CN113584075B - 一种利用转基因大豆生产α-淀粉酶的方法及表达载体 - Google Patents

一种利用转基因大豆生产α-淀粉酶的方法及表达载体 Download PDF

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CN113584075B
CN113584075B CN202111008204.6A CN202111008204A CN113584075B CN 113584075 B CN113584075 B CN 113584075B CN 202111008204 A CN202111008204 A CN 202111008204A CN 113584075 B CN113584075 B CN 113584075B
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曹瑱艳
沈志成
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Abstract

本发明公开了一种利用转基因大豆生产α‑淀粉酶的方法及表达载体，所述方法是将含α‑淀粉酶表达框的表达载体导入到大豆基因组中，获得能够生产α‑淀粉酶的转基因大豆；所述α‑淀粉酶表达框包括启动子、α‑淀粉酶编码基因、终止子。本发明转基因大豆在种子中实现淀粉酶的高表达，所生产的淀粉酶的活性极高，最高每克种子10000酶活单位以上；本发明的转基因大豆生产的淀粉酶在作为饲料添加剂、食品加工添加剂等很多领域的应用时无需经过分离浓缩，可以直接应用，降低了生产成本；本发明利用大豆种子生物反应器生产淀粉酶比传统微生物发酵方法节省能源，是一种更加环境友好型的生产方式。

Description

一种利用转基因大豆生产α-淀粉酶的方法及表达载体
(一)技术领域
本发明涉及一种利用转基因大豆生产α-淀粉酶的方法及表达载体。
(二)背景技术
世界上对酶的需求逐步增加，从2003年到2008年，世界上的酶需求基本上以2倍的速度在增长。从2009年酶市场约51亿美元，到2018年全球酶市场规模达到93亿美元，预计2019-2025年，酶市场将以年复合增长率7.1％的速度增长，达到149亿美元的规模。而这其中淀粉酶在全球酶市场中约占30％，被广泛地应用于工业领域。
淀粉酶根据酶水解产物异构类型的不同可分为α-淀粉酶和β-淀粉酶。α-淀粉酶(α-1,4-glucan-4-glucanohydrolase,EC 3.2.1.1)是外切酶，主要分布在动物、植物、微生物中，它以随机作用方式切断淀粉、糖原、寡聚或多聚糖分子内的α-1,4-糖苷键，将淀粉水解为糊精和一系列以葡萄糖为组成单位的低聚糖。食品工业中α-淀粉酶主要应用于制造果糖和果葡糖浆、提高烘培发酵速率和烘培食品的保质期和柔软度等。在洗涤剂业中，α-淀粉酶可以将含淀粉的食物颗粒消化成较小的水溶性寡糖，用水冲洗后达到去除污渍的目的。在造纸业和纺织业中应用于产品的上浆、脱浆。在燃料乙醇的生产，α-淀粉酶通过将淀粉液化水解后经过糖化，在复合糖化酶的作用下产生葡萄糖，而葡萄糖又作为酿酒酵母的底物，经过发酵后产生酒精。α-淀粉酶也作为饲料添加剂，以提高饲料的利用率。
目前淀粉酶的工业生产一般采用微生物发酵法，但这种生产方法工艺比较复杂、生产设备要求较高，提纯过程费时费力，运输储存过程中对温度要求严格。随着分子农业的发展，农作物作为一种绿色生物反应器在生产外源蛋白方面已获得了大量使用。从20世纪80年代早期第一株转基因植物诞生以来，利用植物生物反应器表达蛋白质就应运而生。1997年，Hood等(Hood EE,Withcher DR,Maddock S,et al.Commercial production ofavidin from transgenic maize：characterization of transformant,production,processing,extaction and purification[J].Molecular Breeding,1997,39(4):291-306)报道了在植物中表达巧白素，并将其提取纯化上市销售。接下来的十几年中研究者们陆续在各种植物中表达了各种外源蛋白，例如在烟草中表达了乙肝病毒表面抗原和疟原虫表面抗原决定簇；在马铃薯中表达了热不稳定肠毒素，其功能与大肠杆菌表达的热不稳定肠毒素功能相同(Obembe OO,Popola JO,Leelavathi S,et al.Advances in plantmolecular farming[J].Biotechnology Advance,2011,29(2):210-222)。尽管转基因动物、细菌和真菌都可以用来生产蛋白，但在植物上往往可以获得更高的经济价值。利用高等植物生产外源蛋白具有以下几点优势：1)与转基因动物饲养、转基因微生物发酵相比显著地降低了生产成本；2)植物具有现成的耕种、收割以及加工的基础设施和专门技术；3)植物不含有已知的可能污染终产物的人类病原体；4)高等植物能合成正确的折叠、糖基化并具有活性的来自真核生物的蛋白；5)蛋白表达在植物细胞中避免了降解，增加了稳定性。有研究表明于休眠期的种子含水量一般比较低，此时种子中的蛋白水解酶活性很低，因此外源蛋白可以长时间地在种子中贮存且保持活性。如将重组疫苗抗原CTB于水稻种子中进行表达，放置在常温条件下2年以上，经检测该重组抗原仍保持较高的活性(Boothe J,NykifoukC,Shen Y,et al.Seed-based expression systems for plant molecular farming[J].Plant Biotechnology Journal,2010,8(5):588-606)。因此种子生物反应器不仅为重组蛋白的保存提供了理想载体，还为后续的分离和纯化提供了便利条件。
在植物中表达生产外源淀粉酶的研究先前也有报道，例如烟草(Pen et al,1992)、苜蓿(Austin et al,1995)、玉米等，但其单位重量中的酶活性仍然普遍比较低。2002年美国专利US7102057B2披露了一种利用转基因玉米表达育种高温α-淀粉酶来生产生物酒精的方法，该发明利用一种玉米内源的胚乳蛋白质启动子控制外源淀粉酶基因在玉米胚乳中表达的方法，但转化得到的表达淀粉酶的玉米表达水平相对比较低。特别是由于玉米种子富含淀粉，在其胚乳中过表达在常温下具有活性的淀粉酶会导致玉米种子自身淀粉的降解，使种子变得干瘪、发育不良、胚乳液化、发芽率等问题。因此需要寻求新的植物淀粉酶生物反应器技术体系，大幅度提高植物中淀粉酶的表达量具有重要应用价值。
本发明披露了一种利用转基因大豆高效生产淀粉酶的方法。大豆是常见的农作物，生产成本低，产量较高。利用转基因大豆生产淀粉酶，可以获得高质量的淀粉酶，降低淀粉酶的生产成本，具有广泛的应用场景和较高的经济价值。特别是本发明发现，表达高水平中温淀粉酶的大豆种子在形态上、发芽率、百粒重等方面基本正常。利用本发明方法获得的表达淀粉酶的大豆种子在碾磨后能直接作为淀粉酶使用于粮食加工、饲料添加剂、食品工业、纺织、燃料酒精的生产等工业过程中。相比微生物发酵产生的淀粉酶，本发明生产成更低，无需提纯加工、便于运输和储存、容易扩大生产规模。本发明利用大豆生物反应器生产淀粉酶是一种环境友好型的生产方式。
(三)发明内容
本发明目的是提供一种利用转基因大豆生产淀粉酶的方法及表达载体，该方法能够稳定、高效、低成本地生产各种用途的淀粉酶，直接运用在粮食加工、饲料生产、食品工业、纺织、燃料酒精的生产等工业过程中。
本发明采用的技术方案是：
本发明提供一种利用转基因大豆生产淀粉酶的方法，所述方法是将含α-淀粉酶表达框的表达载体导入到大豆基因组中，获得能够生产α-淀粉酶的转基因大豆；所述α-淀粉酶表达框包括启动子、α-淀粉酶编码基因、终止子。
进一步，所述α-淀粉酶表达框的启动子能够启动α-淀粉酶在种子中高水平表达。所述启动子包括但不限于种子特异型启动子，优选豆类蛋白的启动子，包括大豆2S球蛋白的启动子(GM2S，核苷酸序列如SEQ ID NO:1所示)、大豆7S球蛋白的启动子(CGY，核苷酸序列如SEQ ID NO:2所示)，大豆11S球蛋白的启动子(GY5，核苷酸序列如SEQ ID NO:3所示)，菜豆贮藏蛋白β-Phaseolin启动子(核苷酸序列如SEQ ID NO:4所示)。其它能够启动α-淀粉酶基因在大豆种子中高效表达的启动子也可以使用，克隆和测试启动子功能的方法目前已经是成熟的方法。
进一步，所述α-淀粉酶能够催化水解直链淀粉中的α-1,4-葡萄糖苷键，生成α-麦芽糖、葡萄糖等物质。α-淀粉酶可以选自NCBI基因库收集的α-淀粉酶，优选以下六种中的任意一种：来源于地衣芽孢杆菌(Bacillus Lichenifarmis)的淀粉酶，其核苷酸序列如SEQID NO:5所示，氨基酸序列如SEQ ID NO:11所示；来源于强烈火球菌(Pyrococcusfuriosus)的淀粉酶，其核苷酸序列如SEQ ID NO:6所示，氨基酸序列如SEQ ID NO:12所示；来源于枯草芽孢杆菌(Bacillus subtilis)的淀粉酶，其核苷酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:13所示；来源于嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的淀粉酶，其核苷酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:14所示；来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的淀粉酶，其核苷酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:15所示；来源于米曲霉菌(Aspergillus oryzae)的淀粉酶，其核苷酸序列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:16所示。
进一步，所述α-淀粉酶基因表达框包括启动子、α-淀粉酶基因及增强α-淀粉酶表达的元件以及终止子；所述增强α-淀粉酶表达的元件包括在α-淀粉酶基因序列5’端加入烟草蚀纹病毒5'非翻译区(TEV 5'UTR)编码基因(核苷酸序列如SEQ ID NO:18所示)和拟南芥2S白蛋白信号肽(At2S2signal peptide)编码基因(核苷酸序列如SEQ ID NO:19所示)，在3’端加入烟草Tobacco RB7核基质结合区调控序列(Tobacco RB7 MAR)(核苷酸序列如SEQID NO:20所示)。
进一步，所述终止子包括但不限于花椰菜花叶病毒35s(CaMV35S)终止子(GenBank:AY560326.1)、拟南芥heat shock protein 18.2基因终止子(GenBank:LR782546.1)，CaMV35S和拟南芥heat shock protein 18.2终止子均为常见终止子，用其他常见终止子替换也均可达到同样的效果。
进一步，所述表达载体包括α-淀粉酶基因表达框和筛选标记基因表达框；筛选标记基因表达框优选由启动子、抗除草剂基因、终止子组成，其中启动子终止子均为花椰菜花叶病毒35s的启动子和终止子，用其他常见终止子替换也均可达到同样的效果。更优选耐草甘膦表达框(Clemente TE.Progeny analysis of glyphosate selected transgenicsoybeans derived from Agrobacterium-mediated transformation.Crop Science,2000,40(3):797-803)和耐草铵膦表达框(Zhang Z,Xing A,Staswick P,et al.The useof glufosinate as a selective agent in Agrobacterium-mediated transformationof soybean.Plant Cell,Tissue and Organ Culture,1999,56(1):37-46)以及耐啶嘧磺隆表达框(专利申请200810164131.8)，其中耐啶嘧磺隆表达框中啶嘧磺隆抗性基因CdP450的核苷酸序列如SEQ ID NO:21所示，启动子为花椰菜花叶病毒35s(CaMV35S)的启动子，终止子为花椰菜花叶病毒35s(CaMV35S)终止子。
进一步，本发明所述表达载体的基础载体是pCambia1300(NCBI序列编号AF234296)。
进一步，本发明还涉及所述方法构建的表达载体，所述表达载体按如下方法构建：(1)将pCambia1300上的潮霉素筛选基因hptII(位于两个XhoI位点之间)替换为从狗牙根克隆获得的啶嘧磺隆抗性基因CdP450(核苷酸序列如SEQ ID NO:21所示)，并用花椰菜花叶病毒35s启动子和终止子控制抗除草剂基因CdP450，改造后的载体命名为p1300-CdP450；(2)用HindIII/KpnI双酶切载体p1300-CdP450；用HindIII/BamHI双酶切启动子质粒，回收启动子片段；用BamHI/KpnI双酶切加了增强基因表达元件的α-淀粉酶编码基因，将上述三个片段过夜连接，将连接产物转入大肠杆菌TG1菌株中，酶切鉴定后获得所述的表达载体。
进一步，优选本发明所述表达载体的T-DNA由种子特异型启动子、α-淀粉酶编码基因、增强α-淀粉酶编码基因表达的元件和终止子、花椰菜花叶病毒35s(CaMV35S)启动子、啶嘧磺隆抗性基因CdP450、花椰菜花叶病毒35s(CaMV35S)终止子组成，核苷酸序列如SEQ IDNO:17所示。
