CN112961852B - 一种启动子及其在棘孢曲霉基因自克隆表达中的应用 - Google Patents
一种启动子及其在棘孢曲霉基因自克隆表达中的应用 Download PDFInfo
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Abstract
本发明属于基因工程和微生物改造技术领域，具体提供了一种来源于棘孢曲霉的启动子及其在棘孢曲霉基因自克隆表达中的应用。所述启动子的编码核苷酸序列如SEQ ID NO：1或SEQ ID NO：2或SEQ ID NO：3或SEQ ID NO：4所示，可显著提高棘孢曲霉内源基因的自克隆表达效率，从而降低棘孢曲霉产酶的生产成本，应用前景广阔。
Description
技术领域
本发明属于基因工程与微生物改造技术领域，具体内容涉及一种启动子及其在棘孢曲霉基因自克隆表达中的应用。
背景技术
基因经过转录、翻译，产生具有特异生物学功能的蛋白质分子或RNA分子的过程称为基因表达(gene expression)。基因表达是受内源及外源信号调控的，这个调控的过程称为基因表达调控(gene regulation or regulation of gene expression)。启动子是基因表达调控的重要顺式作用元件，在基因的表达调控中转录环节是最主要的调控位点，而启动子的调控在转录环节中又占据着十分重要的地位，因此研究启动子的功能序列对于基因调控机制的研究具有十分重要的意义。启动子也是基因工程表达载体的一个重要元件，弄清启动子的分子本质是提高克隆基因表达效率的重要途径。
启动子是位于结构基因5’端上游的一段非编码DNA序列，由核苷酸组成，能活化RNA聚合酶，使之与模板DNA准确地结合并具有转录起始的特异性，是基因转录调控的核心区域。启动子重要组成部分包括许多短的核苷酸序列，即转录因子结合位点，主要分为核心区和上游调控区。
真菌的启动子按功能分为组成型启动子和诱导型启动子。组成型启动子是指能够使基因在所有组织中都能启动表达的启动子，又称为非特异型启动子。其调控不受外界条件的影响，所启动的基因表达大体恒定在一定水平上，具有持续性，但不表现时空特异性。诱导型启动子在某些特定的物理或化学信号的诱导刺激下，可以大幅度地提高基因的转录水平，没有诱导物存在时基因表达水平很低甚至没有。
曲霉中蛋白的表达调控主要发生在转录水平，因此，在构建表达载体时使用高活性的真菌转录区域是进行高效表达的关键，即开发出一些强的，组成型的或者可调节型的启动子来调控蛋白的表达。曲霉表达系统中常用的启动子有cbh1(外切葡聚糖纤维二糖水解酶I)，glaA(葡萄糖淀粉酶)，gpdA(3-磷酸甘油醛脱氢酶)，amy(淀粉酶)。adhA(乙醛脱氢酶)，tpiA(磷酸丙糖异构酶)，pkiA蛋白激酶启动子，ghdA(泡盛曲霉谷氨酸脱氧酶)，oliC(线粒体合成酶ATP合成酶)和转录延伸因子。
棘孢曲霉(Aspergillus aculeatus)属曲霉属，在自然界中分布较为广泛，常见于高等植物和土壤中，具有良好的蛋白分泌能力，棘孢曲霉可产生糖苷酶、蛋白酶和氧化酶等多种酶类，已广泛应用于产品发酵和制备工业酶制剂等方面，是重要的工业微生物。已有研究将棘孢曲霉β-葡萄糖苷酶基因克隆到大肠杆菌-乳酸菌穿梭载体p MG36e中，通过电转化将载体转化至乳酸乳球菌进行表达，实现在L.lactis MG1363中食品级分泌表达，并利用该酶的高活性水解能力水解风味物质前体，实现其在食品领域的应用。另外，有学者构建了含棘孢曲霉β-葡萄糖苷酶I基因的毕赤酵母分泌多拷贝表达载体，成功在毕赤酵母中高表达分泌棘孢曲霉β-葡萄糖苷酶I，可降低纤维素酶的处理成本。另有一株产纤维素酶的棘孢曲霉L22，它所产生的纤维素酶系具有外切酶酶活很低而内切酶酶活较高的特点，学者将其中一个酶组份内切葡聚糖苷酶II(EGII)基因在酿酒酵母中表达，达到了较高的表达效率，实现其在酶法脱墨领域中的应用。
虽然棘孢曲霉的基因已在多个表达系统中实现了高效表达，但其在棘孢曲霉中的产量一直比较低。本发明通过筛选棘孢曲霉内源的启动子，构建得到高效表达棘孢曲霉基因的自克隆菌株。由于棘孢曲霉自克隆菌株没有引入任何其他的外源DNA片段，所以更容易被企业接受和应用到生产实践中，因此更有研究和应用价值。
发明内容
本发明为解决现有技术问题，提供了一种来源于棘孢曲霉的启动子及其在棘孢曲霉基因自克隆表达中的应用。所述启动子可显著提高棘孢曲霉内源基因的自克隆表达效率，从而降低棘孢曲霉产酶的生产成本，应用前景广阔。
本发明一方面提供了一种启动子，其编码核苷酸序列如SEQ ID NO：1或SEQ IDNO：2或SEQ ID NO：3或SEQ ID NO：4所示。
所述启动子在棘孢曲霉内源基因自克隆表达中的应用。
本发明一方面提供了一种自克隆表达盒，包含上述启动子和棘孢曲霉的内源基因。
所述内源基因包括鼠李聚糖半乳糖醛酸水解酶、内切-1，5-阿拉伯糖苷酶、果胶甲酯酶、果胶裂解酶、鼠李糖苷酶、阿拉伯呋喃糖苷酶、纤维素酶、β-葡萄糖苷酶、β-葡聚糖酶、葡萄糖氧化酶、酸性蛋白酶中的任意一种。
所述内源基因优选鼠李聚糖半乳糖醛酸水解酶基因。
所述鼠李聚糖半乳糖醛酸水解酶基因的编码核苷酸序列为SEQ ID NO：12。
所述内源基因优选内切-1，5-阿拉伯糖苷酶基因。
所述内切-1，5-阿拉伯糖苷酶基因的编码核苷酸序列为SEQ ID NO：14。
本发明还提供了一种棘孢曲霉自克隆菌株，包含上述自克隆表达盒。
本发明进一步提供了一种突变菌株棘孢曲霉7177-2(Aspergillus aculeatus7177-2)，已于2019年11月4日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2019891。
所述棘孢曲霉突变菌株在生产鼠李聚糖半乳糖醛酸水解酶中的应用。
本发明进一步提供了一种突变菌株棘孢曲霉ALA-2(Aspergillus aculeatusALA-2)，已于2019年11月25日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2019969。
所述棘孢曲霉突变菌株在生产内切-1，5-阿拉伯糖苷酶中的应用。
本发明提供的P422、P7177、P8175、P8515四个启动子可广泛应用于棘孢曲霉内源基因的自克隆表达，能显著提高基因的表达效率，尤其是P7177启动子的表达效率最高。其中，转入P7177启动子表达盒的自克隆菌株棘孢曲霉7177发酵上清液中鼠李聚糖半乳糖醛酸水解酶的酶活为53u/ml，比棘孢曲霉DSM2344提高了169倍；转入P7177启动子表达盒的自克隆菌株棘孢曲霉ALA-1发酵上清液中内切-1，5-阿拉伯糖苷酶的酶活最高，为7.40u/ml，比棘孢曲霉DSM2344提高了121倍。
为了进一步提高鼠李聚糖半乳糖醛酸水解酶和内切-1，5-阿拉伯糖苷酶的产量，申请人分别以棘孢曲霉7177和棘孢曲霉ALA-1为出发菌株，通过紫外诱变筛选获得突变菌棘孢曲霉7177-2和棘孢曲霉ALA-2。其中，棘孢曲霉7177-2液体摇瓶发酵5d后，发酵上清液中鼠李聚糖半乳糖醛酸水解酶酶活达到79u/ml，比出发菌提高了49.1％；30L罐发酵160h后，发酵上清液中鼠李聚糖半乳糖醛酸水解酶酶活达到212u/ml，比出发菌提高了35.9％；棘孢曲霉ALA-2摇瓶发酵上清液中内切-1，5-阿拉伯糖苷酶的酶活达到10.77u/ml，比出发菌提高了45.5％，30L罐发酵160h后，棘孢曲霉ALA-2发酵上清液中的酶活力达到34.53u/ml，较出发菌提高了68.9％，取得了意料不到的技术效果。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING:ALABORATORY MANUAL,3nd Ed.(Sambrook,2001)和CURRENTPROTOCOLS IN MOLECULAR BIOLOGY(Ausubel,2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。
下面结合具体实施方式对本发明进行详细描述。
实施例1棘孢曲霉DSM2344全基因组测序及蛋白质谱分析
将棘孢曲霉DSM2344接种到新鲜的PDA平板，30℃培养5d。
吸取5ml无菌水洗脱，获得孢子液，接种50ml液体CSL-果糖种子培养基(麦芽糖10％；果糖5％；葡萄糖1％；玉米浆10％；硫酸镁0.05％；磷酸二氢钠0.1％；pH 5.8)，30℃培养2d。
培养1d后，滤纸过滤收集菌丝体，烘干后用液氮研磨破壁。
破壁后加入10ml 1％CTAB于60℃水浴中处理1h，10000rpm离心10min收集上清。
上清用酚氯仿抽提(苯酚：氯仿：异戊醇＝25：24：1)2次。
抽提后的水溶液用2倍体积的异丙醇于-20℃沉淀1h，10000rpm离心收集沉淀。
沉淀用70％乙醇洗2次，自然晾干后加200ul水溶解即得到基因组DNA。
基因组DNA送苏州金唯智进行全基因组测序，测序信息如表1所示。
表1棘孢曲霉DSM2344全基因组测序信息
| 名称 | Bases | Min | Max | Average | N50 | (G+C)％ |
| 测序数据 | 5954561255 | 50 | 91058 | 11954.41 | 17021 | 45.79 |
| 组装 | 40362997 | 1067 | 6135690 | 247625.75 | 2671377 | 48.15 |
| 编码基因 | 16619093 | 85 | 22041 | 1579.01 | 1845 | 57.63 |
吸取5ml无菌水洗脱，获得孢子液，接种50ml液体CSL-果糖种子培养基(麦芽糖10％；果糖5％；葡萄糖1％；玉米浆10％；硫酸镁0.05％；磷酸二氢钠0.1％；pH 5.8)，30℃培养5d。
取胞外上清及菌丝体破壁上清进行蛋白电泳，送样至苏州普泰进行蛋白质谱分析，蛋白质谱分析结果如表2所示，选取g422、g1256、g5665、g6925、g7177、g7561、g8175、g8515、g10045、g10328进行后续自克隆表达盒的构建。
表2蛋白质谱分析
实施例2棘孢曲霉启动子的扩增
以棘孢曲霉DSM2344基因组为模板，利用引物1和引物2扩增出g422基因启动子P422，大小为933bp，其核苷酸序列为SEQ ID NO：1。
引物1(F)：TTTCCCGACTGAGGCCTCCCA；
引物2(R)：GATGTTCTTTGGGGGTTGTTC。
以棘孢曲霉DSM2344基因组为模板，利用引物3和引物4扩增出g7177基因启动子P7177，大小为1080bp，其核苷酸序列为SEQ ID NO：2。
