtctaaatt gtctccaaat caaaacggca agaccatctc tagagttaga 720
gttgtttgtg ccgttatcgt taaggctttg atgggtttag aaagagctaa gaccagagat 780
ttcatgatct gccaaggtat caacatgaga gaaagaacaa aagccccatt gcaaaaacat 840
gcttgtggta atttggccgt ttcttcatac actagaagag ttgctgctgc tgaagcagaa 900
gaattgcaat ctttggttaa cttgatcggt gactccatcg aaaagtctat tgctgattac 960
gccgatatct tgtcctctga tcaagatggt agacatatca tctccaccat gatgaagtct 1020
ttcatgcaat ttgctgcccc agatattaag gctatttctt tcactgactg gtccaagttt 1080
ggtttctacc aagttgattt tggtttcggt aaaccagttt ggactggtgt tagaccagaa 1140
agaccaattt tttccgctgc cattttgatg tctaacagag aaggtgatgg tattgaagct 1200
tggttgcatt tggataagaa cgacatgttg atcttcgaac aggacgaaga aatcaagttg 1260
ttgattacca cctaa 1275
<210> 11
<211> 411
<212> DNA
<213> 黄花蒿(Artemisia annua)
<400> 11
atggcgagtg atccaaaaaa tttcgttttt gacgatatta gcaaacataa caaaactaag 60
gattgctggt tgattattga tggtaaagtt tatgatgtta ccccatttat ggaagatcat 120
ccaggtggtg atgaagtttt gttggctgct accggtaaag atgctaccga tgattttgaa 180
gatgttggtc attctgatga tgctagagaa atgatgcata aatattatat tggtgaagtt 240
gataaagcta ccgttccaaa aaaaagagct tatgttccac cagctgatag gcattacaat 300
ccggacaaaa cccaagattt catagttaaa attctccaat ttttggttcc attggttatt 360
ttgggtttgg cttttgctgt tagatcttat accaaagaaa gatctgctta a 411
<210> 12
<211> 915
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 12
atgcaacaag caactgggaa cgaattactg ggtatcctag atctggataa cgatatagac 60
tttgaaactg cttaccaaat gctcagcagt aacttcgacg accaaatgtc tgcgcacata 120
catgaaaaca cgtttagtgc aacttcccct cctctgttaa cacacgagct cggcataatt 180
cctaacgtag caaccgtgca accctctcac gtagaaacta tacctgccga taaccaaact 240
catcatgctc ctttgcatac tcatgcacac tatctaaatc acaaccctca tcaaccaagc 300
atgggttttg atcaagcgct tggtctcaag ttgtctcctt ccagttcggg gttgttgagc 360
acgaatgaat cgaatgccat tgaacagttt ttagacaatc taatatcaca ggatatgatg 420
tcttccaacg cttccatgaa ctccgaatca catctacata taagatcacc aaaaaagcag 480
cataggtata ccgaattaaa tcaaagatat cctgaaacac atccacacag taacacaggg 540
gagttaccca caaacacagc agatgtgcca actgagttca ccacgaggga aggacctcat 600
cagcctatcg gcaatgacca ctacaacccg ccaccgtttt cagtacctga gatacgaatc 660
ccagactctg atattccagc caatatcgag gacgaccctg tgaaggtacg gaaatggaaa 720
cacgttcaaa tggagaagat acgaagaata aacaccaaag aagcctttga aaggctcatt 780
aaatcagtaa ggaccccacc gaaggaaaac gggaaaagaa ttcccaagca tattctttta 840
acttgtgtaa tgaacgatat caagtccatt agaagcgcaa atgaagcact acagcacata 900
ctggatgatt cctga 915
<210> 13
<211> 1518
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 13
atgagtgaag gccccgtcaa attcgaaaaa aataccgtca tatctgtctt tggtgcgtca 60
ggtgatctgg caaagaagaa gacttttccc gccttatttg ggcttttcag agaaggttac 120
cttgatccat ctaccaagat cttcggttat gcccggtcca aattgtccat ggaggaggac 180
ctgaagtccc gtgtcctacc ccacttgaaa aaacctcacg gtgaagccga tgactctaag 240
gtcgaacagt tcttcaagat ggtcagctac atttcgggaa attacgacac agatgaaggc 300
ttcgacgaat taagaacgca gatcgagaaa ttcgagaaaa gtgccaacgt cgatgtccca 360
caccgtctct tctatctggc cttgccgcca agcgtttttt tgacggtggc caagcagatc 420
aagagtcgtg tgtacgcaga gaatggcatc acccgtgtaa tcgtagagaa acctttcggc 480
cacgacctgg cctctgccag ggagctgcaa aaaaacctgg ggcccctctt taaagaagaa 540
gagttgtaca gaattgacca ttacttgggt aaagagttgg tcaagaatct tttagtcttg 600
aggttcggta accagttttt gaatgcctcg tggaatagag acaacattca aagcgttcag 660
atttcgttta aagagaggtt cggcaccgaa ggccgtggcg gctatttcga ctctataggc 720
ataatcagag acgtgatgca gaaccatctg ttacaaatca tgactctctt gactatggaa 780
agaccggtgt cttttgaccc ggaatctatt cgtgacgaaa aggttaaggt tctaaaggcc 840
gtggccccca tcgacacgga cgacgtcctc ttgggccagt acggtaaatc tgaggacggg 900
tctaagcccg cctacgtgga tgatgacact gtagacaagg actctaaatg tgtcactttt 960
gcagcaatga ctttcaacat cgaaaacgag cgttgggagg gcgtccccat catgatgcgt 1020
gccggtaagg ctttgaatga gtccaaggtg gagatcagac tgcagtacaa agcggtcgca 1080
tcgggtgtct tcaaagacat tccaaataac gaactggtca tcagagtgca gcccgatgcc 1140
gctgtgtacc taaagtttaa tgctaagacc cctggtctgt caaatgctac ccaagtcaca 1200
gatctgaatc taacttacgc aagcaggtac caagactttt ggattccaga ggcttacgag 1260
gtgttgataa gagacgccct actgggtgac cattccaact ttgtcagaga tgacgaattg 1320
gatatcagtt ggggcatatt caccccatta ctgaagcaca tagagcgtcc ggacggtcca 1380
acaccggaaa tttaccccta cggatcaaga ggtccaaagg gattgaagga atatatgcaa 1440
aaacacaagt atgttatgcc cgaaaagcac ccttacgctt ggcccgtgac taagccagaa 1500
gatacgaagg ataattag 1518
<210> 14
<211> 1470
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 14
atgtctgctg atttcggttt gattggtttg gccgtcatgg gtcaaaattt gatcttgaac 60
gctgctgacc acggtttcac tgtttgtgct tacaacagaa ctcaatccaa ggtcgaccat 120
ttcttggcca atgaagctaa gggcaaatct atcatcggtg ctacttccat tgaagatttc 180