本发明所述将含α-淀粉酶表达框的表达载体导入到大豆基因组中的方法如农杆菌介导方法(Hinchee MW,Connor-Ward DV,Newell CAet al.Production of transgenicsoybean plants using Agrobacterium-mediated DNA transfer.NatureBiotechnology,1988,6(8):915-921)，基因枪方法等(Mccabe DE,Swain WF,MartinellBJ,et al.Stable transformation of Soybean(Glycine Max)by particleacceleration.Nature Biotechnology,1988,6(8):923-926)。这些方法都是成熟的方法，本领域的一般技术人员就能够利用这些方法获得转基因大豆。这些不同的方法都可以实现本发明的获得表达淀粉酶的转基因大豆的目的。比较常用的转化方法是农杆菌介导方法。这种方法的特点是构建一种T-DNA转化体载体，然后通过农杆菌介导将载体中的T-DNA导入到大豆基因组。
本发明还涉及一种表达载体构建的转基因植物细胞。
与现有技术相比，本发明有益效果主要体现在：(1)本发明转基因大豆在种子中实现淀粉酶的高表达，所生产的淀粉酶的活性极高，最高每克种子10000酶活单位以上(酶活单位定义为每小时产生1g的还原性末端所需的酶量)；(2)本发明的转基因大豆生产的淀粉酶在作为饲料添加剂、食品加工添加剂等很多领域的应用时无需经过分离浓缩，可以直接应用，降低了生产成本；(3)本发明利用大豆种子生物反应器生产淀粉酶比传统微生物发酵方法节省能源，是一种更加环境友好型的生产方式。
(四)附图说明
图1、用于大豆遗传转化的载体GM2S-AS-3X示意图。
图2、麦芽糖标准曲线。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
若未特别指明，以下实施例中所用生化试剂、材料均为市售均设置三次重复实验，结果取平均值。
实施例1、α-淀粉酶基因的合成
通过NCBI数据库，搜索不同来源的α-淀粉酶基因，由上海生工公司通过人工方法合成了6种α-淀粉酶基因并增强α-淀粉酶表达的元件和终止子：(1)来源于地衣芽孢杆菌(Bacillus Lichenifarmis)的淀粉酶(GenBank:WP_095325322.1)，其核苷酸序列如SEQ IDNO:5所示，氨基酸序列如SEQ ID NO:11所示；(2)来源于强烈火球菌(Pyrococcusfuriosus)的淀粉酶(GenBank:AAL80601.1)，其氨基酸序列如SEQ ID NO:6所示，氨基酸序列如SEQ ID NO:12所示；(3)来源于枯草芽孢杆菌(Bacillus subtilis)的淀粉酶(GenBank:ATQ64034.1)，其核苷酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:13所示；(4)来源于嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的淀粉酶(GenBank:ABH10675.1)，其核苷酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:14所示；(5)来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的淀粉酶(GenBank:WP_013352208.1)，其核苷酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:15所示；(6)来源于米曲霉菌(Aspergillus oryzae)的淀粉酶(GenBank:AAA32708.1)，其核苷酸序列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:16所示，再在各个α-淀粉酶基因5’端加上一个带有BamHI位点的烟草蚀纹病毒5'非翻译区(TEV 5’UTR，核苷酸序列如SEQ ID NO:18所示)和拟南芥2S白蛋白信号肽(At2S2 signalpeptide，核苷酸序列如SEQ ID NO:19所示)，在3’端加上一个带有KpnI位点的烟草Tobacco RB7核基质结合区调控序列基因(TobaccoRB7MAR，核苷酸序列如SEQ ID NO:20所示)和拟南芥heat shock protein 18.2基因(HSP,At5g59720)终止子(GenBank:LR782546.1)。
实施例2、构建大豆遗传转化载体
本发明实施例1合成的六种添加了增强α-淀粉酶表达的元件和终止子的α-淀粉酶编码基因的遗传转化载体的构建方式完全一致，各个遗传转化载体之间的区别仅在于α-淀粉酶编码基因的不同，以实施例1合成的核苷酸序列如SEQ ID NO:8所示的α-淀粉酶基因为例，按如下步骤构建：
(1)基础载体：本发明中的大豆遗传转化载体是基于pCambia1300(NCBI序列编号AF234296)载体改造后构建的。将原始pCambia1300上的潮霉素筛选基因hptII(位于两个XhoI位点之间)替换为从狗牙根克隆获得的啶嘧磺隆抗性基因CdP450(核苷酸序列如SEQID NO:21所示)，并用花椰菜花叶病毒35s(CaMV35S)启动子和终止子(GenBank:AY560326.1))控制抗除草剂基因CdP450。改造后的载体命名为p1300-CdP450。
(2)启动子：本发明所述的四种种子特异型启动子的获取方式完全一致，各个启动子之间的区别仅在于种子特异型启动子的编码基因的不同。根据NCBI上大豆2S球蛋白启动子GM2S(GLYMA_13G288100)序列设计特异性引物，以大豆品种Williams 82为基因组模板扩增GM2S启动子片段，引物序列如下：GM2S-F:5’-CAGATTAAACGACGCCGTTTCG-3’；GM2S-R:5’-GAAGTGAAGGTGGGGGCATTTATTG-3’。将PCR产物回收后与pGEM-T Easy载体连接，用EcoRI酶切鉴定，阳性质粒命名为pGEM-GM2S送上海生工进行测序，GM2S启动子核苷酸序列如SEQ IDNO:1所示。
(3)转化载体
用HindIII/KpnI双酶切载体p1300-CdP450；用HindIII/BamHI双酶切pGEM-GM2S质粒，回收GM2S启动子片段；用BamHI/KpnI双酶切实施例1由上海生工合成的增强α-淀粉酶表达的元件+SEQ ID NO:8所示编码α-淀粉酶+终止子的DNA序列。将上述三个片段过夜连接，将连接产物转入大肠杆菌TG1菌株中，经酶切鉴定正确的质粒命名为GM2S-AS-3X，该质粒的T-DNA序列为SEQ ID NO 17。将该质粒电击转入农杆菌EHA105感受态细胞，酶切鉴定正确后以甘油菌保存，获得重组农杆菌，用于大豆转化。
实施例3、转基因大豆的获得
大豆转化流程主要参照Paz的农杆菌介导的half-seed转化法(Paz MM,MartinezJC,Kalvig AB,et al.(2006),Improved cotyledonary node method using analternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation.Plant Cell Reports,25(3):206-213)。具体如下：
①大豆种子灭菌：挑选品质优良的大豆种子(品种：皖豆28)，放入干燥器里，用100mL质量浓度30％次氯酸钠水溶液和8mL纯盐酸反应所产生的氯气处理16小时。
②种子萌发：将步骤①灭过菌的大豆种子在无菌蒸馏水中浸泡16小时，获得萌发的大豆种子。
③农杆菌菌液的制备：将实施例2制备的重组农杆菌在含50mg/L Kan(卡那霉素)和25mg/L利福平的YEP固体培养基上划线，28℃培养2天。刮取YEP培养板上的菌落，均匀悬浮在侵染培养基中，将其浓度调整为OD650＝0.5-0.6。
④外植体的准备和侵染：将步骤②萌发的大豆种子沿着种脐纵向切割均匀切成两瓣，将带有胚轴和胚芽的那一瓣去掉种皮后迅速放入步骤③制备好的农杆菌菌液中，室温下侵染30min，取出放到衬有滤纸无菌培养皿中，封口后于22℃黑暗条件下共培养3天。
⑤芽诱导培养：步骤④共培养后的外植体转入芽诱导培养基中，于26℃、16h光照/8h黑暗条件下培养3周。
⑥芽伸长和筛选：将步骤⑤诱导后的外植体切掉子叶，带丛生芽的部分转到加入0.1mg/L啶嘧磺隆的芽伸长培养基中，于26℃、16h光照/8h黑暗条件下培养21天。
⑦生根和移苗：将步骤⑥伸长后的丛生芽转移到加入含0.025mg/L啶嘧磺隆的生根培养基生根。当根长出后，洗去根上附着的培养基，将T0代幼苗移栽到土壤中培养。单株植株成活2～3周后，用25％啶嘧磺隆水分散粒剂(购自郑州莱恩坪安生物科技有限责任公司)进行喷施，根据说明书喷施浓度为100mg/L，7天后叶片发黄，枯死的为阴性；和喷清水对照长势一样的为阳性植株。将阳性植株保留并编号，以便后续的鉴定和检测。筛选获得阳性株系，并分别记为ASW2、ASW5、ASW7、ASW15、ASW20。
实施例4、转基因大豆种子α-淀粉酶活性测定
α-淀粉酶活性的测定采用DNS法(Miller GL(1959).Using of dinitrosalicylicacid reagent for determination of reducing sugar.Analytical Chemistry,31:426-489)。其中3,5-二硝基水杨酸(DNS)溶液的配制如下：185g酒石酸钾溶于500mL的水中，45℃加热溶解后加入6.3g DNS以及262ml 2MNaOH水溶液，5g结晶酚，5g亚硫酸钠，搅拌溶解，冷却后用水定容至1000ml。麦芽糖标准溶液的配制及标准曲线的制作：用水配制1mg/ml的麦芽糖标准液，按表1加入不同的试剂，混合均匀后于沸水中煮5min，于540nm波长下测吸光度，以麦芽糖含量为横坐标，吸光度为纵坐标绘制标准曲线。
表1、麦芽糖标准曲线制作
酶液的配置：取实施例3表达α-淀粉酶的大豆种子(ASW2、ASW5、ASW7、ASW15、ASW20株系的T0代种子)每20mg加入1ml乙酸钠-氯化钠缓冲液(20mM乙酸钠，250mM氯化钠，溶剂为水，pH 5.4)，研磨后的混合液即为酶液。
底物准备：1g可溶性淀粉溶于5ml乙酸钠缓冲液(20mM乙酸钠，溶剂为水，pH 5.4)中，另取95ml乙酸钠缓冲液加热至沸腾，将溶解好的可溶性淀粉缓缓加入煮沸的乙酸钠缓冲液中，搅拌至透明。
酶活性的测定：将酶液用乙酸钠-氯化钠缓冲液(20mM乙酸钠，250mM氯化钠，溶剂为水，pH 5.4)稀释100体积倍后，取50μl加到450μl的底物中，65℃反应1小时后加入1mlDNS溶液终止反应，沸水中煮5min后测OD540，以在65℃每小时产生1克的还原性末端所需的酶量为一个酶单位(unit)，根据前面绘制的麦芽糖标准曲线计算酶活性。分别取鉴定为阳性的ASW2、ASW5、ASW7、ASW15、ASW20株系(通过载体GM2S-AS-3X转化获得的转化体)的T0代种子进行酶活测定，每个株系测定3枚种子，测定结果如表2所示，其中ASW7酶活最高，达到了10004unit/g。
表2、T0代种子进行酶活
实施例5、转基因大豆种子发芽率、百粒重测定
选择实施例3制备的3个表达淀粉酶较高的转基因大豆转化体ASW7、ASW15、ASW20种子中纯合子后代的T2和T3代大豆进行α-淀粉酶活性测定(同实施例4方法)，其α-淀粉酶的活性均在每克种子8000单位以上。并对ASW7、ASW15、ASW20种子中纯合子种子的T3代的百粒重和发芽率进行测量，采用SPSS对百粒重和发芽率分别进行单因素方差分析，采用T-test进行差异显著性分析。结果见表3，表达不同水平的淀粉酶表达量与非转基因大豆在发芽率和百粒重方面没有显著性差异(P＞0.05)。表明该性状能够稳定遗传且不会影响后代的发芽率，具有广阔的生产应用前景。
表3、不同种子的百粒重和发芽率
淀粉酶活性(单位/克)* 百粒重(g) 发芽率(％)
转化体1 8750 32.6a 94b
转化体2 5085 34.8a 90b
转化体3 11356 31.3a 95b
对照 574 35.3a 95b
*：相同小写字母表示这些转化体的百粒重和发芽率没有显著性差异(P＞0.05)。
活性单位的定义是为每小时产生1克的还原性末端所需的酶量。
序列表
<110> 浙江大学
<120> 一种利用转基因大豆生产α-淀粉酶的方法及表达载体
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<170> SIPOSequenceListing 1.0
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ctgtagaggg ggaccattca gggtgcctgc aattttggtg ttgttcatgt acggttgcag 1380
atataaacga agcatagctt atgtatgagg tgtaacaaaa ttggaaacaa tagccatgca 1440
aggtgaagaa tgtcaccaac tcagaaaccc ttcttcattg acgtgtccct cactcactct 1500
cctctcttca ctataaatcg ccactcttcg tgttctccac 1540
<210> 4
<211> 1476
<212> DNA
<213> 未知(Unknown)
<400> 4
aagcttattg tactcccagt atcattatag tgaaagtttt ggctctctcg ccggtggttt 60
tttacctcta tttaaagggg ttttccacct aaaaattctg gtatcattct cactttactt 120
gttactttaa tttctcataa tctttggttg aaattatcac gcttccgcac acgatatccc 180
tacaaattta ttatttgtta aacattttca aaccgcataa aattttatga agtcccgtct 240
atctttaatg tagtctaaca ttttcatatt gaaatatata atttacttaa ttttagcgtt 300
ggtagaaagc ataaagattt attcttattc ttcttcatat aaatgtttaa tatacaatat 360