引物3(F)：ATAGGGTAGGTAGTCATCGCC；
引物4(R)：GGCGAGGCAAGGGAAAAACGG。
以棘孢曲霉DSM2344基因组为模板，利用引物5和引物6扩增出g8175基因启动子P8175，大小为891bp，其核苷酸序列为SEQ ID NO：3。
引物5(F)：GGTGACCCGTGCGACCCCTGA；
引物6(R)：TTTGAAGGATGTGGATGATGA。
以棘孢曲霉DSM2344基因组为模板，利用引物7和引物8扩增出g8515基因启动子P8515，大小为883bp，其核苷酸序列为SEQ ID NO：4。
引物7(F)：TGGCGATGGTGGGATTGGTGA；
引物8(R)：TGTTTTTTGTTGGTTCGATGA。
以棘孢曲霉DSM2344基因组为模板，利用引物9和引物10扩增出g1256基因启动子P1256，大小为906bp，其核苷酸序列为SEQ ID NO：5。
引物9(F)：GATGGAACTTGCTAGCGAATG；
引物10(R)：GGTGGTGGACGCTTCGACTGA。
以棘孢曲霉DSM2344基因组为模板，利用引物11和引物12扩增出g5665基因启动子P5665，大小为913bp，其核苷酸序列为SEQ ID NO：6。
引物11(F)：GGTATATTGTCCTAAATGGGA；
引物12(R)：TGTGATAGATGAGTATGAAAA。
以棘孢曲霉DSM2344基因组为模板，利用引物13和引物14扩增出g6925基因启动子P6925，大小为930bp，其核苷酸序列为SEQ ID NO：7。
引物13(F)：TGAGTATCGATTCCGAGGTCA；
引物14(R)：GGTGAATGAGGGAGGGAAGAG。
以棘孢曲霉DSM2344基因组为模板，利用引物15和引物16扩增出g7561基因启动子P7561，大小为928bp，其核苷酸序列为SEQ ID NO：8。
引物15(F)：GCCTTTCACTATAGCCTCCCA；
引物16(R)：GGTGGGCGTGGGAGATTCGGG。
以棘孢曲霉DSM2344基因组为模板，利用引物17和引物18扩增出g10045基因启动子P10045，大小为899bp，其核苷酸序列为SEQ ID NO：9。
引物17(F)：TCGTAGGCAGAGTGCCCCTGA；
引物18(R)：GACTGGCTTGGATCAATGGGA。
以棘孢曲霉DSM2344基因组为模板，利用引物19和引物20扩增出g10328基因启动子P10328，大小为901bp，其核苷酸序列为SEQ ID NO：10。
引物19(F)：CCGACAGGCACGTTGTTGTGA；
引物20(R)：GGTGTTTAAGCGTCTGAGATG。
实施例3棘孢曲霉尿嘧啶缺陷型宿主筛选及筛选标记扩增
以棘孢曲霉DSM2344基因组为模板，利用引物21和引物22扩增出pyrG基因(包含启动子及终止子)，大小为2159bp，其核苷酸序列为SEQ ID NO：11。
引物21(F)：TGGAGAACTCCTCCCAGACTA
引物22(R)：TCAACATTAATGGCATGGCCA
将棘孢曲霉DSM2344接种到新鲜的PDA平板，30℃培养5d。
吸取5ml无菌水洗脱，获得孢子液，离心后用无菌水重悬，用血球计数板计数。取一个90mm培养皿，加入5ml稀释好的孢子悬液(浓度约为1×107个/mL)，加入转子并在磁力搅拌器上搅拌使孢子液处于均匀状态。在无菌超净工作台中，用功率为9w的紫外灯于垂直距离20cm的上方照射120s，取照射后的孢子液稀释10、100、1000倍，取100ul涂布PDA平板(含有0.1％尿苷、0.1％5-氟乳清酸)，30℃培养2-3d后，挑取长出的单菌落分别接种到PDA、PDA+U平板。在PDA平板上不生长，PDA+U平板上生长的菌株即为尿嘧啶缺陷型菌株。
申请人挑取一株尿嘧啶缺陷型菌株作为宿主进行后续转化，命名为棘孢曲霉ACU-1(Aspergiulls aculeatus ACU-1)。
实施例4鼠李聚糖半乳糖醛酸水解酶基因自克隆表达盒的构建
根据全基因组测序结果分析，选取棘孢曲霉DSM2344中的鼠李聚糖半乳糖醛酸水解酶基因g10045进行自克隆表达，其核苷酸序列(去除内含子)为SEQ ID NO：12，其编码的氨基酸序列为SEQ ID NO：13。
以棘孢曲霉DSM2344基因组为模板，利用引物23(TCTAGA为XbaI酶切位点)和引物24扩增出鼠李聚糖半乳糖醛酸水解酶基因片段(含内含子及终止子)。
PCR引物和反应条件如下：
引物23(F)：TCTAGAATGCGTGGTCTTTTCCTTCTT；
引物24(R)：TCGACAAGGAAGGGATGACGC。
反应条件为：94℃变性5min；然后94℃变性30s，58℃复性30s，72℃延伸90s，30个循环后，72℃保温10min。琼脂糖电泳结果显示，扩增得到的鼠李聚糖半乳糖醛酸水解酶基因及终止子大小为1844bp。
将扩增得到的鼠李聚糖半乳糖醛酸水解酶基因及终止子连接到pMD19T-simple载体上，连接后的载体命名为pMD19T-RG。鼠李聚糖半乳糖醛酸水解酶基因起始密码子ATG之前留有XbaI酶切位点，用于连接不同的启动子序列。
将实施例2中扩增得到的10个启动子(P422、P7177、P8175、P8515、P1256、P5665、P6925、P7561、P10045、P10328)序列，通过XbaI酶切位点分别连接到pMD19T-RG载体上，构建得到10个含有不同启动子的鼠李聚糖半乳糖醛酸水解酶表达载体。
以上述构建得到的10个含有不同启动子的鼠李聚糖半乳糖醛酸水解酶表达载体为模板，分别以引物1/引物24、引物3/引物24、引物5/引物24、引物7/引物24、引物9/引物24、引物11/引物24、引物13/引物24、引物15/引物24、引物17/引物24、引物19/引物24扩增含有对应启动子(P422、P7177、P8175、P8515、P1256、P5665、P6925、P7561、P10045、P10328)、鼠李聚糖半乳糖醛酸水解酶基因、终止子序列的基因表达盒片段。
实施例5鼠李聚糖半乳糖醛酸水解酶不同启动子表达盒的筛选
原生质体制备：接种实施例3所述宿主菌棘孢曲霉ACU-1于PDA+U平板，30℃培养5-7d。割取2cm×2cm大小的菌块，接种100ml液体PDA+U培养基中，30℃培养24h生长菌丝体，用于转化。将长好的菌丝体过滤后，用20ml 1.2M硫酸镁溶液重悬，加入0.2g溶菌酶。30℃、100rpm培养2-3h。将裂解好的菌丝用2层擦镜纸过滤，3000rpm离心10min获得原生质体。
转化：原生质体用1.2M山梨醇溶液清洗2遍，再用适量的山梨醇溶液重悬，使原生质体浓度达到108个/mL。200ul原生质体中分别加入实施例4中PCR扩增得到的10个启动子表达盒基因片段20ug，再加入实施例3中PCR扩增得到的pyrG基因片段5ug，再加入50ul25％的PEG6000，冰浴20min，再加入2ml 25％的PEG6000室温放置5min，加入4ml山梨醇溶液颠倒混匀。倒入50ml转化上层培养基后，倾倒入4个转化下层平板中，待上层培养基凝固后，于30℃培养箱中倒置培养5d。
转化子筛选：培养5d后，挑取长出的菌落，点种到转化下层平板进行复筛，30℃培养2d。将正常生长的转化子分别接种到新鲜的PDA平板，30℃培养5-7d。每个转化子割取2cm×2cm大小的菌块，分别接种50ml液体摇瓶培养基中发酵，32℃培养5d，每天加入适量氨水，控制pH在4.5左右。培养5d后，离心菌体获得上清液即为粗酶液，进行蛋白电泳检测。
电泳检测结果显示，本发明选用的10个启动子中，只有分别转入P422、P7177、P8175、P8515四个启动子表达盒的棘孢曲霉重组菌株能够表达鼠李聚糖半乳糖醛酸水解酶。申请人检测了所述棘孢曲霉重组菌株发酵上清液中鼠李聚糖半乳糖醛酸水解酶的酶活，分别为25.1u/ml、53u/ml、29.8u/ml、37.6u/ml。
可见，转入P7177启动子表达盒的棘孢曲霉重组菌株发酵上清液中鼠李聚糖半乳糖醛酸水解酶的酶活最高，为53u/ml，比棘孢曲霉DSM2344的产量提高了169倍，表达效率得到显著提升。申请人将该重组菌株命名为棘孢曲霉7177(Aspergillus aculeatus7177)。
鼠李聚糖半乳糖醛酸水解酶酶活测定方法
(1)酶活单位定义
在40℃，pH为4.0条件下，每分钟水解7mg/ml鼠李聚糖半乳糖醛酸底物，生成等同于1微摩尔半乳糖醛酸的还原糖的酶量定义为一个活力单位(IU)。
(2)酶活测定方法
(2.1)试剂和溶液：
底物：AZCL-Rhamnogalacturonan(Megazyme，T-RHAM)
50mM乙酸钠缓冲液：秤取冰醋酸3.05g，溶于900ml蒸馏水，用1M NaOH溶液调节pH到4.0(约12ml)，加入0.2g叠氮化钠，定容至1L，4℃储存6个月；
终止液2％磷酸三钠溶液：秤取20g磷酸三钠溶于900ml蒸馏水中，用1M HCL调节pH到11.0，定容至1L，室温储存6个月；
(2.2)样品溶液的制备：
准确吸取酶液1ml，用19ml乙酸钠缓冲液稀释，该溶液当做母液，再用乙酸钠缓冲液稀释到适当倍数测酶活，使590nm吸光值在0.2～1.4范围。
(2.3)酶活力测定：
取经过适当稀释的酶液0.5ml于试管中，40℃放置5min；
加入AZCL-Rhamnogalacturonan片剂，静置不要搅拌，计时10min后，加入10ml磷酸三钠终止液，涡旋震荡后立即放到室温；
室温放置5min，冷却后，再次涡旋混匀，用Whatman No.1(9cm)滤纸过滤，过滤后590nm比色读数；
空白管中酶液先加入终止液，再加入AZCL-Rhamnogalacturonan片剂。
酶活计算公式：
标准曲线：mU/ml＝53×Abs+0.08
式中：Abs为样品管减去空白管吸光值之差；
酶活力(U/ml)：X＝Y×(1/1000)×2×n
式中：Y――标准曲线计算出的酶活mU/ml；
1/1000――mU到U的转换系数；
2――酶液0.5ml到1ml的转换系数；
n――稀释倍数。
实施例6内切-1，5-阿拉伯糖苷酶基因自克隆表达盒构建
根据全基因组测序结果分析，选取棘孢曲霉中内切-1，5-阿拉伯糖苷酶基因g10328进行自克隆表达，其核苷酸序列(去除内含子)为SEQ ID NO：14，其编码的氨基酸序列为SEQ ID NO：15。
以棘孢曲霉基因组为模板，利用引物25(TCTAGA为XbaI酶切位点)和引物26扩增出内切-1，5-阿拉伯糖苷酶基因片段(含内含子及终止子)。
PCR引物和反应条件如下：
引物25(F)：TCTAGAATGTACTCCCTCCTCACTGCA；