atctccaaat tgaagagacc tagaaaggtc atgcttttgg ttaaagctgg tgctccagtt 240
gacgctttga tcaaccaaat cgtcccactt ttggaaaagg gtgatattat catcgatggt 300
ggtaactctc acttcccaga ttctaataga cgttacgaag aattgaagaa gaagggtatt 360
cttttcgttg gttctggtgt ctccggtggt gaggaaggtg cccgttacgg tccatctttg 420
atgccaggtg gttctgaaga agcttggcca catattaaga acatcttcca atccatctct 480
gctaaatccg acggtgaacc atgttgcgaa tgggttggcc cagccggtgc tggtcactac 540
gtcaagatgg ttcacaacgg tattgaatac ggtgatatgc aattgatttg tgaagcttat 600
gacatcatga agagattggg tgggtttacc gataaggaaa tcagtgacgt ttttgccaaa 660
tggaacaatg gtgtcttgga ttccttcttg gtcgaaatta ccagagatat tttgaaattc 720
gacgacgtcg acggtaagcc attagttgaa aaaatcatgg atactgctgg tcaaaagggt 780
actggtaagt ggactgccat caacgccttg gatttgggta tgccagttac tttgattggt 840
gaagctgtct ttgcccgttg tctatctgct ttgaagaacg agagaattag agcctccaag 900
gtcttaccag gcccagaagt tccaaaagac gccgtcaagg acagagaaca atttgtcgat 960
gatttggaac aagctttgta tgcttccaag attatttctt acgctcaagg tttcatgttg 1020
atccgtgaag ctgctgctac ttatggctgg aaactaaaca accctgccat cgctttgatg 1080
tggagaggtg gttgtatcat tagatctgtt ttcttgggtc aaatcacaaa ggcctacaga 1140
gaagaaccag atttggaaaa cttgttgttc aacaagttct tcgctgatgc cgtcaccaag 1200
gctcaatctg gttggagaaa gtcaattgcg ttggctacca cctacggtat cccaacacca 1260
gccttttcca ccgctttgtc tttctacgat gggtacagat ctgaaagatt gccagccaac 1320
ttactacaag ctcaacgtga ctactttggt gctcacactt tcagagtgtt gccagaatgt 1380
gcttctgaca acttgccagt agacaaggat atccatatca actggactgg ccacggtggt 1440
aatgtttctt cctctacata ccaagcttaa 1470
<210> 15
<211> 1503
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 15
atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60
acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120
aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180
accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240
tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300
gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360
ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420
gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480
gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540
tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600
acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660
gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720
agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780
tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840
gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900
aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960
gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020
gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080
tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140
gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200
gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260
ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320
ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380
tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440
gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500
taa 1503
<210> 16
<211> 1959
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 16
atgagtcaaa cacataaaca cgcaatccct gccaacatag ccgacagatg cttaatcaat 60
cctgaacaat acgaaaccaa atacaaacaa tctattaatg atccagacac tttttggggt 120
gaacaaggta aaatcttgga ttggatcaca ccataccaaa aggttaagaa tacctcattc 180
gctcctggta acgtctccat taagtggtac gaagatggta ctttgaattt ggctgcaaac 240
tgcttggata gacatttgca agaaaatggt gacagaacag ctattatatg ggaaggtgac 300
gacgcatcac aatccaagca tatatcatac agagaattac acagagatgt ttgcagattt 360
gcaaacactt tgttggattt gggtattaaa aagggtgacg ttgtcgccat atacatgcca 420
atggttcctg aagccgctgt cgccatgtta gcatgtgcca gaatcggtgc tgtccatagt 480
gtaatttttg gtggtttctc tccagaagct attgcaggta gaatcattga ttcttcatcc 540
agattggtca taacagcaga cgaaggtgta agagccggta gatcaatccc attgaagaaa 600
aatgttgatg acgcattgaa aaatcctaac gttacttccg tcgaacacgt aattgttttg 660
aagagaactg gtaacgatat agactggcaa gaaggtagag atttgtggtg gagagactta 720
attgaaaaag cttcaccaga acatcaacct gaagccatga acgctgaaga tccattgttt 780
atattgtaca ctagtggttc tacaggtaaa cctaagggtg ttttacacac tacaggtggt 840
tatttggtct acgcagccac cacttttaag tatgtattcg attaccatcc aggtgacatc 900
tattggtgta ctgcagatgt aggttgggtt acaggtcact cctatttgtt atacggtcca 960
ttagcatgcg gtgccacaac cttgatgttc gaaggtgttc ctaattggcc aactcctgct 1020
agaatgtgtc aagtagttga taagcatcaa gttaacatct tgtacacagc tccaaccgca 1080
attagagcct tgatggctga aggtgacaag gctatagaag gtacagacag aagttctttg 1140
agaatattag gttctgttgg tgaaccaatc aaccctgaag catgggaatg gtactggaaa 1200
aagattggta aagaaaagtg tcctgtcgta gatacctggt ggcaaactga aacaggtggt 1260