aaacaaattc tttaccttaa gaaggatttc ccattttata ttttaaaaat atatttatca 420
aatatttttc aaccacgtaa atctcataat aataagttgt ttcaaaagta ataaaattta 480
actccataat ttttttattc gactgatctt aaagcaacac ccagtgacac aactagccat 540
ttttttcttt gaataaaaaa atccaattat cattgtattt tttttataca atgaaaattt 600
caccaaacaa tcatttgtgg tatttctgaa gcaagtcatg ttatgcaaaa ttctataatt 660
cccatttgac actacggaag taactgaaga tctgctttta catgcgagac acatcttcta 720
aagtaatttt aataatagtt actatattca agatttcata tatcaaatac tcaatattac 780
ttctaaaaaa ttaattagat ataattaaaa tattactttt ttaattttaa gtttaattgt 840
tgaatttgtg actattgatt tattattcta ctatgtttaa attgttttat agatagttta 900
aagtaaatat aagtaatgta gtagagtgtt agagtgttac cctaaaccat aaactataac 960
atttatggtg gactaatttt catatatttc ttattgcttt taccttttct tggtatgtaa 1020
gtccgtaact agaattacag tgggttgcca tggcactctg tggtcttttg gttcatgcat 1080
gggtcttgcg caagaaaaag acaaagaaca aagaaaaaag acaaaacaga gagacaaaac 1140
gcaatcacac aaccaactca aattagtcac tggctgatca agatcgccgc gtccatgtat 1200
gtctaaatgc catgcaaagc aacacgtgct taacatgcac tttaaatggc tcacccatct 1260
caacccacac acaaacacat tgcctttttc ttcatcatca ccacaaccac ctgtatatat 1320
tcattctctt ccgccacctc aatttcttca cttcaacaca cgtcaacctg catatgcgtg 1380
tcatcccatg cccaaatctc catgcatgtt ccaaccacct tctctcttat ataataccta 1440
taaatacctc taatatcact cacttctttc ggatcc 1476
<210> 5
<211> 1539
<212> DNA
<213> 地衣芽孢杆菌(Bacillus Lichenifarmis)
<400> 5
atgaaacaac aaaaacggct ttacgcccga ttgctgccgc tgttatttgc gctcatcttc 60
ttgctgcctc attctgcagc agcggcggca aatcttaaag ggacgctgat gcagtatttt 120
gaatggtaca tgcccaatga cggccaacat tggaagcgct tgcaaaacga ctcggcatat 180
ttggctgaac acggtattac tgccgtctgg attcccccgg catataaggg aacgagccaa 240
gcggatgtgg gctacggtgc ttacgacctt tatgatttag gggagtttca tcaaaaaggg 300
acggttcgga caaagtacgg cacaaaagga gagctgcaat ctgcgatcaa aagtcttcat 360
tcccgcgaca ttaacgttta cggggatgtg gtcatcaacc acaaaggcgg cgctgatgcg 420
accgaagatg taaccgcggt tgaagtcgat cccgctgacc gcaaccgcgt aatttcagga 480
gaacaccgaa ttaaagcctg gacacatttt cattttccgg ggcgcggcag cacatacagc 540
gattttaaat ggcattggta ccattttgac ggaaccgatt gggacgagtc ccgaaagctg 600
aaccgcatct ataagtttca aggaaaggct tgggattggg aagtttccaa tgaaaacggc 660
aactatgatt atttgatgta tgccgacatc gattatgacc atcctgatgt cgcagcagaa 720
attaagagat ggggcacttg gtatgccaat gaactgcaat tggacggttt ccgtcttgat 780
gctgtcaaac acattaaatt ttcttttttg cgggattggg ttaatcatgt cagggaaaaa 840
acggggaagg aaatgtttac ggtagctgaa tattggcaga atgacttggg cgcgctggaa 900
aactatttga acaaaacaaa ttttaatcat tcagtgtttg acgtgccgct tcattatcag 960
ttccatgctg catcgacaca gggaggcggc tatgatatga ggaaattgct gaacggtacg 1020
gtcgtttcca agcatccgtt gaaagcggtt acatttgtcg ataaccatga tacacagccg 1080
gggcaatcgc ttgagtcgac tgtccaaaca tggtttaagc cgcttgctta cgcttttatt 1140
ctcacaaggg aatctggata ccctcaggtt ttctacgggg atatgtacgg gacgaaagga 1200
gactcccagc gcgaaattcc tgccttgaaa cacaaaattg aaccgatctt aaaagcgaga 1260
aaacagtatg cgtacggagc acagcatgat tatttcgacc accatgacat tgtcggctgg 1320
acaagggaag gcgacagctc ggttgcaaat tcaggtttgg cggcattaat aacagacgga 1380
cccggtgggg caaagcgaat gtatgtcggc cggcaaaacg ccggtgagac atggcatgac 1440
attaccggaa accgttcgga gccggttgtc atcaattcgg aaggctgggg agagtttcac 1500
gtaaacggcg ggtcggtttc aatttatgtt caaagatag 1539
<210> 6
<211> 2627
<212> DNA
<213> 强烈火球菌(Pyrococcusfuriosus)
<400> 6
ttcttggtaa aacttcctct tcaagcatcg ccttcatttc tcttggcatg cctgggagca 60
caaagatttt agtgcctcta tattctatat aggccccagg tgctgcccca acagagtttt 120
tcaatggaat ggctcctttg ggaaggtaag ccatcttttt ccttgcatca ttcagcgtag 180
gatcatcaat tagcccctcc ctatataatc tctcataaaa ttcctttatt tcctctaagc 240
atttttcaca gagcactaat tcttttccca gcgccttagc gacggccacc attgtaacgt 300
catcgtgagt tggcccgagg cctcccgaaa taataagaac atcgggtttt ctgttaaggg 360
attctaaaat tacagacctt atatcctcca cgtcatcgcc aacagtagtt attctcttaa 420
cgaggtatcc tctctccgtt aatttcttcg ctatgtgtgc tgagttgctg tttacagtat 480
tccctgttaa aagttcatca cctacggtaa ttatttccgc gaacattggt tctccccagg 540
aattgttttt atcaagagtt tattagattt tgacgtgcgt tgatgaacat ttatgttcac 600
atgatcataa cagaaaattt atatgtatca tcaccagtga tacattatga gactttggtg 660
tatggaggtg atcacgtgaa cataaagaaa ttaacacccc tcctaactct attactgttt 720
tttatagtac tagcaagtcc agtaagtgca gcaaaatact tggagcttga agagggagga 780
gttataatgc aagcattcta ttgggatgtt ccagggggag gaatttggtg ggatcatata 840
agatcgaaga ttcctgaatg gtatgaagct ggaatctctg caatatggct acctccacca 900
agcaagggga tgagtggagg atattcaatg ggctacgatc cctatgatta ctttgatctc 960
ggcgagtact accagaaggg aactgtagag acgcgttttg gatcaaaaga agaactagtg 1020
agattgatac aaactgccca tgcctatgga ataaaggtaa tcgccgatgt agttataaac 1080
cacagggctg gtggtgacct agaatggaac cccttcgttg gagattacac atggacagac 1140
ttttctaaag ttgcctcagg gaaatataca gctaactatc tggacttcca tccaaacgag 1200
cttcattgtt gtgacgaagg aacctttgga ggatttccag atatatgtca tcacaaagag 1260
tgggatcagt actggctatg gaagagcaat gagagttatg ctgcttattt aagaagcata 1320
ggatttgatg gttggagatt tgactatgtt aagggctatg gagcttgggt tgtcagagac 1380
tggcttaatt ggtggggagg ttgggcagtt ggagagtact gggacacaaa tgtagatgca 1440
ctactaagct gggcatatga gagtggtgca aaggtctttg acttcccgct ctactataaa 1500
atggatgaag catttgacaa taacaacatt ccagcattag tctatgccct acaaaacgga 1560
caaactgtag tttcgagaga tccatttaag gcagtaactt tcgttgccaa tcatgacaca 1620
gatataatat ggaacaagta tccagcatat gcgttcatat tgacatatga gggacagcca 1680
gtaatattct acagggactt tgaggaatgg ctgaacaagg ataagctaat taacctcatt 1740
tggatccatg atcatttggc aggaggaagc acaacaattg tctactacga caacgatgag 1800
ctcatatttg tgagaaatgg agattctaga aggcctgggc ttataactta cattaacttg 1860
agccctaact gggttggtag gtgggtatac gttccaaagt ttgcaggggc ttgtattcat 1920
gaatacactg gaaacctagg aggatgggta gataaaagag tagatagtag cggatgggta 1980
tacctagagg caccacctca cgatccagct aacggctact atgggtactc cgtatggagt 2040
tattgtggtg ttgggtgact ttttcttttt tctttttaac aatgggagaa gtgcaaatac 2100
tgcgacaatt cctgggccgc atacaggagt ttctggagcg gcttcattca atattactgt 2160
tttatttcca attccataca acgtgacatt tgttggttgg agaggttccc agggaagtcc 2220
cttatatagt tgaactgtaa aagttttgtt tctggggagg ggaaccacgg caaagtagtg 2280
tcccgttgga gatttcaaga gggggtctcg gctctgagag ttctggtatg ttccccttga 2340
cccaaaggta accagtgtag tttccttgta ggactattag tgctgggata tcattgctag 2400