引物26(R)：CGAGCGTTGAACGGACGGGCA。
反应条件为：94℃变性5min；然后94℃变性30s，58℃复性30s，72℃延伸90s，30个循环后，72℃保温10min。琼脂糖电泳结果显示，扩增得到的内切-1，5-阿拉伯糖苷酶基因及终止子大小为1569bp。
将扩增得到的内切-1，5-阿拉伯糖苷酶基因及终止子连接到pMD19T-simple载体上，连接后的载体命名为pMD19T-ALA。内切-1，5-阿拉伯糖苷酶基因起始密码子ATG之前留有XbaI酶切位点，用于连接不同的启动子序列。
将实施例2中扩增得到的10个启动子(P422、P7177、P8175、P8515、P1256、P5665、P6925、P7561、P10045、P10328)序列，通过XbaI酶切位点分别连接到pMD19T-ALA载体上，构建得到10个含有不同启动子的内切-1，5-阿拉伯糖苷酶表达载体。
以上述构建得到的10个含有不同启动子的内切-1，5-阿拉伯糖苷酶表达载体为模板，分别以引物1/引物24、引物3/引物24、引物5/引物24、引物7/引物24、引物9/引物24、引物11/引物24、引物13/引物24、引物15/引物24、引物17/引物24、引物19/引物24扩增含有对应启动子(P422、P7177、P8175、P8515、P1256、P5665、P6925、P7561、P10045、P10328)、内切-1，5-阿拉伯糖苷酶基因、终止子序列的基因表达盒片段。
实施例7内切-1，5-阿拉伯糖苷酶不同启动子表达盒的筛选
原生质体制备：接种实施例3所述宿主菌棘孢曲霉ACU-1于PDA+U平板，30℃培养5-7d。割取2cm×2cm大小的菌块，接种100ml液体PDA+U培养基中，30℃培养24h生长菌丝体，用于转化。将长好的菌丝体过滤后，用20ml 1.2M硫酸镁溶液重悬，加入0.2g溶菌酶。30℃、100rpm培养2-3h。将裂解好的菌丝用2层擦镜纸过滤，3000rpm离心10min获得原生质体。
转化：原生质体用1.2M山梨醇溶液清洗2遍，再用适量的山梨醇溶液重悬，使原生质体浓度达到108个/ml。200ul原生质体中分别加入实施例6中PCR扩增得到的不同启动子表达盒基因片段20ug，再加入实施例3中PCR扩增得到的pyrG基因片段5ug，再加入50ul25％的PEG6000，冰浴20min，再加入2ml 25％的PEG6000室温放置5min，加入4ml山梨醇溶液颠倒混匀。倒入50ml转化上层培养基后，倾倒入4个转化下层平板中，待上层培养基凝固后，于30℃培养箱中倒置培养5d。
转化子筛选：培养5d后，挑取长出的菌落，点种到转化下层平板进行复筛，30℃培养2d。将正常生长的转化子分别接种到新鲜的PDA平板，30℃培养5-7d。每个转化子割取2cm×2cm大小的菌块，分别接种于50ml液体摇瓶培养基中发酵，32℃培养5d，每天加入适量氨水，控制pH在4.5左右。培养5d后，离心菌体获得上清液即为粗酶液，进行蛋白电泳检测，筛选出有明显蛋白条带表达的转化子。
电泳检测结果显示，只有分别转入P422、P7177、P8175、P8515四个启动子表达盒的棘孢曲霉重组菌株能够表达内切-1，5-阿拉伯糖苷酶。申请人检测了所述棘孢曲霉重组菌株发酵上清液中内切-1，5-阿拉伯糖苷酶的酶活，分别为2.61u/ml、7.40u/ml、5.32u/ml、2.98u/ml。
可见，转入P7177启动子表达盒的棘孢曲霉重组菌株发酵上清液中内切-1，5-阿拉伯糖苷酶的酶活最高，为7.40u/ml，比棘孢曲霉DSM2344的产量提高了121倍，表达效率得到显著提升。申请人将该重组菌株命名为棘孢曲霉ALA-1(Aspergillus aculeatus ALA-1)。
内切-1，5-阿拉伯糖苷酶酶活测定方法
(1)酶活单位定义
在40℃，pH为4.0条件下，每分钟水解2mg/ml 1-5-a-L-阿拉伯聚糖底物，生成等同于1微摩尔阿拉伯糖的还原糖的酶量定义为一个活力单位(IU)。
(2)酶活测定方法
(2.1)试剂和溶液：
底物：AZCL-Arabinan(Megazyme，T-ARZ200)；
50mM乙酸钠缓冲液：秤取冰醋酸3.05g，溶于900ml蒸馏水，用1M NaOH溶液调节pH到4.0(约12ml)，加入0.2g叠氮化钠，定容至1L，4℃储存6个月；
终止液2％磷酸三钠溶液：秤取20g磷酸三钠溶于900ml蒸馏水中，用1M HCL调节pH到11.0，定容至1L，室温储存6个月；
(2.2)样品溶液的制备：
准确吸取酶液1ml，用19ml乙酸钠缓冲液稀释，该溶液当做母液，再用乙酸钠缓冲液稀释到适当倍数测酶活，使590nm吸光值在0.1～1.5范围。
(2.3)酶活力测定：
取经过适当稀释的酶液0.5ml于试管中，40℃放置5min；
加入AZCL-Arabinan片剂，静置不要搅拌，计时10min后，加入10ml Trizma Base溶液(2％w/v)终止反应，涡旋震荡后立即放到室温；
室温放置5min，冷却后，再次涡旋混匀，用Whatman No.1(9cm)滤纸过滤，过滤后590nm比色读数；
空白管中酶液先加入终止液，再加入AZCL-Arabinan片剂。
酶活计算公式：
标准曲线：mU/ml＝23.7×Abs+0.1
式中：Abs为样品管减去空白管吸光值之差；
酶活力(U/ml)：X＝Y×(1/1000)×2×n
式中：Y――标准曲线计算出的酶活mU/ml；
1/1000――mU到U的转换系数；
2――酶液0.5ml到1ml的转换系数；
n――稀释倍数。
除了鼠李聚糖半乳糖醛酸水解酶和内切-1，5-阿拉伯糖苷酶，本发明提供的棘孢曲霉P422、P7177、P8175、P8515四个启动子还可广泛应用于果胶甲酯酶、果胶裂解酶、鼠李糖苷酶、阿拉伯呋喃糖苷酶、纤维素酶、β-葡萄糖苷酶、β-葡聚糖酶、葡萄糖氧化酶、酸性蛋白酶等棘孢曲霉内源基因的自克隆表达，使基因的表达效率普遍提高80％以上，其中P7177启动子的表达效率最高，表达效率可以普遍提高150％以上。
实施例8诱变筛选
紫外诱变导致的突变随机性很强，突变产生的效果也是随机的，难以预测。因此，为了获得有效的正突变，技术人员通常需要进行多轮紫外诱变，筛选的工作量较大，而且存在无法获得有效正突变的可能性。但因为紫外诱变所需设备简单、费用少，并且可在短时间内获得大量突变体，因此，它现在仍是一种常用的诱变选育方法。
申请人以棘孢曲霉7177为出发菌株，通过紫外诱变方法对其进行遗传学改造，进一步提高其鼠李聚糖半乳糖醛酸水解酶的产量。
1、确定致死率
将出发菌株棘孢曲霉7177接种于PDA平板，30℃培养5d。待菌落表面产生大量孢子时，吸取5ml无菌水洗脱，获得孢子液，离心后用无菌水重悬，用血球计数板计数。取一个90mm培养皿，加入5ml稀释好的孢子悬液(浓度约为1×107个/mL)，加入转子并在磁力搅拌器上搅拌使孢子液处于均匀状态。在无菌超净工作台中，用功率为9w的紫外灯于垂直距离20cm的上方照射，分别照射30s、60s、90s、120s、150s、180s，取照射后的孢子液稀释10、100、1000倍，取100ul涂布PDA平板，30℃培养2-3d后计数，以未照射的孢子液为对照，计算致死率。其中照射150s时，致死率为99％，选取该照射时间进行后续诱变实验。
2、第一轮诱变筛选：
取一个90mm培养皿，加入5ml稀释好的孢子悬液(浓度为1×107个/mL)，加入转子并在磁力搅拌器上搅拌使孢子液处于均匀状态。在无菌超净工作台中，用功率为9w的紫外灯于垂直距离20cm的上方照射，照射150s后稀释1000倍，取100ul涂布PDA平板，30℃培养2-3d。
第一轮诱变共涂布120块PDA平板，30℃培养2-3d后，每个平板长出10-20个菌落。首先通过观察菌落形态，筛选出菌落形态发生显著变化的突变菌共83个，分别接种到PDA平板，30℃培养5d；每个转化子割取2cm×2cm大小的菌块，分别接种50ml液体摇瓶培养基中发酵，32℃培养5d，每天加入适量氨水，控制pH在4.5左右。培养5d后，离心菌体获得上清液即为粗酶液，分别进行鼠李聚糖半乳糖醛酸水解酶活力检测，同时以出发菌株棘孢曲霉7177作为对照组。
结果显示，第一轮紫外诱变筛选获得的83株突变菌中，没有一株突变菌发酵上清液中鼠李聚糖半乳糖醛酸水解酶的酶活高于出发菌；其中，78株突变菌的酶活与出发菌基本相当，其余5株突变菌的酶活甚至比出发菌普遍降低了5-10％。
申请人按照上述方法继续进行了14轮诱变筛选，最终获得一株鼠李聚糖半乳糖醛酸水解酶产量显著高于出发菌的突变菌株，命名为棘孢曲霉7177-2(Aspergillusaculeatus7177-2)。棘孢曲霉7177-2摇瓶发酵上清液中鼠李聚糖半乳糖醛酸水解酶的酶活最高，达79u/ml，比出发菌提高了49.1％，取得了意料不到的技术效果。
参照上述同样的方法，申请人以棘孢曲霉ALA-1(Aspergillus aculeatus ALA-1)为出发菌株，通过12轮紫外诱变，最终筛选获得一株内切-1，5-阿拉伯糖苷酶产量最高的突变菌株，命名为棘孢曲霉ALA-2(Aspergiulls aculeatus ALA-2)。棘孢曲霉ALA-2摇瓶发酵上清液中内切-1，5-阿拉伯糖苷酶的酶活达到10.77u/ml，比出发菌提高了45.5％，取得了意料不到的技术效果。
实施例9 30L罐发酵放大
将出发菌棘孢曲霉7177和突变菌棘孢曲霉7177-2分别在30升发酵罐中进行发酵，发酵使用的培养基配方为：麦芽糖2.5g/L、硫酸铵0.9％、磷酸氢二钠0.15g/L、氯化钙0.1g/L、硫酸钾0.5g/L、硫酸镁16.4g/L、柠檬酸钠0.2g/L、磷酸二氢钾0.25％、豆粉0.5％、消泡剂0.05％。补料培养基：麦芽糖浓度为400g/L，pH调至4.0-5.0之间。
发酵生产工艺：pH值4.0、温度30℃、搅拌速率300-700rpm、通风量1.0-1.5(v/v)、溶氧控制在20％以上。
整个发酵过程分为三个阶段：第一阶段为菌体培养阶段，按7％比例接入种子，30℃培养15-25h，以溶氧回升为标志；第二阶段为饥饿阶段，当底糖耗完之后，不流加任何碳源，溶氧上升至60％以上表明该阶段结束，为期约30-120min；第三阶段为产酶阶段，流加补料培养基进行产酶培养，期间保持溶氧在20％以上且维持发酵液中还原糖浓度不低于1g/L，发酵周期在140-170h之间。发酵结束后，发酵液通过板框过滤机处理后获得粗酶液。
发酵160h后，出发菌棘孢曲霉7177发酵上清液中鼠李聚糖半乳糖醛酸水解酶的酶活力达到156u/ml，而突变菌棘孢曲霉7177-2的酶活力达到212u/ml，较出发菌提高了35.9％，取得了意料不到的技术效果。