tttatgataa ctccattgcc tggtgctatc gaattaaaag caggtagtgc cactagacca 1320
tttttcggtg tacaacctgc tttagttgat aatgaaggtc atccacaaga aggtgcaacc 1380
gagggtaact tagttattac tgattcttgg cctggtcaag ctagaacatt gtttggtgac 1440
catgaaagat tcgaacaaac ctacttctca actttcaaaa acatgtactt ctccggtgac 1500
ggtgcaagaa gagatgaaga cggttattac tggattacag gtagagtcga tgacgtatta 1560
aacgttagtg gtcatagatt gggtaccgct gaaatcgaat ctgctttagt tgcacatcca 1620
aaaattgcag aagctgcagt tgtcggtatt cctcacgcca taaagggtca agccatctat 1680
gcttacgtta ccttaaatca tggtgaagaa ccatctcctg aattgtatgc cgaagtcaga 1740
aactgggtta gaaaagaaat tggtccattg gctacacctg atgttttaca ctggacagac 1800
tcattgccaa aaaccagatc cggtaaaatc atgagaagaa ttttgagaaa gatagccgct 1860
ggtgacacta gtaatttggg tgacacctct actttggccg accctggtgt agttgaaaaa 1920
cctttagaag aaaaacaagc aatagcaatg ccaagttaa 1959
<210> 17
<211> 1580
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga 60
tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata 120
cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag 180
cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga 240
aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc 300
ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaaaagcctg 360
aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 420
tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 480
gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 540
ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 600
agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 660
aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 720
ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 780
ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 840
tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 900
ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 960
tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 1020
cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 1080
agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 1140
atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 1200
atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 1260
ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 1320
tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taatcagtac 1380
tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt 1440
agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat 1500
ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat 1560
gctggtcgct atactgctgt 1580
<210> 18
<211> 1457
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
agctgaagct tcgtacgctg caggtcgacg gatccccggg ttaattaagg cgcgccagat 60
ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata 120
ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca 180
tttagcccat acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg 240
cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca 300
cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt 360
tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc 420
acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt cgaggccgcg 480
attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg 540
gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct 600
gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg 660
gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc 720
atggttactc accactgcga tccccggcaa aacagcattc caggtattag aagaatatcc 780
tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat 840
tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc 900
acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc 960
tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt 1020
cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg 1080
tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa 1140
ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga 1200
taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcagt 1260
actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt 1320
gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc 1380
atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga 1440
atgctggtcg ctatact 1457
<210> 19
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atgctttctg aaaacacgac tattct 26
<210> 20
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gatgtaagga ttcgcggaag ctatctactg agatttctgg ct 42
<210> 21
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agccagaaat ctcagtagat agcttccgcg aatccttaca tcacac 46
<210> 22
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atctaaagat gcaaccattt tgtaattaaa acttagatta g 41
<210> 23
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