aatagtacct aagcttaacc tcattgcttc tagttccatt aacaacaacc caaacaggaa 2460
tccacgcatt catagtttta atctcgggaa cctctacaag gatggaattt tcttctttct 2520
caataattct tgctctccta cctcctatat acaccttgat gtttccattg aggcctgctc 2580
cggctataag gattttttgc ccaggttggg ctatgtatgg agttata 2627
<210> 7
<211> 1981
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 7
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gatgggatgt cacgcagaat 540
tcattgctcg ggctgtatga ctggaataca caaaatacac aagtacagtc ctatctgaaa 600
cggttcttag aaagggcatt gaatgacggg gcagacggtt ttcgatttga tgccgccaaa 660
catatagagc ttccagatga tggcagttac ggcagtcaat tttggccgaa tatcacaaat 720
acatctgcag agttccaata cggagaaatc ctgcaggata gtgcctccag agatgctgca 780
tatgcgaatt atatggatgt gacagcgtct aactatgggc attccataag gtccgcttta 840
aagaatcgta atctgggcgt gtcgaatatc tcccactatg catctgatgt gtctgcggac 900
aagctagtga catgggtaga gtcacatgat acgtatgcca atgatgatga agagtcgaca 960
tggatgagcg atgatgatat ccgtttaggc tgggcggtga tagcttctcg ttcaggcagt 1020
acgcctcttt tcttttccag acctgaggga ggcggaaatg gtgtgaggtt cccggggaaa 1080
agccaaatag gcgatcgcgg gagtgcttta tttgaagatc aggctatcac tgcggtcaat 1140
agatttcaca atgtgatggc tggacagcct gaggaactct cgaacccgaa tggaaacaac 1200
cagatattta tgaatcagcg cggctcacat ggcgttgtgc tggcaaatgc aggttcatcc 1260
tctgtctcta tcaatacggc aacaaaattg cctgatggca ggtatgacaa taaagctgga 1320
gcgggttcat ttgaagtgaa cgatggtaaa ctgacaggca cgatcaatgc caggtctgta 1380
gctgtgcttt atcctgatga tattgcaaaa gcgcctcatg ttttccttga gaattacaaa 1440
acaggtgtaa cacattcttt caatgatcaa ctgacgatta ccttgcgtgc agatgcgaat 1500
acaacaaaag ccgtttatca aatcaataat ggaccagaga cggcgtttaa ggatggagat 1560
caattcacaa tcggaaaagg agatccattt ggcaaaacat acaccatcat gttaaaagga 1620
acgaacagtg atggtgtaac gaggaccgag aaatacagtt ttgttaaaag agatccagcg 1680
tcggccaaaa ccatcggcta tcaaaatccg aatcattgga gccaggtaaa tgcttatatc 1740
tataaacatg atgggagccg agtaattgaa ttgaccggat cttggcctgg aaaaccaatg 1800
actaaaaatg cagacggaat ttacacgctg acgctgcctg cggacacgga tacaaccaac 1860
gcaaaagtga tttttaataa tggcagcgcc caagtgcccg gtcagaatca gcctggcttt 1920
gattacgtgc taaatggttt atataatgac tcgggcttaa gcgcttctct tcccgattga 1980
a 1981
<210> 8
<211> 1650
<212> DNA
<213> 嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)
<400> 8
gtgctaacgt ttcaccgcat cattcgaaaa ggatggatgt tcctgctcgc gtttttgctc 60
actgccttgc tgttctgccc aaccggacag cccgccaagg ctgccgcacc gtttaacggc 120
accatgatgc agtattttga atggtacttg ccggatgatg gcacgttatg gaccaaagtg 180
gccaatgaag ccaacaactt atccagcctt ggcatcaccg ctctttggct gccgcccgct 240
tacaaaggaa caagccgcag cgacgtaggg tacggagtat acgacttgta tgacctcggt 300
gaattcaatc aaaaaggggc cgtccgcaca aaatacggaa caaaagctca atatcttcaa 360
gccattcaag ccgcccacgc cgctggaatg caagtgtacg ccgatgtcgt gttcgaccat 420
aaaggcggcg ccgacggcac ggaatgggtg gacgccgtcg aagtcaatcc gtccgaccgc 480
aaccaagaaa tctcgggcac ctatcaaatc caagcatgga cgaaatttga ttttcccggg 540
cggggcaaca cctactccag ctttaagtgg cgctggtacc attttgatgg cgttgattgg 600
gacgaaagcc gaaaattgag ccgcatttac aaattccgcg gcatcggcaa agcgtgggat 660
tgggaagtag acacggaaaa cggaaactat gactacttaa tgtatgccga ccttgatatg 720
gatcatcccc aagtcgtgac tgagctgaaa agctggggga aatggtatgt caacacaacg 780
aacattgatg ggttcccgct tgatgccgtc aagcatatta agttcagttt tttccctgat 840
tgggtgtcgg acgtgcgttc tcagacggcc aagccgttat ttaccgtcgg ggagtatggg 900
agttatgaca tcaacaaatt tcacaattac tttatgaaaa caaacggaac gatgtctttg 960
tttgatgccc cgttacacaa caaattttat aacgctccca aatcaggggg cacatttgat 1020
atgcgcacgt taatgaccaa tactctcatg aaagatcaac caacattggc cgtcaccttc 1080
gttgataatc atgacaccga acccgcccaa gctccgcaat catgggtcgt cccatggttc 1140
aaaccgttgg cttacgcctt tattctaact cggcaggaag gatatccgtg cgtcttttat 1200
ggggactatt atggcattcc ccaatataac attccttcgc tgaaaagcaa aatcgatccg 1260
ctcctcatcg cgcgcaggga ttatgcttac ggaactcaac atgattatct tgatcactcc 1320
gacatcatcg ggtggacaag ggaaggggtc actgaaaaac caggatccgg actggccgca 1380
ttgatcaccg atgggccggg aggaagcaaa tggatgtacg ttggcaaaca acacgccgga 1440
aaagtgttct atgaccttac cggcaaccgg agtgacaccg tcaccatcaa cagtgatgga 1500
tggggggaat tcaaagtcaa tggcggttcg gtttcggtct gggttcctag aaaaacgacc 1560
gtttctacca tcactcggcc gatcacaacc cgaccgtgga ctggtgaatt cgtccgttgg 1620
accgaaccac ggttggtggc atggccttga 1650
<210> 9
<211> 2084
<212> DNA
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 9
gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg 60
ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc 120
atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat aaggaataaa 180
ggggggttgt tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg 240
agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc 300
acgctgttat ttgtcagttt gccgattaca aaaacatcag ccgtaaatgg cacgctgatg 360
cagtattttg aatggtatac gccgaacgac ggccagcatt ggaaacgatt gcagaatgat 420
gcggaacatt tatcggatat cggaatcact gccgtctgga ttcctcccgc atacaaagga 480
ttgagccaat ccgataacgg atacggacct tatgatttgt atgatttagg agaattccag 540
caaaaaggga cggtcagaac gaaatacggc acaaaatcag agcttcaaga tgcgatcggc 600
tcactgcatt cccggaacgt ccaagtatac ggagatgtgg ttttgaatca taaggctggt 660
gctgatgcaa cagaagatgt aactgccgtc gaagtcaatc cggccaatag aaatcaggaa 720
acttcggagg aatatcaaat caaagcgtgg acggattttc gttttccggg ccgtggaaac 780
acgtacagtg attttaaatg gcattggtat catttcgacg gagcggactg ggatgaatcc 840
cggaagatca gccgcatctt taagtttcgt ggggaaggaa aagcgtggga ttgggaagta 900
tcaagtgaaa acggcaacta tgactattta atgtatgctg atgttgacta cgaccaccct 960
gatgtcgtgg cagagacaaa aaaatggggt atctggtatg cgaatgaact gtcattagac 1020
ggcttccgta ttgatgccgc caaacatatt aaattttcat ttctgcgtga ttgggttcag 1080
gcggtcagac aggcgacggg aaaagaaatg tttacggttg cggagtattg gcagaataat 1140
gccgggaaac tcgaaaacta cttgaataaa acaagcttta atcaatccgt gtttgatgtt 1200
ccgcttcatt tcaatttaca ggcggcttcc tcacaaggag gcggatatga tatgaggcgt 1260
ttgctggacg gtaccgttgt gtccaggcat ccggaaaagg cggttacatt tgttgaaaat 1320
catgacacac agccgggaca gtcattggaa tcgacagtcc aaacttggtt taaaccgctt 1380
gcatacgcct ttattttgac aagagaatcc ggttatcctc aggtgttcta tggggatatg 1440
tacgggacaa aagggacatc gccaaaggaa attccctcac tgaaagataa tatagagccg 1500
attttaaaag cgcgtaagga gtacgcatac gggccccagc acgattatat tgaccacccg 1560