参照上述同样的方法，申请人将出发菌棘孢曲霉ALA-1和突变菌棘孢曲霉ALA-2分别在30升发酵罐中进行发酵，发酵160h后，出发菌棘孢曲霉ALA-1发酵上清液中内切-1，5-阿拉伯糖苷酶酶活达到20.45u/ml，突变菌棘孢曲霉ALA-2的酶活力达到34.53u/ml，较出发菌提高了68.9％，取得了意料不到的技术效果。
申请人已于2019年11月4日将突变菌棘孢曲霉7177-2(Aspergillus aculeatus
7177-2)保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCCNO:M 2019891。该突变菌株可广泛应用于鼠李聚糖半乳糖醛酸水解酶的生产，有利于降低该酶的生产成本，促进其在食品行业中的推广与应用。
申请人已于2019年11月25日将突变菌棘孢曲霉ALA-2(Aspergillus aculeatusALA-2)保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M2019969。该突变菌株可广泛应用于内切-1，5-阿拉伯糖苷酶的生产，有利于降低该酶的生产成本，促进其在食品行业中的推广与应用。
序列表
<110> 青岛蔚蓝生物集团有限公司
<120> 一种启动子及其在棘孢曲霉基因自克隆表达中的应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 933
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 1
tttcccgact gaggcctccc attatacata gaagctagga agacaataac taaagtatac 60
cagtatacca gtataccagc cctgaggtga acagagatca tgtgactata ctctgacccc 120
cgcggactac agtactccct cggagctcat agttgcctgc catgacaatg tagagcatat 180
cataggctag tattcgaaag tcatcatgat cgtcgtattg attgactcac ccagtcaatc 240
atgtacttta ctccttgggt tcaccggtct accggtctaa acatctccgg gcagaatcgg 300
agggacagga agttaaatga tcgtcgagga ggagaaggag gagcatgtca tggtcattac 360
gtcttctggc ctgcctgtct atttaagctg cccctccttc cctcctctca accccgcttc 420
ttttcttctc tttcttttct ccccaacaca atcactcgtc ccatcttcat ccatcttcat 480
ccatcttcat ccatcttcat ccatcttcga cagatactac cactatctcc agaagtcgtg 540
atccctttca ttcgattgaa tctcccccag tcatgggaaa agcttcaact aacctatcta 600
caggaactca attcgtccaa tgtaaacacc acctaccacg gcccctcgcg ggtcttgacg 660
gtggaagtca tgcaatgcct tgaagctggg caactcattg atcaggtacc tcaggtaact 720
ttggccatgg accggtgttc gttcctatct gccagcgtaa ttcgcgctgg tggctctggg 780
ttcgttctga gattatacgg ttaaacttga tctggataat accagcgaaa ggatcatgcc 840
ttccctcgtg cccccttcat tgatggaata actaacaatc ctcagtcact accgtgactt 900
gatccctcac ttgaacaacc cccaaagaac atc 933
<210> 2
<211> 1080
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 2
atagggtagg tagtcatcgc ccttgcaatg gaatgtttgc aagcacgttc ttgtctgagc 60
attggtggac ggggttgctc ggtccctggg cttcagctgg gatgtattgc accacaagat 120
gtcagaccat cttggctcat cactctagtc aagttctccg gtgggattcc gtgcttagct 180
tgtgacccat agacctgtac tgtacctagc gtattcttgg tctatggttt agtgcatttt 240
tcggcagtcc tgttctcgat ggcccaaccg tcttttgaag tgccttcgtc gagtcaatgg 300
aaattccgag gcggcgctgg ctagatagcc caaggggtgt agcttgtcga gactggctgg 360
agggggaaat gaagacaagc agcggaggaa aggtggtcga ccatctccat tggtcgacgg 420
ctgccatctt cattaatacc tcatttccgg cccaggcggg cttccctctt ttcccttcgc 480
tctttcgctg cgtctcccca ccggcgaggt aagcgatgac cctttcgtct cttccccacc 540
tccttcacga tattcagcag catcatcaag gaatcaagac tgacggactt gtatttagcg 600
tggcactagt ggacctcact aacgcggttg ccgtgatgaa tcgtccactt tgactccttg 660
acggcctgac ccccccgaga agaaaagccg taaccgagcc aggggcggcg attctgcggg 720
gaaatcagag ccctctcgtt ggaggaaacg gacttggctc cccggatttg acctcatcgg 780
agaagctggt ctgctggtca cagtgtgcag aacgcccgcg caggggctca tgcaggtcac 840
acctcgtcta acaaggtaca agccagatgc cgacaaagcg attggatgcc tgtggccaat 900
gcatgttcat gtccgcttca catgcagtcg agtcgaacct ctccccctca cccggtgctg 960
gacggccgcc tcatgcggtc tataaaaggc agtgactttc ctactgtcta gcgattcttt 1020
ttttcactcc agtctcatcc tctgttctat tcattatccc cgtttttccc ttgcctcgcc 1080
<210> 3
<211> 891
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 3
ggtgacccgt gcgacccctg actccaggaa agagaccacg aggggcaacc ggaccttttc 60
aaccgcggat agagatttga gaacggttcc gtgcagttgt ccatccttga actggataga 120
ggagggttcg agttcgtaag gggaggacca agttgagccc ttcgcggtga catcgtcggc 180
gaacgctcgg ttccgcttac agaaccccga ttgtgagcat gtcttgaaac tctcgtgttt 240
cacggcaact atacgttgca attaggcata agcacgatac cataaagagc cttgagagag 300
aacttacacc ctgggatgat gagacatcca aggacaatca ccaacgataa taagagggtc 360
catcgggtgg acgaaggccg ggagccggcc atgttgcgag cttaacagat aagatgcaat 420
cagagacact caatctctga aaacaataaa cttcaccagt cactaggggt ggggacctgc 480
atctaccaga ggcgaaggtc aaatcaagca gtggcaggct ctgggacgtt tgaataagag 540
tgacatgtga acatgaacct tgaatagttc cagcctggcc agattggcga tgaccgtgac 600
ggaagctccc tggattgggg ttagtgtgcc tctaaactcg atcgggaggt tggggttaga 660
cacctcaggc ggtgaggcgg tcaggccatt cagctggccg ctccaggcca caaggccctc 720
aaaggcattg ccaggtgccc ctccttttgc gtccctttcc gtcgagcccc acacagcgcg 780
tttgcggtcc ctccttatat ttttcgccca actcacgccg gctctgcttc ctttcccttt 840
tcctcctctc attcgaatct ccgtgaatcg tcatcatcca catccttcaa a 891
<210> 4
<211> 883
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 4
tggcgatggt gggattggtg acggggggac aggaaggtag tcgtttggtt tccacgaggg 60
aggcatcatc atgtgataga aagggttgcc ggccggcttt ctcgaggaga aatatcagcg 120
tgcccaggtg ttaaaacagg ataaatcttc gtgtgtcatg ggctggcatt gtgttgctag 180
caccgagtga tatatatgct aacggcaagt cgtgtccggt gttgtatgcg ctagtctgta 240