agttttaatt acaaaatggt tgcatcttta gatgctttg 39
<210> 24
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ataagaaatt cgcttaaaca actggttcaa acaaaac 37
<210> 25
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tgaaccagtt gtttaagcga atttcttatg atttatg 37
<210> 26
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ctaacattca acgctagtat agatcatgat acataaaagc 40
<210> 27
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gcttttatgt atcatgatct atactagcgt tgaatgttag 40
<210> 28
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atttaactgc agctgacatt ttgtttgttt atgtgtg 37
<210> 29
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
cataaacaaa caaaatgtca gctgcagtta aatgttcatt gact 44
<210> 30
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
cattaaagta acttaaggag ttaaatttaa tttgaaacac aagacatc 48
<210> 31
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gtgtttcaaa ttaaatttaa ctccttaagt tactttaatg 40
<210> 32
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tacaacgtcg tgactgggaa agcggcgaaa agccaattag t 41
<210> 33
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
actaattggc ttttcgccgc tttcccagtc acgacgttgt a 41
<210> 34
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ggtcgtcaca gtagctgaca tacgaattcg agctcggtac c 41
<210> 35
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ggtaccgagc tcgaattcgt atgtcagcta ctgtgacgac c 41
<210> 36
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gcgtaatgcc caatttttcg c 21
<210> 37
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
caagagtgag aaactcctac atcaac 26
<210> 38
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gtcttgagtt gaagtcagga atctaaaata caaagaatct gaatcactca tcttttg 57
<210> 39
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tccaaaagat gagtgattca gattctttgt attttagatt cctgacttca actcaagac 59
<210> 40
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
attggttaaa actggcattg ttttatattt gttgtaaaaa gtag 44
<210> 41
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
caacaaatat aaaacaatgc cagttttaac caataaaaca gtc 43
<210> 42
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ctatcgattt caattcaatt caatttagga tttaatgcag gtgac 45
<210> 43
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gtcacctgca ttaaatccta aattgaattg aattgaaatc gatag 45
<210> 44
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
taacattcaa cgctagtatc actatactgg atctaaagag tacaatag 48
<210> 45
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ctctttagat ccagtatagt gatactagcg ttgaatgtta gcg 43
<210> 46
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
atcttggcct ccattttgtt tgtttatgtg tgtttattc 39
<210> 47
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
cataaacaaa caaaatggag gccaagatag atgag 35
<210> 48
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
tgaagccata gaaccaccac ccaattcgga taagtggtct at 42
<210> 49
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
ggtggtggtt ctatggcttc agaaaaagaa att 33
<210> 50
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
tcggcgacca agcagtaagc ctgcaacaac tc 32
<210> 51
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
caggcttact gcttggtcgc cgat 24
<210> 52
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
gctcaccaag ctcttaaaac gataaaatcc ttggactagt cacgtgg 47
<210> 53
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
ccacgtgact agtccaagga ttttatcgtt ttaagagctt ggtgagc 47
<210> 54
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
aatccatttg ttagtctgct tgcatcgagt tcaagagaaa aaaaaagaaa aagc 54
<210> 55
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
gctttttctt tttttttctc ttgaactcga tgcaagcaga ctaacaaatg gatt 54
<210> 56
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
acgaccagat gaatcacatg tga 23
<210> 57
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
gtccgataaa cacagttgtg atctg 25
<210> 58
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ttaatggtgg ctcgctgtct gtcgtttgct atgatttaat tctgattctt tg 52
<210> 59
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
caaagaatca gaattaaatc atagcaaacg acagacagcg agccaccatt aa 52
<210> 60
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
aagtttttcg atagtactct ccatgtttag ttaattatag ttcgttgacc gtatattct 59
<210> 61
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ggtagatact tgagagatgt ctggtgagcg gatctcttat gtctttacga ttt 53
<210> 62
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
aattaaaagt ccaacgcgcc ttatatgagg gtgtgtacat tgcagt 46
<210> 63
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
atacggtcaa cgaactataa ttaactaaac atggagagta ctatcgaaaa actt 54
<210> 64