gatgtgatcg gatggacgag ggaaggtgac agctccgccg ccaaatcagg tttggccgct 1620
ttaatcacgg acggacccgg cggatcaaag cggatgtatg ccggcctgaa aaatgccggc 1680
gagacatggt atgacataac gggcaaccgt tcagatactg taaaaatcgg atctgacggc 1740
tggggagagt ttcatgtaaa cgatgggtcc gtctccattt atgttcagaa ataaggtaat 1800
aaaaaaacac ctccaagctg agtgcgggta tcagcttgga ggtgcgttta ttttttcagc 1860
cgtatgacaa ggtcggcatc aggtgtgaca aatacggtat gctggctgtc ataggtgaca 1920
aatccgggtt ttgcgccgtt tggctttttc acatgtctga tttttgtata atcaacaggc 1980
acggagccgg aatctttcgc cttggaaaaa taagcggcga tcgtagctgc ttccaatatg 2040
gattgttcat cgggatcgct gcttttaatc acaacgtggg atcc 2084
<210> 10
<211> 3366
<212> DNA
<213> 米曲霉(Aspergillus oryzae)
<400> 10
ttcatggtgt tttgatcatt ttaaattttt atatggcggg tggtgggcaa ctcgcttgcg 60
cgggcaactc gcttaccgat tacgttaggg ctgatattta cgtaaaaatc gtcaagggat 120
gcaagaccaa agtagtaaaa ccccggagtc aacagcatcc aagcccaagt ccttcacgga 180
gaaaccccag cgtccacatc acgagcgaag gaccacctct aggcatcgga cgcaccatcc 240
aattagaagc agcaaagcga aacagcccaa gaaaaaggtc ggcccgtcgg ccttttctgc 300
aacgctgatc acgggcagcg atccaaccaa caccctccag agtgactagg ggcggaaatt 360
taaagggatt aatttccact caaccacaaa tcacagtcgt ccccggtatt gtcctgcaga 420
atgcaattta aactcttctg cgaatcgctt ggattccccg cccctggccg tagagcttaa 480
agtatgtccc ttgtcgatgc gatgtatcac aacatataaa tactagcaag ggatgccatg 540
cttggaggat agcaaccgac aacatcacat caagctctcc cttctctgaa caataaaccc 600
cacagaaggc atttatgatg gtcgcgtggt ggtctctatt tctgtacggc cttcaggtcg 660
cggcacctgc tttggctgca acgcctgcgg actggcgatc gcaatccatt tatttccttc 720
tcacggatcg atttgcaagg acggatgggt cgacgactgc gacttgtaat actgcggatc 780
aggtgtgttg ttacctacta gctttcagaa agaggaatgt aaactgactt gatatagaaa 840
tactgtggtg gaacatggca gggcatcatc gacaaggtaa attgcccctt tatcaaaaaa 900
aaagaaggaa aagcagaaga aaaataaaat aaaaagaact ctagtcctaa ccatcacata 960
gttggactat atccagggaa tgggcttcac agccatctgg atcacccccg ttacagccca 1020
gctgccccag accaccgcat atggagatgc ctaccatggc tactggcagc aggatatgta 1080
agtcgatttc tttaaatatc tacctgtcat cttttacatc aatatgaact aacttgatgg 1140
ttttagatac tctctgaacg aaaactacgg cactgcagat gacttgaagg cgctctcttc 1200
ggcccttcat gagaggggga tgtatcttat ggtcgatgtg gttgctaacc atatggttcg 1260
tggtcctttg caactgactt cgcggatatg gttcatttca gtactgacaa tgagtaatat 1320
cagggctatg atggagcggg tagctcagtc gattacagtg tgtttaaacc gttcagttcc 1380
caagactact tccacccgtt ctgtttcatt caaaactatg aagatcagac tcaggttgag 1440
gattgctggc taggagataa cactgtctcc ttgcctgatc tcgataccac caaggatgtg 1500
gtcaagaatg aatggtacga ctgggtggga tcattggtat cgaactactc cagtaagata 1560
tttctccctc attctacaac ttggctgatc gatgatactt acgaaatcag ttgacggcct 1620
ccgtatcgac acagtaaaac acgtccagaa ggacttctgg cccgggtaca acaaagccgc 1680
aggcgtgtac tgtatcggcg aggtgctcga cggtgatccg gcctacactt gtccctacca 1740
gaacgtcatg gacggcgtac tgaactatcc catgtatggt tcctccaacc atgagccttc 1800
ttgcaagtct catctcctaa cgaaacggct aaaaccagtt actatccact cctcaacgcc 1860
ttcaagtcaa cctccggcag catggacgac ctctacaaca tgatcaacac cgtcaaatcc 1920
gactgtccag actcaacact cctgggcaca ttcgtcgaga accacgacaa cccacggttc 1980
gcttcgtaag tcttcccttt tattttccgt tcccaatttc cacacagaac cccacctaac 2040
aagagcaaag ttacaccaac gacatagccc tcgccaagaa cgtcgcagca ttcatcatcc 2100
tcaacgacgg aatccccatc atctacgccg gccaagaaca gcactacgcc ggcggaaacg 2160
accccgcgaa ccgcgaagca acctggctct cgggctaccc gaccgacagc gagctgtaca 2220
agttaattgc ctccgcgaac gcaatccgga actatgccat tagcaaagat acaggattcg 2280
tgacctacaa ggtaagcaca acctctaagc ataccctaat ggcctatctt cagagtatct 2340
gacacaagag actaatcact ggcaatacag aactggccca tctacaaaga cgacacaacg 2400
atcgccatgc gcaagggcac agatgggtcg cagatcgtga ctatcttgtc caacaagggt 2460
gcttcgggtg attcgtatac cctctccttg agtggtgcgg gttacacagc cggccagcaa 2520
ttgacggagg tcattggctg cacgaccgtg acggttggtt cggatggaaa tgtgcctgtt 2580
cctatggcag gtgggctacc tagggtattg tatccgactg agaagttggc aggtagcaag 2640
atctgtagta gctcgtgaag ggtggagagt atatgatggt actgctattc aatctggcat 2700
tggacagtga gtttgagttt gatgtacagt tggagtcgtt actgctgtca tccccttata 2760
ctcttcgatt gtttttcgaa ccctaatgcc aagcacgcta gtctattata ggaaaggatc 2820
cggattaatg tgttttcata acgcggtact gtatggtact tctgtattat atcaccgaag 2880
ctcatgtatc ttacatgtat atattataca gacacaacct tggttacccc accatgatgt 2940
ttcctgcaga taatctcctg acgatcaatc ttaccacagg gatatgatgg cacccaacct 3000
ggcgccttcg caacatcaaa ttatttgtga tatgatcaag tgcgatccat cacttactaa 3060
tgcccagata gctgaagctg ctaactgcag cacacgcgca attcctagga ttcggtcaaa 3120
tctccggcta ttcggcagta gcaaagcccc tccaaataaa ggtggacgcc cacgaagcat 3180
ctcaccaata atgctggagg ctctttgtga tcatcttctt gaaaagcctg atctatacct 3240
tgacgaaatg gccatctttc tatgggatga gttccaaata tacgcaacta catctagtat 3300
caggcgggct ctgtcttcta aaggttggtc caaaaaggca gctcggcaga aagcaaagga 3360
acggaa 3366
<210> 11
<211> 512
<212> PRT
<213> 地衣芽孢杆菌(Bacillus Lichenifarmis)
<400> 11
Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe
1 5 10 15
Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu
20 25 30
Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro Asn Asp Gly
35 40 45
Gln His Trp Lys Arg Leu Gln Asn Asp Ser Ala Tyr Leu Ala Glu His
50 55 60
Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln
65 70 75 80
Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe
85 90 95
His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu
100 105 110
Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr Gly
115 120 125
Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp Val
130 135 140
Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser Gly
145 150 155 160
Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg Gly
165 170 175
Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr
180 185 190
Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln Gly
195 200 205
Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp Tyr
210 215 220
Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Ala Ala Glu
225 230 235 240
Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp Gly
245 250 255
Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg Asp
260 265 270
Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr Val
275 280 285
Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu Asn
290 295 300
Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr Gln
305 310 315 320
Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys Leu
325 330 335
Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr Phe
340 345 350
Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val
355 360 365
Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu
370 375 380
Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly
385 390 395 400
Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro Ile
405 410 415
Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr Phe
420 425 430
Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser Val
435 440 445
Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ala
450 455 460
Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His Asp
465 470 475 480
Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly Trp
485 490 495
Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210> 12
<211> 460
<212> PRT
<213> 强烈火球菌(Pyrococcusfuriosus)
<400> 12
Met Asn Ile Lys Lys Leu Thr Pro Leu Leu Thr Leu Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Ala Ser Pro Val Ser Ala Ala Lys Tyr Leu Glu Leu Glu
20 25 30
Glu Gly Gly Val Ile Met Gln Ala Phe Tyr Trp Asp Val Pro Gly Gly
35 40 45
Gly Ile Trp Trp Asp His Ile Arg Ser Lys Ile Pro Glu Trp Tyr Glu
50 55 60
Ala Gly Ile Ser Ala Ile Trp Leu Pro Pro Pro Ser Lys Gly Met Ser
65 70 75 80
Gly Gly Tyr Ser Met Gly Tyr Asp Pro Tyr Asp Tyr Phe Asp Leu Gly
85 90 95
Glu Tyr Tyr Gln Lys Gly Thr Val Glu Thr Arg Phe Gly Ser Lys Glu
100 105 110
Glu Leu Val Arg Leu Ile Gln Thr Ala His Ala Tyr Gly Ile Lys Val
115 120 125
Ile Ala Asp Val Val Ile Asn His Arg Ala Gly Gly Asp Leu Glu Trp
130 135 140
Asn Pro Phe Val Gly Asp Tyr Thr Trp Thr Asp Phe Ser Lys Val Ala
145 150 155 160
Ser Gly Lys Tyr Thr Ala Asn Tyr Leu Asp Phe His Pro Asn Glu Leu
165 170 175
His Cys Cys Asp Glu Gly Thr Phe Gly Gly Phe Pro Asp Ile Cys His
180 185 190
His Lys Glu Trp Asp Gln Tyr Trp Leu Trp Lys Ser Asn Glu Ser Tyr
195 200 205
Ala Ala Tyr Leu Arg Ser Ile Gly Phe Asp Gly Trp Arg Phe Asp Tyr
210 215 220
Val Lys Gly Tyr Gly Ala Trp Val Val Arg Asp Trp Leu Asn Trp Trp
225 230 235 240
Gly Gly Trp Ala Val Gly Glu Tyr Trp Asp Thr Asn Val Asp Ala Leu
245 250 255
Leu Ser Trp Ala Tyr Glu Ser Gly Ala Lys Val Phe Asp Phe Pro Leu
260 265 270
Tyr Tyr Lys Met Asp Glu Ala Phe Asp Asn Asn Asn Ile Pro Ala Leu
275 280 285
Val Tyr Ala Leu Gln Asn Gly Gln Thr Val Val Ser Arg Asp Pro Phe
290 295 300
Lys Ala Val Thr Phe Val Ala Asn His Asp Thr Asp Ile Ile Trp Asn
305 310 315 320
Lys Tyr Pro Ala Tyr Ala Phe Ile Leu Thr Tyr Glu Gly Gln Pro Val
325 330 335
Ile Phe Tyr Arg Asp Phe Glu Glu Trp Leu Asn Lys Asp Lys Leu Ile
340 345 350
Asn Leu Ile Trp Ile His Asp His Leu Ala Gly Gly Ser Thr Thr Ile
355 360 365
Val Tyr Tyr Asp Asn Asp Glu Leu Ile Phe Val Arg Asn Gly Asp Ser
370 375 380
Arg Arg Pro Gly Leu Ile Thr Tyr Ile Asn Leu Ser Pro Asn Trp Val
385 390 395 400
Gly Arg Trp Val Tyr Val Pro Lys Phe Ala Gly Ala Cys Ile His Glu
405 410 415
Tyr Thr Gly Asn Leu Gly Gly Trp Val Asp Lys Arg Val Asp Ser Ser
420 425 430
Gly Trp Val Tyr Leu Glu Ala Pro Pro His Asp Pro Ala Asn Gly Tyr
435 440 445
Tyr Gly Tyr Ser Val Trp Ser Tyr Cys Gly Val Gly
450 455 460
<210> 13
<211> 659
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 13
Met Phe Ala Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly
1 5 10 15
Phe Leu Leu Leu Phe His Leu Val Leu Ala Gly Pro Ala Ala Ala Ser
20 25 30
Ala Glu Thr Ala Asn Lys Ser Asn Glu Leu Thr Ala Pro Ser Ile Lys
35 40 45
Ser Gly Thr Ile Leu His Ala Trp Asn Trp Ser Phe Asn Thr Leu Lys
50 55 60
His Asn Met Lys Asp Ile His Asp Ala Gly Tyr Thr Ala Ile Gln Thr
65 70 75 80
Ser Pro Ile Asn Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met
85 90 95
Ser Asn Trp Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn
100 105 110
Arg Tyr Leu Gly Thr Glu Gln Glu Phe Lys Glu Met Cys Ala Ala Ala
115 120 125
Glu Glu Tyr Gly Ile Lys Val Ile Val Asp Ala Val Ile Asn His Thr
130 135 140
Thr Ser Asp Tyr Ala Ala Ile Ser Asn Glu Val Lys Ser Ile Pro Asn
145 150 155 160
Trp Thr His Gly Asn Thr Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp
165 170 175
Val Thr Gln Asn Ser Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn
180 185 190
Thr Gln Val Gln Ser Tyr Leu Lys Arg Phe Leu Glu Arg Ala Leu Asn
195 200 205
Asp Gly Ala Asp Gly Phe Arg Phe Asp Ala Ala Lys His Ile Glu Leu
210 215 220
Pro Asp Asp Gly Ser Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn
225 230 235 240
Thr Ser Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser
245 250 255
Arg Asp Ala Ala Tyr Ala Asn Tyr Met Asp Val Thr Ala Ser Asn Tyr
260 265 270
Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn Leu Gly Val Ser
275 280 285
Asn Ile Ser His Tyr Ala Ser Asp Val Ser Ala Asp Lys Leu Val Thr
290 295 300
Trp Val Glu Ser His Asp Thr Tyr Ala Asn Asp Asp Glu Glu Ser Thr
305 310 315 320
Trp Met Ser Asp Asp Asp Ile Arg Leu Gly Trp Ala Val Ile Ala Ser
325 330 335
Arg Ser Gly Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly
340 345 350
Asn Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser
355 360 365
Ala Leu Phe Glu Asp Gln Ala Ile Thr Ala Val Asn Arg Phe His Asn
370 375 380
Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn
385 390 395 400
Gln Ile Phe Met Asn Gln Arg Gly Ser His Gly Val Val Leu Ala Asn
405 410 415
Ala Gly Ser Ser Ser Val Ser Ile Asn Thr Ala Thr Lys Leu Pro Asp
420 425 430
Gly Arg Tyr Asp Asn Lys Ala Gly Ala Gly Ser Phe Glu Val Asn Asp
435 440 445
Gly Lys Leu Thr Gly Thr Ile Asn Ala Arg Ser Val Ala Val Leu Tyr
450 455 460
Pro Asp Asp Ile Ala Lys Ala Pro His Val Phe Leu Glu Asn Tyr Lys
465 470 475 480
Thr Gly Val Thr His Ser Phe Asn Asp Gln Leu Thr Ile Thr Leu Arg
485 490 495
Ala Asp Ala Asn Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn Gly Pro
500 505 510
Glu Thr Ala Phe Lys Asp Gly Asp Gln Phe Thr Ile Gly Lys Gly Asp
515 520 525
Pro Phe Gly Lys Thr Tyr Thr Ile Met Leu Lys Gly Thr Asn Ser Asp
530 535 540
Gly Val Thr Arg Thr Glu Lys Tyr Ser Phe Val Lys Arg Asp Pro Ala
545 550 555 560
Ser Ala Lys Thr Ile Gly Tyr Gln Asn Pro Asn His Trp Ser Gln Val
565 570 575
Asn Ala Tyr Ile Tyr Lys His Asp Gly Ser Arg Val Ile Glu Leu Thr