gctgcatcgc atccatgcag gcagcagtcc acgcggtatt gacggccccg tatctgccat 300
gttcccaatt gagtaactcc tggaaattaa gtgctcattt gacctctatg ataccaagta 360
actaaatttc tatcggtttg aacgcgaccg agtcccaatt cttggaattt gcggggttca 420
ctgttgccac cgaattgagg ttgggtgcat gcttaagcat aatggacgat tgcgtgatat 480
atatttggct ggcagataag gtgggcggcg agtcaccacc tttccacaat ataattaatc 540
accgctttac tatctcacct ccctttacag agctaatcag aacccgaccg tcctatcatc 600
catccccaaa gctagctatc ccaaattgaa tcagcgagta aaccaactta ctaggtacaa 660
tacgtagtaa ctcggactaa aatctgacat ccagccccaa caaaacaagc ctgcctacat 720
gcgtagcacg atcacatgac cataagcatc acattaccac gagtcaacca caacaattaa 780
tgtaatctcc gaactgtaag ccagtgctcc tcctcatcaa ccacgcatct cttatcccaa 840
gtgcatctca gccaatataa gctcatcgaa ccaacaaaaa aca 883
<210> 5
<211> 906
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 5
gatggaactt gctagcgaat ggttttggca aggaagagcc cccccccatt cccctcaatc 60
catctcggaa aaagtctcgg ggcaatttcc actacgcatg ccatggccaa ctgggtcact 120
cagatacgat gaacacacta gtcaaaaatc gactcatcgc ctccggctcc tccatccagg 180
gacctgaatc tctgaaaaag cggggctgca agtacccagc ggagaccaag tgcgccatat 240
ctgagtgaat cgtggaaccc tcggcttcgg acttaacggg tctggccctg aaaaactgcg 300
tgaaacgata ggctatcaag ctttttttac gagctctggg gagcaggcac cccccggcag 360
ttaatataaa cacacaggat gtgattgtgc ccacagaaca caagccgctg atctttagtc 420
cacagctgac gatttcgcca ggggtcctga agctcaggtc cacccagtca ctcgctatca 480
ttcgccttcg tcgggctgac attccttttc gtcttgcgca ggatcggttg atcagactac 540
attgcaatca tgattccgtc ctggctgctc aatgcgggct gtttcttcgg atttataacc 600
tgccatacgc ccccaggtgt ttggcctgat catggtgcga cggtcatttc atccactgca 660
gctcctttga caacttcaac cattggagtt tcaggaacaa ttgttcctgg cactacttca 720
attgttcatt cgtttactac caccgactct ctcgctttgt ctactcccac tgctgcacct 780
actactttac tcacttcgtt cagctctact gctacaatcg ttgcatcgct ttcttcttcc 840
tctctttcct cctcttcctc ctcttcttcg tcttcctctg ttgtgtcagt cgaagcgtcc 900
accacc 906
<210> 6
<211> 913
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 6
ggtatattgt cctaaatggg aagcatcacc tccaaagaat cattcgagaa cggttaaatt 60
cggttttaat ggcggatcag ggggatcctg gaaccggaga accatcttgc ctcgctattc 120
cgagtgctcc gtaatgagct ggaacgtagc gggcaaaggc cacgggagcg aagacaaaga 180
ccgcagaaca agccgaagag gggcaaccga ggggtaaacg ggaacggtag gggaatactg 240
ctccgtactg aatactccgt atgctgttgt acctatgacc atgcagtaag cccacagctg 300
ctgcagcggc agctccaaca cgctggggaa gaaacctccg gtgaagcaaa aagggcctgg 360
atggggtcgt gattagccgg gcgtgagtgg accgcccggc gctgtcttgc cggcccagtg 420
tgctccggtc tgtaccaacc aacaatgcat gaattgaagt atgtgtatgt atggggaggg 480
ggagtaggat gtgagaatga gaggtgatac aggcactgag taggactagt gcgcctcgtg 540
ttaagtaaga gctataacga tcatggatag tggtagtaat ccgtaggtgg tatgtacgga 600
gtatgcattg tggatggaga aggaagatag ttaagtggaa ggaaatatta acaacaacag 660
aaacgagcag aaaggcatag cgtcatcggg gagggaagct gtttgcccca ccattcgctc 720
atccagtttc ccttcccaat cccctccccc tcccacttcc ctctccgctg acttccctcc 780
cccctactta ttcttaccct attccgcctc tgatcctttt cttcttcctt caatttcttc 840
aatctctcca aaagttcttc ttattcactt cttaatcttc caactcccca ccttttcata 900
ctcatctatc aca 913
<210> 7
<211> 930
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 7
tgagtatcga ttccgaggtc acgtgccttc gatgggcctg cagggagagg cttggcatgc 60
atttcacatc gtacagtata tgtacatgat gggttgggaa ggggagcaga ccagaagtcc 120
accactacta ctactactac tactactgaa caatagtcaa tgaacccaag gacagtccgg 180
agtagactac gtacgtttca tccagaaaat ctcttaatcc agctcgctta gagctttgca 240
tcccgagaga ttccttgcca cacgaggatt tcccgggagg actgaaagcg gcgagagggg 300
cgggtttccg ccgatagata acgggcgcag attctgtgac cataaactat cgactaacac 360
tgccccgaac ttcggacgcc cacccggcat tgctttgccg tgattagtcc cgctgccgtt 420
gactgcctta ctacttttcc tctccggcgg ggacttccta cgtctcgtcc gactccggct 480
gttactgtac acctttttgg tgtttctttg attttcccgt ttcctttttt cttttctttc 540
tttttttttt ttttcgtttt gccgtccgga caaccctctt ccgctccgac ccagtggcac 600
ccaccatcca ctgctaccta ccatgactgc taccacaaac ccagtccggg gtcgggggta 660
gaagcaggag aggaggaggg ggaggggagg ggggactgaa acgggaaggg aagggcagag 720
gggggaaacc cggaagaaaa agaggcaatg ggaaagatac ataaacccat tctctctcct 780
ccctcatctc tttctctttc tttcttccca tccccatcct ccatctctct cccagaccag 840
gtcttcaaaa gtcttgtcgg gtatcctttg ggtcattctt cttcttctaa caacatcata 900
ctaaatattc tcttccctcc ctcattcacc 930
<210> 8
<211> 928
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 8
gcctttcact atagcctccc aactgggttg cggcggtgaa cagcaccatg gttcggatga 60
ggatggcaag tctctttctc tctggcttat tgtctggaaa tcgcccgatt gtacagagta 120
cattgatgta ctacattact cttcttgaca ggcaacgtgc tacctaatat ctccacagca 180
gcagcctgca gtgcagtcga catcatggat cacaatgaat tctgtcagac cactccgctt 240