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
aaatcgtaaa gacataagag atccgctcac cagacatctc tcaagtatct acc 53
<210> 65
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
actgcaatgt acacaccctc atataaggcg cgttggactt ttaatt 46
<210> 66
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
gcaacaacaa agtcatggtt tccattttgt aattaaaact tagattagat tgctatgc 58
<210> 67
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
aattatccca ttgaagcctt aaattgaatt gaattgaaat cgatagatca atttttttc 59
<210> 68
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
gtcttgagtt gaagtcagga atctaaaata aaaaccgatt gaccaatata tgtctct 57
<210> 69
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
cagagacata tattggtcaa tcggttttta ttttagattc ctgacttcaa ctcaagac 58
<210> 70
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
aacttgaacc aattccattg ttttatattt gttgtaaaaa gtagataatt acttcctt 58
<210> 71
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
agcatagcaa tctaatctaa gttttaatta caaaatggaa accatgactt tgttgttg 58
<210> 72
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
ttgatctatc gatttcaatt caattcaatt taaggcttca atgggataat tctcaatg 58
<210> 73
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
aattatctac tttttacaac aaatataaaa caatggaatt ggttcaagtt atcgctg 57
<210> 74
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
tatttaataa taaaaatcat aaatcataag aaattcgctc agatggatgg tgggttc 57
<210> 75
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
tccatctgag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaa 59
<210> 76
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
tgacgctaac attcaacgct agtatcgtaa aaaaagcatg cacgtataca c 51
<210> 77
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
gtgtatacgt gcatgctttt tttacgatac tagcgttgaa tgttagcgtc a 51
<210> 78
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
acaacgataa cttcaaccaa ttccattttg tttgtttatg tgtgtttatt cgaaact 57
<210> 79
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
agtttcgaat aaacacacat aaacaaacaa aatggaattg gttgaagtta tcgttgt 57
<210> 80
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
acaaaggaaa aggggcctgt ttagaatgag gggttcagtg gaat 44
<210> 81
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
attccactga acccctcatt ctaaacaggc cccttttcct ttgt 44
<210> 82
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
ttaatggtgg ctcgctgtct ggaaaacaac aacggaagcg a 41
<210> 83
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
tcgcttccgt tgttgttttc cataacaata ctgacagtac taaataattg cctac 55
<210> 84
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
gagattcttt cgaccttcat tttgaatatg tattacttgg ttatggttat atatgaca 58
<210> 85
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
atatataacc ataaccaagt aatacatatt caaaatgaag gtcgaaagaa tctcccg 57
<210> 86
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
aaatcattaa agtaacttaa ggagttaaat ttaggtggta atcaacaact tgatttct 58
<210> 87
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
ccacctaaat ttaactcctt aagttacttt aatgatttag tttttattat taataattc 59
<210> 88
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
ttctattaaa tgcttcctat attatatata tagtaatgtc gtttgaaagg tacgatgctt 60
ggaaac 66
<210> 89
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
atttatttaa gtattgtttg tgcacttgcc cagtgtatta ccaacaaccg ttcg 54
<210> 90
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
cgggatcctc tgatgggtg 19
<210> 91
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
gtttccaagc atcgtacctt tcaaacgaca ttactatata tataatatag gaagcattt 59
<210> 92
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
cgaacggttg ttggtaatac actgggcaag tgcacaaaca atacttaaat aaat 54
<210> 93
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
gcttcggtta cttctaagga agtcc 25
<210> 94
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
gtgtgatgta aggattcgcg gttggaaagt cattaggtga ggttaaca 48
<210> 95
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
tgttaacctc acctaatgac tttccaaccg cgaatcctta catcacac 48
<210> 96
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
acaaaggaaa aggggcctgt ttaatttgaa acacaagaca tcttttcacc 50
<210> 97
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
ggtgaaaaga tgtcttgtgt ttcaaattaa acaggcccct tttcctttgt 50
<210> 98
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
gtcttgagtt gaagtcagga atctaaaata ggaaaacaac aacggaagcg a 51
<210> 99
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
tcgcttccgt tgttgttttc ctattttaga ttcctgactt caactcaaga c 51
<210> 100
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
ggtattctgg gcctccatgt caatccttgg actagtcacg tgg 43
<210> 101