580 585 590
Gly Ser Trp Pro Gly Lys Pro Met Thr Lys Asn Ala Asp Gly Ile Tyr
595 600 605
Thr Leu Thr Leu Pro Ala Asp Thr Asp Thr Thr Asn Ala Lys Val Ile
610 615 620
Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe
625 630 635 640
Asp Tyr Val Leu Asn Gly Leu Tyr Asn Asp Ser Gly Leu Ser Ala Ser
645 650 655
Leu Pro Asp
<210> 14
<211> 537
<212> PRT
<213> 嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)
<400> 14
Met Ala Ser Ile Asn Arg Pro Ile Val Phe Phe Thr Val Cys Leu Phe
1 5 10 15
Leu Leu Cys Asp Gly Ser Leu Ala Ala Ala Ala Pro Phe Asn Gly Thr
20 25 30
Met Met Gln Tyr Phe Glu Trp Tyr Leu Pro Asp Asp Gly Thr Leu Trp
35 40 45
Thr Lys Val Ala Asn Glu Ala Asn Asn Leu Ser Ser Leu Gly Ile Thr
50 55 60
Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly Thr Ser Arg Ser Asp Val
65 70 75 80
Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ala Gln Tyr Leu Gln Ala
100 105 110
Ile Gln Ala Ala His Ala Ala Gly Met Gln Val Tyr Ala Asp Val Val
115 120 125
Phe Asp His Lys Gly Gly Ala Asp Gly Thr Glu Trp Val Asp Ala Val
130 135 140
Glu Val Asn Pro Ser Asp Arg Asn Gln Glu Ile Ser Gly Thr Tyr Gln
145 150 155 160
Ile Gln Ala Trp Thr Lys Phe Asp Phe Asn Gly Arg Gly Asn Thr Tyr
165 170 175
Ser Ser Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Val Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Ser Arg Ile Tyr Lys Phe Arg Gly Ile Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Asp Thr Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Leu Asp Met Asp His Pro Glu Val Val Thr Glu Leu
225 230 235 240
Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr Thr Asn Ile Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Pro Asp Trp
260 265 270
Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Pro Leu Phe Thr Val Gly
275 280 285
Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu His Asn Tyr Ile Thr Lys
290 295 300
Thr Asn Gly Thr Met Ser Leu Phe Asp Ala Pro Leu His Asn Lys Phe
305 310 315 320
Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe Asp Met Ser Thr Leu Met
325 330 335
Asn Asn Thr Leu Met Lys Asp Gln Pro Thr Leu Ala Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Glu Pro Gly Gln Ala Leu Gln Ser Trp Val Asp
355 360 365
Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Gln Glu
370 375 380
Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Gln Tyr
385 390 395 400
Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp Pro Leu Leu Ile Ala Arg
405 410 415
Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp Tyr Leu Asp His Ser Asp
420 425 430
Ile Ile Gly Trp Thr Arg Glu Gly Val Thr Glu Lys Pro Gly Ser Gly
435 440 445
Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys Trp Met Tyr
450 455 460
Val Gly Lys Gln His Ala Gly Lys Val Phe Tyr Asp Leu Thr Gly Asn
465 470 475 480
Arg Ser Asp Thr Val Thr Ile Asn Ser Asp Gly Trp Gly Glu Phe Lys
485 490 495
Val Asn Gly Gly Ser Val Ser Val Trp Val Pro Arg Lys Thr Thr Val
500 505 510
Ser Thr Ile Ala Trp Pro Ile Thr Thr Arg Pro Trp Thr Gly Glu Phe
515 520 525
Val Arg Trp Thr Glu Pro Arg Leu Val
530 535
<210> 15
<211> 659
<212> PRT
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 15
Met Phe Ala Lys Arg Phe Lys Thr Pro Leu Leu Pro Leu Phe Ala Gly
1 5 10 15
Phe Leu Leu Leu Phe His Pro Val Leu Ala Gly Pro Ala Ala Ala Ser
20 25 30
Ala Glu Thr Ala Asn Lys Ser Asn Glu Leu Thr Ala Pro Ser Ile Lys
35 40 45
Ser Gly Thr Ile Leu His Ala Trp Asn Trp Ser Phe Asn Thr Leu Lys
50 55 60
His Asn Met Lys Asp Ile His Asp Ala Gly Tyr Thr Ala Ile Gln Thr
65 70 75 80
Ser Pro Ile Asn Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met
85 90 95
Ser Asn Trp Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn
100 105 110
Arg Tyr Leu Gly Thr Glu Gln Glu Phe Lys Glu Met Cys Ala Ala Ala
115 120 125
Glu Glu Tyr Gly Ile Lys Val Ile Val Asp Ala Val Ile Asn His Thr
130 135 140
Thr Ser Asp Tyr Ala Ala Ile Ser Asn Glu Val Lys Ser Ile Pro Asn
145 150 155 160
Trp Thr His Gly Asn Thr Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp
165 170 175
Val Thr Gln Asn Ser Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn
180 185 190
Thr Gln Val Gln Ser Tyr Leu Lys Arg Phe Leu Glu Arg Ala Leu Asn
195 200 205
Asp Gly Ala Asp Gly Phe Arg Phe Asp Ala Ala Lys His Ile Glu Leu
210 215 220
Pro Asp Asp Gly Ser Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn
225 230 235 240
Thr Ala Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser
245 250 255
Arg Asp Ala Ala Tyr Ala Asn Tyr Met Asn Val Thr Ala Ser Asn Tyr
260 265 270
Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn Leu Gly Val Ser
275 280 285
Asn Ile Ser His Tyr Ala Ser Asp Val Ser Ala Asp Lys Leu Val Thr
290 295 300
Trp Val Glu Ser His Asp Thr Tyr Ala Asn Asp Asp Glu Glu Ser Thr
305 310 315 320
Trp Met Ser Asp Asp Asp Ile Arg Leu Gly Trp Ala Val Ile Ala Ser
325 330 335
Arg Ser Gly Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly
340 345 350
Asn Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser
355 360 365
Ala Leu Phe Glu Asp Gln Ala Ile Thr Ala Val Asn Arg Phe His Asn
370 375 380
Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn
385 390 395 400
Gln Ile Phe Met Asn Gln Arg Gly Ser His Gly Val Val Leu Ala Asn
405 410 415
Ala Gly Ser Ser Ser Val Ser Ile Asn Thr Ala Thr Lys Leu Pro Asp
420 425 430
Gly Arg Tyr Asp Asn Lys Ala Gly Ala Gly Ser Phe Gln Val Asn Asp
435 440 445
Gly Lys Leu Thr Gly Thr Ile Asn Ala Arg Ser Val Ala Val Leu Tyr
450 455 460
Pro Asp Asp Ile Ala Lys Ala Pro His Val Phe Leu Glu Asn Tyr Lys
465 470 475 480
Thr Gly Val Thr His Ser Phe Asn Asp Gln Leu Thr Ile Thr Leu Arg
485 490 495
Ala Asp Ala Asn Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn Gly Pro
500 505 510
Glu Thr Ala Phe Lys Asp Gly Asp Gln Phe Thr Ile Gly Lys Gly Asp
515 520 525
Pro Phe Gly Lys Thr Tyr Thr Ile Met Leu Lys Gly Thr Asn Ser Asp
530 535 540
Gly Val Thr Arg Thr Glu Glu Tyr Ser Phe Ile Lys Arg Asp Pro Ala
545 550 555 560
Ser Ala Lys Thr Ile Gly Tyr Gln Asn Pro Asn His Trp Ser Gln Val
565 570 575
Asn Ala Tyr Ile Tyr Lys His Asp Gly Gly Gln Ala Ile Glu Leu Thr
580 585 590
Gly Ser Trp Pro Gly Lys Pro Met Thr Lys Asn Ala Asp Gly Ile Tyr