actgaccact gcacttaacg ttaccagtga cttctttgtt gctccactgg gtggatcttc 300
tccccctctt ctccgtgcgg ggtaagccca atccggacag cctgttgtcg cctggtgttt 360
caggcactaa tatagatgac ttaggctcca aggcatgagc tgcaccgcct cgtatgttta 420
cgtcatcaat ggcttcaacg gcggcatcct tatccctgct aagccacact tctctgattg 480
gcgccagcca cgcctcaggc agagatttca ctgtccactt ggccaatcat catctcacaa 540
aaaaagtccc ccgccgcccg cctcgatttt tctctcccat ctcattgttt tctctccccc 600
cctcctcctc ctcctccttc ttcttctgac gctgtgtacc ctctccactc ttcttttttc 660
tgtgtttttg gtgtttggaa cgcatcctcc aggaggattg ctgctttgtt gtttggtttg 720
tggttgtata gttctttcca ttcagtcagc ttcttctctt atctccctct ctctctcctg 780
ctccgaacga ggctgaacgc ccctctccgc ccacctccaa aaatccacgg cttacggctt 840
gtgctggttg gaagccttca ccacttcacc acgatagaat acccctcgca tatttagaat 900
gcctcacccc gaatctccca cgcccacc 928
<210> 9
<211> 899
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 9
tcgtaggcag agtgcccctg agagaacaat gacgacattt tgtattactc ggttataaaa 60
ttgcgggaca caggcgtcga atattaagcg attttggcgt cgcgaatgtg atgctgggcc 120
gtcttggtag ggcctgagcg ctgggtgcaa cacgaagaac ccaactggca cgcaactcac 180
tggatactcg gcagtcattc acccgacagc tgcacctgaa caatgtcttc tttcttgctc 240
gcatcaaacc gacccggatt ttcagggcag aagctttatg gtgattgctt gttgcacgca 300
tcaccccgcc gcacgtaccc cacaaagtcc gtcaggcgat gcatctagct gggggaatga 360
gttcttgctt gatatttgcc gagatgaacc ctcctggccg cccctggaac taaatcctcc 420
gcagaaatca ctatcaattt gggatgtgat cagcgcttca agcctcgaga cggatgctca 480
agtagcgctg acactggctt gcgtggcggt ataatcaatc agaattctcc atgaataagg 540
aggagcttgg agtttgaagc ctctgtgagg aagcttgact cctgtcactc cactgggttt 600
cttagcaaaa acagtagaga tcatcatcca aaggggttct gacattttat gtcacggagt 660
aggaagccac aatcaacgtc cgacaggacg gagaatcttc agcctgcttg attcccagcg 720
gctcgagggg ctcgaggata agtgtacgtt gtagattctc cggtggacat gagcccctga 780
ccacaatcac catgaacgct ccttactgcg atataaaagc tgcgattgct gcgtaagttc 840
ttgagaagaa ttccaagaat caagcacgtc gaggttgctc ccattgatcc aagccagtc 899
<210> 10
<211> 901
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 10
ccgacaggca cgttgttgtg actaacccct tcattttcac tatcggctgg actcggttga 60
ggtggtagaa tcaggattcc gatctcttgt agtgcctcaa gcccgtagtt acagggccat 120
ttcagtcgtg ccaataattc tcggaattag ttggatgctt aatctttcgg gtgcctcaca 180
tcctgtcaat ctttcggcag cttcatgttg agaactatag cccacagcaa ctatttaaag 240
atggaacatc cggtgagttt gacagcacaa tagtcggtcc aagtgtttca tcagtgaagt 300
gtgctgactc gaatctgcac ggatgcttcc gaaaatggat aaaatgccgg cacacggacc 360
cctacgaccg tcaatgcgag caatttgtag aggcgtagga tatttgaggc gtataagttc 420
tcagtagcac tttcgtgctg agtgttgtag tattgtggat gtagatgttt ttttttttcg 480
gactcatgtc acagtaattc agtaaaatac cccagagtcc tgcagaattc tgtagacatg 540
ggagcggagc aaggctgtgt gtaatttaga ctgccactac gccgggttac cccacacact 600
aagcagtgcc ggcattcgct agtcgctcgt cacttgccat cagcaccact tctccaccag 660
tcacctgcag catccctccg ggattttgct atttcctcta cgcgttttga aacctccaaa 720
gtatcacgga gccaggtccg tatctgcaaa cgtctcaact attgcacccg tctacacacg 780
ccgcactgat ccggcggcac ggcgacaccc ctgaacaccg aacaatcgac aacctatata 840
acatccaaaa tggccgaaac aaagcaagaa acgatcgtca catctcagac gcttaaacac 900
c 901
<210> 11
<211> 2159
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 11
tggagaactc ctcccagact atgctcttca cgctggtggc cgggctgaaa tacccgcagc 60
tggcggcggg cttgggcgcg gcgtgggtgg tcttgcgggc gctgttcttg cacggatacg 120
tgtactcggg caagtcggag ggtaagggtc ggtacagggg gggtctgttc tggttggtgc 180
agggtgcgtt gtggtggttg acggtctttg gcgtggcgaa ggagttgttc taggggtgca 240
cgggtgaggt tggtgagagt gtggggatgg ggatgggctg ctggtcctgt gtagtctagt 300
taatgttcgt aagggaatct gttaggtttg catcttttat ctgagcaatt ttgtttgttg 360
gagctctggg tgaagactga acggtagtag tggggactgg ttgttgaggg attggtgctc 420
attgtgaggg ttggagtaag tgtacaagta tcagttgcaa tggtaagtag gttatggtca 480
gtggaaagat atctggagac ttagctcagc atcttgagat acgaggcttg gagcagtggc 540
tgtctgtatt caagctcggc ggagaagctc tcagggccgc agcctcggca ttcggcagtg 600
tggctgaccc cacggtcgga ggtggagcaa tctagaagcc gatgtggctg atgaaggatg 660
gagggggaat ctttgggtat gattttatcg tctatcgtgt tttcttccgc aggccttctt 720
ttcgtcaata ccccgccatt gagctcaatt acttgactga tatcattatc ggaggcctct 780
tcacatactg gtcaatccct caatcagccc attccaacaa aaagctccac atcccacccc 840
atccgctacc accccgcaaa aaccccgcca tgtcgtccaa gtcgcaattg acctacactg 900
cccgtgcgag caagcacaac aaccctcagg ccaagcgcct cttcgagatc gccgaggcca 960
agaagaccaa tgtgaccgtc tccgccgacg tgaccactac caaggagctc ctggacctcg 1020
ctgaccgtag gcccgccgaa acctctttca ctccatgata aggaattgac ccccgagata 1080
tgtacatagg cctgggaccc tacatcgccg tgatcaagac gcacatcgac attctgtcgg 1140
acttcagcaa cgagaccatc gagggcctca aggcgctgtc cgagaagcac aacttcctga 1200