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
ccacgtgact agtccaagga ttgacatgga ggcccagaat acc 43
<210> 102
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
agttagtaga tgatagttga tttctattcc aacaacagca gtatagcgac cagc 54
<210> 103
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
gctggtcgct atactgctgt tgttggaata gaaatcaact atcatctact aact 54
<210> 104
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
cacaggcgct accatgaga 19
<210> 105
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
ccggggcacc tgtcactttg gaa 23
<210> 106
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
gtcttgagtt gaagtcagga atctaaaata tttcctctaa tcaggttcca ccaa 54
<210> 107
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
ttggtggaac ctgattagag gaaatatttt agattcctga cttcaactca agac 54
<210> 108
<211> 65
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
gcaacaacaa agtcatggtt tccattgttt tatatttgtt gtaaaaagta gataattact 60
tcctt 65
<210> 109
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
aattatctac tttttacaac aaatataaaa caatggaaac catgactttg ttgttgc 57
<210> 110
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 110
tcctcttctc cacataccac caccaggctt caatgggata attctcaatg 50
<210> 111
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 111
tatcccattg aagcctggtg gtggtatgtg gagaagagga gctggt 46
<210> 112
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 112
aaaacgaaat tttttggatc actcgcacca ccaccccaga cgtccctcaa gtacc 55
<210> 113
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 113
ggtacttgag ggacgtctgg ggtggtggtg cgagtgatcc aaaaaatttc gtttt 55
<210> 114
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 114
actaccagct cctcttctcc aaccaccacc atcttgggtt ttgtccggat tg 52
<210> 115
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 115
caagattaag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaa 59
<210> 116
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 116
aattaaaagt ccaacgcgcc tcgtaaaaaa agcatgcacg tatacac 47
<210> 117
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 117
gtgtatacgt gcatgctttt tttacgaggc gcgttggact tttaatt 47
<210> 118
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 118
aacttgaacc aattccattt tgtaattaaa acttagatta gattgctatg ctttc 55
<210> 119
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 119
gaaagcatag caatctaatc taagttttaa ttacaaaatg gaattggttc aagttatcgc 60
tg 62
<210> 120
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 120
caccgatgga tggtgggttc aaagga 26
<210> 121
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 121
tcctttgaac ccaccatcca tcggtggtgg ttggagaaga ggagctggta gt 52
<210> 122
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 122
aaatcgtaaa gacataagag atccgcttaa tcttgggttt tgtccggatt g 51
<210> 123
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 123
ccggacaaaa cccaagatta agcggatctc ttatgtcttt acgattt 47
<210> 124
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 124
tgacgctaac attcaacgct agtattatat gagggtgtgt acattgcagt 50
<210> 125
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 125
actgcaatgt acacaccctc atataatact agcgttgaat gttagcgtca 50
<210> 126
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 126
actaccagct cctcttctcc aaccaccacc gaatgagggg ttcagtggaa tca 53
<210> 127
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 127
actgaacccc tcattcggtg gtggttggag aagaggagct ggtagt 46
<210> 128
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 128
ttgatctatc gatttcaatt caattcaatt taatcttggg ttttgtccgg attg 54
<210> 129
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 129
tccggacaaa acccaagatt aaattgaatt gaattgaaat cgatagatca atttttttc 59
<210> 130
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 130
ttaatggtgg ctcgctgtct aaaaccgatt gaccaatata tgtctctg 48
<210> 131
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 131
cagagacata tattggtcaa tcggttttag acagcgagcc accattaa 48
<210> 132
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 132
gagattcttt cgaccttcat gtttagttaa ttatagttcg ttgaccgtat attct 55
<210> 133
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 133
agaatatacg gtcaacgaac tataattaac taaacatgaa ggtcgaaaga atctcccg 58
<210> 134
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 134
acaaaggaaa aggggcctgt ttaggtggta atcaacaact tgatttctt 49
<210> 135
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 135
atcaagttgt tgattaccac ctaaacaggc cccttttcct ttgt 44
<210> 136
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 136
agcgtacgaa gcttcagctg gaaaacaaca acggaagcga 40
<210> 137
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 137
tcgcttccgt tgttgttttc cagctgaagc ttcgtacgct 40
<210> 138
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 138
cctttagact tacgtttgct actctcatag tatagcgacc agcattcaca ta 52
<210> 139
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 139
tatgtgaatg ctggtcgcta tactatgaga gtagcaaacg taagtctaaa gg 52
<210> 140
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 140
gcggaaaata cggaaacgcg 20
<210> 141
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 141
ggcaccttgt ccaattgaac a 21
<210> 142
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 142
gtcttgagtt gaagtcagga atctaaaata tctggattgt caccttcaac aaatc 55
<210> 143
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 143
tgaaggtgac aatccagata ttttagattc ctgacttcaa ctcaagac 48
<210> 144
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 144
gttcccagtt gcttgttgca ttgttttata tttgttgtaa aaagtagata attacttcct 60
t 61
<210> 145
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 145
aattatctac tttttacaac aaatataaaa caatgcaaca agcaactggg aac 53
<210> 146
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 146
acaaaggaaa aggggcctgt tcaggaatca tccagtatgt gct 43
<210> 147
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 147
agcacatact ggatgattcc tgaacaggcc ccttttcctt tgt 43
<210> 148
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 148
atgtagatat actagaagtt ctcctcgagg aaaacaacaa cggaagcga 49
<210> 149
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 149
tcgcttccgt tgttgttttc ctcgaggaga acttctagta tatctacata cc 52
<210> 150
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 150
gtttatacat aattttacaa ctcattacgc acactcgact acgtcgttaa ggcc 54
<210> 151
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 151
ggccttaacg acgtagtcga gtgtgcgtaa tgagttgtaa aattatgtat aaac 54
<210> 152
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 152
acgaggagaa ctagtatgtc ctgg 24
<210> 153
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 153
ggaagaggag tagggaatat tactgg 26
<210> 154
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 154
aattaaaagt ccaacgcgcc tctgcaagcc gcaaactttc a 41
<210> 155
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 155
ggcttgcaga ggcgcgttgg acttttaatt 30
<210> 156
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 156
gtgtttatgt gtttgactca ttttgtaatt aaaacttaga ttagattgct atgctttc 58
<210> 157
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 157
gcatagcaat ctaatctaag ttttaattac aaaatgagtc aaacacataa acacgcaa 58
<210> 158
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 158
acaaaggaaa aggggcctgt ttaacttggc attgctattg cttgt 45
<210> 159
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 159
acaagcaata gcaatgccaa gttaaacagg ccccttttcc tttgt 45
<210> 160
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 160
cgattctttg attagtctct tcaaacaaac aggaaaacaa caacggaagc ga 52
<210> 161
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 161
tcgcttccgt tgttgttttc ctgtttgttt gaagagacta atcaaagaat cg 52
<210> 162
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 162
caaagtgtag cttagtcatt gtgtattacg atatagttaa tagttgatag ttgattg 57
<210> 163
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 163
actatcaact attaactata tcgtaataca caatgactaa gctacacttt gacactg 57
<210> 164
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 164
atcattaaag taacttaagg agttaaattt acaacttaat tctgacagct tttacttca 59
<210> 165
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 165
agttgtaaat ttaactcctt aagttacttt aatgatttag tttttattat taataattc 59
<210> 166
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 166
gtcttgagtt gaagtcagga atctaaaata tgaaaggtac gatgcttgga aac 53
<210> 167
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 167
gtttccaagc atcgtacctt tcatatttta gattcctgac ttcaactcaa gac 53
<210> 168
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 168
gacggggcct tcactcattg ttttatattt gttgtaaaaa gtagataatt acttcctt 58
<210> 169
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 169
aattatctac tttttacaac aaatataaaa caatgagtga aggccccgtc 50
<210> 170
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 170
aaatcgtaaa gacataagag atccgcctaa ttatccttcg tatcttctgg cttagt 56
<210> 171
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 171
actaagccag aagatacgaa ggataattag gcggatctct tatgtcttta cgattt 56
<210> 172
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 172
tgacgctaac attcaacgct agtattatat gagggtgtgt acattgcagt 50
<210> 173
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 173
actgcaatgt acacaccctc atataatact agcgttgaat gttagcgtca 50
<210> 174
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 174