tcttcgagga ccgcaagttc atcgacatcg gcaacaccgt ccagaagcag taccacaagg 1260
gcaccctccg gatctcggag tgggcccaca tcatcaactg cagcatcctg cccggcgagg 1320
gcatcgtgga ggccctggcc cagaccgccg ctgccgagga cttcgagtac ggcgccgagc 1380
gcggcctcct gatcctggcc gagatgacct ccaagggatc cctcgccacg ggccagtaca 1440
ccaccgcctc ggtggactac gcgcgcaaat accagaagtt cgtcatgggg ttcgtgtcga 1500
cgcgggcgct gggcgaggtg cagtcggagg cgagctccgc ctcggaggag gaggactttg 1560
tcgtgttcac gacgggcgtc aatatctcct ccaagggcga taagctcggc cagcagtacc 1620
agacccccgc ctctgcgatt ggtcgcggag ccgatttcat catcgccggt cgcgggatct 1680
atgccgcggc ggacccggtg caggcggcgc agcagtacca gaaggagggc tgggaggcgt 1740
accaggcccg tgttggcgga aactaaaatt gagtgagggg tggctgttta ttttagacga 1800
tgtacaggat tagcaacata atgccatgtt acgactaatt tgctttcaag catccaagct 1860
ctcggataaa gacgaggggg tttcgatcct gcagggaaga taagatccct tgaatcagtc 1920
atcttgcctt tcaggcccat tcaggccatg ggttttgtga ttgataacat gggagatgat 1980
aacctaatct agaccgaacg agataagacc tgaaatccag cctctacctt acatgactgt 2040
agaggcaggc agatgatggg tctgtggatc tcccagcata tctcagcata tactttcctc 2100
tggccaagat tgttatcagc caatggatgg atgcaatatg gccatgccat taatgttga 2159
<210> 12
<211> 1323
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 12
atgcgtggtc ttttccttct tgctctgggt gctatcccgg cgctcgtcag cggtcaactc 60
tctggcagtg ttggcccgtt gacctccgct tccaccaaag gtgcgacaaa aacatgcaac 120
atcctcagct acggtgcagt ggccgacaac tcgaccgacg ttgggcctgc catcacatcg 180
gcctgggctg catgcaaaag cggaggtctt gtctacatcc catctggcaa ctatgcccta 240
aatacctggg tcaccctgac tgggggcagc gcgaccgcaa tccagctgga tggtatcatc 300
taccgcacag gtaccgcgag tgggaacatg attgcggtca ctgacaccac cgatttcgag 360
ctgttcagta gcacctccaa aggtgctgtg cagggattcg gctatgtgta ccatgcggag 420
ggtacctacg gagcacgaat tctgcgcttg accgacgtga ctcatttctc cgtgcacgat 480
attatcttgg tggacgcgcc tgcttttcac tttaccatgg atacctgctc cgatggcgag 540
gtgtacaaca tggcgattcg tggtggcaat gagggcggct tggacgggat tgatgtctgg 600
ggaagcaaca tctgggttca cgatgttgaa gtgaccaaca aggatgaatg tgtaacagtc 660
aagagccctg ccaacaatat cctggtggag agtatctatt gcaactggag tggtggttgc 720
gcaatgggat cgctcggggc cgacaccgac gtcaccgaca ttgtctaccg caatgtttac 780
acctggtcgt cgaaccagat gtacatgatc aagagcaatg gtggcagtgg aacggtgtcg 840
aatgttctgc tggaaaactt catcgggcac ggtaatgcgt actcgctcga cattgatggc 900
tactggagca gcatgactgc ggtggccggg gacggagtgc agctgaacaa catcactgtg 960
aagaactgga agggcaccga ggcgaacgga gcgacccgac caccgatccg agttgtgtgt 1020
agtgacacgg cgccttgcac ggacctgacg ttggaagata ttgccatttg gaccgaaagc 1080
ggctcgagtg aattgtacct gtgccgttcc gcttacggat cgggatactg tctgaaggac 1140
agctcttcgc acacatccta caccacgacc agcactgtca cggcggctcc atcagggtat 1200
tcggcgacaa ccatggcagc cgacttggcg accgcatttg gcctcactgc ttccattcct 1260
attccgacca tcccgacctc gttttatccc gggttgaccc cgtacagtgc cttggcaggc 1320
taa 1323
<210> 13
<211> 440
<212> PRT
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 13
Met Arg Gly Leu Phe Leu Leu Ala Leu Gly Ala Ile Pro Ala Leu Val
1 5 10 15
Ser Gly Gln Leu Ser Gly Ser Val Gly Pro Leu Thr Ser Ala Ser Thr
20 25 30
Lys Gly Ala Thr Lys Thr Cys Asn Ile Leu Ser Tyr Gly Ala Val Ala
35 40 45
Asp Asn Ser Thr Asp Val Gly Pro Ala Ile Thr Ser Ala Trp Ala Ala
50 55 60
Cys Lys Ser Gly Gly Leu Val Tyr Ile Pro Ser Gly Asn Tyr Ala Leu
65 70 75 80
Asn Thr Trp Val Thr Leu Thr Gly Gly Ser Ala Thr Ala Ile Gln Leu
85 90 95
Asp Gly Ile Ile Tyr Arg Thr Gly Thr Ala Ser Gly Asn Met Ile Ala
100 105 110
Val Thr Asp Thr Thr Asp Phe Glu Leu Phe Ser Ser Thr Ser Lys Gly
115 120 125
Ala Val Gln Gly Phe Gly Tyr Val Tyr His Ala Glu Gly Thr Tyr Gly
130 135 140
Ala Arg Ile Leu Arg Leu Thr Asp Val Thr His Phe Ser Val His Asp
145 150 155 160
Ile Ile Leu Val Asp Ala Pro Ala Phe His Phe Thr Met Asp Thr Cys
165 170 175
Ser Asp Gly Glu Val Tyr Asn Met Ala Ile Arg Gly Gly Asn Glu Gly
180 185 190
Gly Leu Asp Gly Ile Asp Val Trp Gly Ser Asn Ile Trp Val His Asp
195 200 205
Val Glu Val Thr Asn Lys Asp Glu Cys Val Thr Val Lys Ser Pro Ala
210 215 220
Asn Asn Ile Leu Val Glu Ser Ile Tyr Cys Asn Trp Ser Gly Gly Cys
225 230 235 240
Ala Met Gly Ser Leu Gly Ala Asp Thr Asp Val Thr Asp Ile Val Tyr
245 250 255
Arg Asn Val Tyr Thr Trp Ser Ser Asn Gln Met Tyr Met Ile Lys Ser
260 265 270
Asn Gly Gly Ser Gly Thr Val Ser Asn Val Leu Leu Glu Asn Phe Ile
275 280 285
Gly His Gly Asn Ala Tyr Ser Leu Asp Ile Asp Gly Tyr Trp Ser Ser
290 295 300
Met Thr Ala Val Ala Gly Asp Gly Val Gln Leu Asn Asn Ile Thr Val
305 310 315 320
Lys Asn Trp Lys Gly Thr Glu Ala Asn Gly Ala Thr Arg Pro Pro Ile
325 330 335
Arg Val Val Cys Ser Asp Thr Ala Pro Cys Thr Asp Leu Thr Leu Glu
340 345 350
Asp Ile Ala Ile Trp Thr Glu Ser Gly Ser Ser Glu Leu Tyr Leu Cys