aatcaaaccg aaatcagcag acattttgtt tgtttatgtg tgtttattcg aaact 55
<210> 175
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 175
agtttcgaat aaacacacat aaacaaacaa aatgtctgct gatttcggtt tgatt 55
<210> 176
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 176
ataataaaaa tcataaatca taagaaattc gcttaagctt ggtatgtaga ggaagaaaca 60
<210> 177
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 177
ccaagcttaa gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa 60
<210> 178
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 178
ttcgaaccct tgcatccgac gtaaaaaaag catgcacgta tacac 45
<210> 179
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 179
gtgtatacgt gcatgctttt tttacgtcgg atgcaagggt tcgaa 45
<210> 180
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 180
ctactcagta ataacctatt tcttagcaac tgttctttga tgttagaaca atttaggt 58
<210> 181
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 181
ttctaacatc aaagaacagt tgctaagaaa taggttatta ctgagtagta tttattt 57
<210> 182
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 182
cattgatgag gcaacgctaa ttatca 26

Claims (4)

1.生产毛喉素的重组酿酒酵母菌的构建方法，其特征是包括如下步骤：
(1)向酿酒酵母中导入含有优化的泪柏醚合酶编码基因tCfTPS2和优化的泪柏醚合酶编码基因tCfTPS3的表达盒，得到重组菌1；
所述优化的泪柏醚合酶编码基因tCfTPS2的核苷酸序列如SEQ ID No.1所示；
所述优化的泪柏醚合酶编码基因tCfTPS3的核苷酸序列如SEQ ID No.2所示；
(2)向重组菌1中导入截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1、牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1和法呢基焦磷酸合酶编码突变基因ERG20F96C表达盒，得到重组菌2；
所述截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1的核苷酸序列如SEQ IDNo.3所示；
所述牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1的核苷酸序列如SEQ ID No.4所示；
所述法呢基焦磷酸合酶编码突变基因ERG20F96C的核苷酸序列如SEQ ID No.5所示；
(3)向重组菌2中导入含有
优化的细胞色素P450酶编码基因CfCYP76AH15、优化的细胞色素P450酶编码基因CfCYP76AH11、优化的细胞色素P450酶编码基因CfCYP76AH16，优化的细胞色素P450还原酶编码基因CfCPR和优化的乙酰基转移酶编码基因CfACT1-8的表达盒，得到重组菌3；所述优化的细胞色素P450酶编码基因CfCYP76AH15的核苷酸序列如SEQ ID No.6所示；所述优化的细胞色素P450酶编码基因CfCYP76AH11的核苷酸序列如SEQ ID No.7所示；所述优化的细胞色素P450酶编码基因CfCYP76AH16的核苷酸序列如SEQ ID No.8所示；所述优化的细胞色素P450还原酶编码基因CfCPR的核苷酸序列如SEQ ID No.9所示；所述优化的乙酰基转移酶编码基因CfACT1-8的核苷酸序列如SEQ ID No.10所示；
(4)向重组菌3中导入含优化的泪柏醚合酶编码基因tCfTPS2、优化的泪柏醚合酶编码基因tCfTPS3、截短的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1、牻牛儿基牻牛儿基焦磷酸合酶编码基因BTS1和法呢基焦磷酸合酶编码突变基因ERG20F96C的表达盒得到重组菌4；
(5)向重组菌4中导入含有CfCYP76AH15～t66CPR～t30CYB5、fCYP76AH11～t66CPR～t30CYB5、CfCYP76AH16～t66CPR～t30CYB5和CfACT1-8表达盒，得到重组菌5；
所述t66CPR是CfCPR从N端截短了60个氨基酸后获得；所述t30CYB5是细胞色素CYB5从C端截短了30个氨基酸后获得，细胞色素CYB5的核苷酸序列如SEQ ID No.11所示；
(6)向重组菌5中导入内质网调控因子INO2表达盒，得到重组菌6；
所述内质网调控因子INO2的核苷酸序列如SEQ ID No.12所示；
(7)向重组菌6中导入含有6-磷酸葡萄糖脱氢酶编码基因ZWF1，磷酸葡萄糖酸脱氢酶编码基因GND1，乙醛脱氢酶编码基因ALD6和乙酰辅酶A合酶编码基因ACS的表达盒，得到重组菌7；
所述6-磷酸葡萄糖脱氢酶编码基因ZWF1的核苷酸序列如SEQ ID No.13所示；所述磷酸葡萄糖酸脱氢酶编码基因GND1的核苷酸序列如SEQ ID No.14所示；所述乙醛脱氢酶编码基因ALD6的核苷酸序列如SEQ ID No.15所示；所述乙酰辅酶A合酶编码基因ACS的核苷酸序列如SEQ ID No.16所示。
2.根据权利要求1所述生产毛喉素的重组酿酒酵母菌的构建方法，其特征是所述酿酒酵母为酿酒酵母Saccharomyces cerevisiae W303-1a。
3.权利要求1或2的方法构建的生产毛喉素的重组酿酒酵母菌。
4.权利要求3的重组酿酒酵母菌发酵生产毛喉素的用途。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005519A1 (es) * 2002-07-08 2004-01-15 Universidade De Santiago De Compostela Construcción de una cepa de levadura enológica de saccharomyces cerevisiae recombinante que sobreexpresa una endopoligalacturonasa, para ser utilizada en la elaboracion de vinos
CN107746815A (zh) * 2017-09-11 2018-03-02 天津大学 生产13r‑泪柏醚的重组酿酒酵母菌及其构建方法
CN111334522A (zh) * 2020-02-24 2020-06-26 天津大学 生产龙涎香醇的重组酿酒酵母菌及构建方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005519A1 (es) * 2002-07-08 2004-01-15 Universidade De Santiago De Compostela Construcción de una cepa de levadura enológica de saccharomyces cerevisiae recombinante que sobreexpresa una endopoligalacturonasa, para ser utilizada en la elaboracion de vinos
CN107746815A (zh) * 2017-09-11 2018-03-02 天津大学 生产13r‑泪柏醚的重组酿酒酵母菌及其构建方法
CN111334522A (zh) * 2020-02-24 2020-06-26 天津大学 生产龙涎香醇的重组酿酒酵母菌及构建方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAIYAN JU等: "Progress in heterologous biosynthesis of forskolin", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY》 *
HAIYAN JU等: "Progress in heterologous biosynthesis of forskolin", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY》, vol. 2021, no. 48, 4 February 2021 (2021-02-04) *
XIAOYAN GUO等: "Stepwise increase in the production of 13R-manoyl oxide through metabolic engineering of Saccharomyces cerevisiae", 《BIOCHEMICAL ENGINEERING JOURNAL》 *
XIAOYAN GUO等: "Stepwise increase in the production of 13R-manoyl oxide through metabolic engineering of Saccharomyces cerevisiae", 《BIOCHEMICAL ENGINEERING JOURNAL》, vol. 2019, no. 144, 31 December 2019 (2019-12-31) *

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