355 360 365
Arg Ser Ala Tyr Gly Ser Gly Tyr Cys Leu Lys Asp Ser Ser Ser His
370 375 380
Thr Ser Tyr Thr Thr Thr Ser Thr Val Thr Ala Ala Pro Ser Gly Tyr
385 390 395 400
Ser Ala Thr Thr Met Ala Ala Asp Leu Ala Thr Ala Phe Gly Leu Thr
405 410 415
Ala Ser Ile Pro Ile Pro Thr Ile Pro Thr Ser Phe Tyr Pro Gly Leu
420 425 430
Thr Pro Tyr Ser Ala Leu Ala Gly
435 440
<210> 14
<211> 966
<212> DNA
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 14
atgtactccc tcctcactgc attgtcggtg ccgctcctgg caggcctggc tcatggctac 60
gccaaccccg gctcctgctc cggttcctgc aacgtccatg acccagcctt gatcgtccgc 120
gagtcggacg gcaaatactt ccgtttctcg accggcaacg agatttccta tgcctctgcc 180
tcctccatca acggtccgtg gaccgccatt ggatccgtgg tgcctgccgg atctaagatc 240
gacctgtccg gcaacactga cctctgggcc cccgatctta gctacgtcga tgggacctac 300
tactgcctct actccgtctc gacctttggc tcccaggact ctgccattgg agtggcctcg 360
tccaccacga tggagctgaa cacctggacc gaccacgggt ccgtgggcgt cgcctcctcg 420
tcctctaaga actacaacgc catcgacggc aacctcctcg tggacggcag ctcatattac 480
ctccagttcg gctccttctg gggcgatatc taccaggtca agatggcctc gcccctcaag 540
acggccggct cggcctccta caacatcgcc tacaacgcga cgggcaccca ctcggaggag 600
ggctcctact tgttcaagta cggcagctac tactatctct tcttctcgtc gggcacctgc 660
tgcggctacg acacctcccg cccggcccag ggcgaggagt acaagatcat ggtctgccgc 720
tccaccagcg cgaccggcgg atttgtggac aagaatggca atgcttgcac ggaaagtggc 780
ggcacgattg tgctcgccag tcacggcacc gtctatggac cgggtggaca gggcgtgtat 840
gacgacccga cctacggccc tgtgctctac taccactatg tcgacaccac cattggttac 900
gccgatgacc agaagctgtt tgggtggaac accattgact tctcgagtgg ctggcctgtt 960
gtgtag 966
<210> 15
<211> 321
<212> PRT
<213> 棘孢曲霉(Aspergillus aculeatus)
<400> 15
Met Tyr Ser Leu Leu Thr Ala Leu Ser Val Pro Leu Leu Ala Gly Leu
1 5 10 15
Ala His Gly Tyr Ala Asn Pro Gly Ser Cys Ser Gly Ser Cys Asn Val
20 25 30
His Asp Pro Ala Leu Ile Val Arg Glu Ser Asp Gly Lys Tyr Phe Arg
35 40 45
Phe Ser Thr Gly Asn Glu Ile Ser Tyr Ala Ser Ala Ser Ser Ile Asn
50 55 60
Gly Pro Trp Thr Ala Ile Gly Ser Val Val Pro Ala Gly Ser Lys Ile
65 70 75 80
Asp Leu Ser Gly Asn Thr Asp Leu Trp Ala Pro Asp Leu Ser Tyr Val
85 90 95
Asp Gly Thr Tyr Tyr Cys Leu Tyr Ser Val Ser Thr Phe Gly Ser Gln
100 105 110
Asp Ser Ala Ile Gly Val Ala Ser Ser Thr Thr Met Glu Leu Asn Thr
115 120 125
Trp Thr Asp His Gly Ser Val Gly Val Ala Ser Ser Ser Ser Lys Asn
130 135 140
Tyr Asn Ala Ile Asp Gly Asn Leu Leu Val Asp Gly Ser Ser Tyr Tyr
145 150 155 160
Leu Gln Phe Gly Ser Phe Trp Gly Asp Ile Tyr Gln Val Lys Met Ala
165 170 175
Ser Pro Leu Lys Thr Ala Gly Ser Ala Ser Tyr Asn Ile Ala Tyr Asn
180 185 190
Ala Thr Gly Thr His Ser Glu Glu Gly Ser Tyr Leu Phe Lys Tyr Gly
195 200 205
Ser Tyr Tyr Tyr Leu Phe Phe Ser Ser Gly Thr Cys Cys Gly Tyr Asp
210 215 220
Thr Ser Arg Pro Ala Gln Gly Glu Glu Tyr Lys Ile Met Val Cys Arg
225 230 235 240
Ser Thr Ser Ala Thr Gly Gly Phe Val Asp Lys Asn Gly Asn Ala Cys
245 250 255
Thr Glu Ser Gly Gly Thr Ile Val Leu Ala Ser His Gly Thr Val Tyr
260 265 270
Gly Pro Gly Gly Gln Gly Val Tyr Asp Asp Pro Thr Tyr Gly Pro Val
275 280 285
Leu Tyr Tyr His Tyr Val Asp Thr Thr Ile Gly Tyr Ala Asp Asp Gln
290 295 300
Lys Leu Phe Gly Trp Asn Thr Ile Asp Phe Ser Ser Gly Trp Pro Val
305 310 315 320
Val
Claims (4)
1.一种启动子，其特征在于，所述启动子的编码核苷酸序列为SEQ ID NO：2。
2.权利要求1所述的启动子在棘孢曲霉内源基因自克隆表达中的应用。
3.一种自克隆表达盒，其特征在于，所述自克隆表达盒包含权利要求1所述的启动子和棘孢曲霉的内源基因；所述的内源基因选自鼠李聚糖半乳糖醛酸水解酶、内切-1，5-阿拉伯糖苷酶、果胶甲酯酶、果胶裂解酶、鼠李糖苷酶、阿拉伯呋喃糖苷酶、纤维素酶、β-葡萄糖苷酶、β-葡聚糖酶、葡萄糖氧化酶、酸性蛋白酶中的任意一种。
4.如权利要求3所述的自克隆表达盒，其特征在于，所述的内源基因为鼠李聚糖半乳糖醛酸水解酶基因或内切-1，5-阿拉伯糖苷酶基因。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0498137A1 (en) * | 1991-02-06 | 1992-08-12 | Novo Nordisk A/S | Novel expression systems |
| CN107988085A (zh) * | 2017-12-14 | 2018-05-04 | 青岛蔚蓝生物集团有限公司 | 一种高产酸性果胶酶的棘孢曲霉菌株及其应用 |
| CN107988086A (zh) * | 2017-12-14 | 2018-05-04 | 青岛蔚蓝生物集团有限公司 | 一种高产鼠李聚糖半乳糖醛酸水解酶的菌株 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001627A (en) * | 1991-05-02 | 1999-12-14 | Novo Nordisk A/S | Rhamnogalacturonase, corresponding DNA sequence, rhamnogalacturonase containing enzyme preparation and use of the enzyme preparation |
| JP6950873B2 (ja) * | 2017-06-07 | 2021-10-13 | ピーティーティー グローバル ケミカル パブリック カンパニー リミテッド | セルラーゼ及びキシラナーゼを生産するための変異株アスペルギルス・アクレアツス並びにその調製方法 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0498137A1 (en) * | 1991-02-06 | 1992-08-12 | Novo Nordisk A/S | Novel expression systems |
| CN107988085A (zh) * | 2017-12-14 | 2018-05-04 | 青岛蔚蓝生物集团有限公司 | 一种高产酸性果胶酶的棘孢曲霉菌株及其应用 |
| CN107988086A (zh) * | 2017-12-14 | 2018-05-04 | 青岛蔚蓝生物集团有限公司 | 一种高产鼠李聚糖半乳糖醛酸水解酶的菌株 |
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