595 600 605
Thr Leu Thr Leu Pro Ala Asp Thr Asp Thr Thr Asn Ala Lys Val Ile
610 615 620
Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe
625 630 635 640
Asp Tyr Val Gln Asn Gly Leu Tyr Asn Asp Ser Gly Leu Ser Gly Ser
645 650 655
Leu Pro His
<210> 16
<211> 498
<212> PRT
<213> 米曲霉(Aspergillus oryzae)
<400> 16
Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala Ala
1 5 10 15
Pro Ala Leu Ala Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr
20 25 30
Phe Leu Leu Thr Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala
35 40 45
Thr Cys Asn Thr Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly
50 55 60
Ile Ile Asp Lys Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile
65 70 75 80
Trp Ile Thr Pro Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly
85 90 95
Asp Ala Tyr His Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu
100 105 110
Asn Tyr Gly Thr Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His
115 120 125
Glu Arg Gly Met Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly
130 135 140
Tyr Asp Gly Ala Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe
145 150 155 160
Ser Ser Gln Asp Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu
165 170 175
Asp Gln Thr Gln Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser
180 185 190
Leu Pro Asp Leu Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr
195 200 205
Asp Trp Val Gly Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg
210 215 220
Ile Asp Thr Val Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn
225 230 235 240
Lys Ala Ala Gly Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro
245 250 255
Ala Tyr Thr Cys Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr
260 265 270
Pro Ile Tyr Tyr Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser
275 280 285
Met Asp Asp Leu Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro
290 295 300
Asp Ser Thr Leu Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg
305 310 315 320
Phe Ala Ser Tyr Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala
325 330 335
Phe Ile Ile Leu Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu
340 345 350
Gln His Tyr Ala Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp
355 360 365
Leu Ser Gly Tyr Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser
370 375 380
Ala Asn Ala Ile Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val
385 390 395 400
Thr Tyr Lys Asn Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met
405 410 415
Arg Lys Gly Thr Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys
420 425 430
Gly Ala Ser Gly Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr
435 440 445
Thr Ala Gly Gln Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr
450 455 460
Val Gly Ser Asp Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro
465 470 475 480
Arg Val Leu Tyr Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser
485 490 495
Ser Ser
<210> 17
<211> 8090
<212> DNA
<213> 未知(Unknown)
<400> 17
taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta 60
aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc 120
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 180
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 240
cgacgttgta aaacgacggc cagtgccaag cttcagatta aacgacgccg tttcgttcct 300
ttcgcttcac ggcttaacga tgtcgtttct gtctgtgccc aaaaaataaa ggcatttgtt 360
atttgcacca gatatttact aagtgcaccc tagtttgaca agtaggcgat aattacaaat 420
agatgcggtg caaataataa attttgaagg aaataattac aaaagaacag aacttatatt 480
tactttattt taaaaaacta aaatgaaaga acaaaaaaaa gtaaaaaata caaaaaatgt 540
gctttaacca ctttcattat ttgttacaga aagtatgatt ctactcaaat tgatctgttg 600
tatctggtgc tgccttgtca cactggcgat ttcaatcccc taaagatatg gtgcaaactg 660
cgaagtgatc aatatctgct cggttaattt agattaatta ataatattca acgtgatgta 720
ccaaaaaaag acaatttttt gctccattga caaattaaac ctcatcaagg taatttccaa 780
acctataagc aaaaaaattt cacattaatt ggcccgcaat cctattagtc ttattatact 840
agagtaggaa aaaaaacaat tacacaactt gtcttattat tctctatgct aatgaatatt 900
tttccctttt gttagaaatc agtgtttcct aatttattga gtattaattc cactcaccgc 960
atatatttac cgttgaataa gaaaatttta cacataattc tttttaaaat aaataatttt 1020
tttatactag atcttatatg attacgtgaa gccaagtggg ttatactaat gatatataat 1080
gtttgatagt aatcagttta taaaccaaat gcatggaaat gttacgtgga agcacgtaaa 1140
ttaacaagca ttgaagcaaa tgcagccacc gcaccaaaac caccccactt cacttccacg 1200
taccatattc catgcaacta caacacccta aaacttcaat aaatgccccc accttcactt 1260
ctcaacacaa catatacaaa acaaacgaat ctcaagcaat caagcattct acttctattg 1320
cagcaattta aatcatttct tttaaagcaa aagcaatttt ctgaaaattt tcaccattta 1380
cgaacgatag ccatggctaa caagctcttc cttgtttgcg ctactttcgc tctttgcttc 1440
cttcttacta acgctgcagc tgcaccgttc aacggcacca tgatgcagta cttcgagtgg 1500
tatctgccgg acgacggcac cctgtggacc aaggtggcca acgaggccaa caacctgagc 1560
agcctgggca tcaccgctct gtggctgcct ccagcctaca agggcaccag caggagcgac 1620
gtgggctacg gcgtgtacga cctgtacgac ctgggcgagt tcaaccagaa gggcaccgtg 1680
aggaccaagt acggcaccaa ggcccagtac cttcaggcca tccaggctgc ccacgcagcc 1740
ggcatgcagg tgtacgccga cgtggtgttc gaccacaagg gtggcgcaga cggcaccgag 1800
tgggtggacg ccgtggaggt gaacccgagc gacaggaacc aggagatcag cggcacctac 1860
cagatccagg cctggaccaa gttcgacttc aacggcaggg gcaacaccta cagcagcttc 1920
aagtggaggt ggtatcactt cgacggcgtg gactgggacg agagcaggaa gctgagcagg 1980
atctacaagt tcaggggcat cggcaaggcc tgggactggg aggtggacac cgagaacggc 2040
aactacgact acctgatgta cgccgacctg gacatggacc acccggaggt ggtgaccgag 2100
ctgaagaact ggggcaagtg gtacgtgaac accaccaaca tcgacggctt caggctggac 2160
gccgtgaagc acatcaagtt cagcttcttc ccggactggc tgagctacgt gaggagccag 2220
accggcaagc cgctgttcac cgtgggcgag tactggagct acgacatcaa caagctgcac 2280
aactacatca ccaagaccaa cggcaccatg agcctgttcg acgctccgct gcacaacaag 2340
ttctacaccg ccagcaagag cggtggagcc ttcgacatga gcaccctgat gaacaacacc 2400
ctgatgaagg accagccgac cctggccgtg accttcgtgg acaaccacga caccgagcca 2460
ggccaggccc tgcagagctg ggtggacccg tggttcaagc cgctggccta cgccttcatc 2520
ctgaccaggc aggagggcta cccgtgcgtg ttctacggcg actactacgg catcccgcag 2580
tacaacatcc cgagcctgaa gagcaagatc gacccgctgc tgatcgccag gagggactac 2640
gcctacggca cccagcacga ctacctggac cacagcgaca tcatcggctg gaccagggag 2700
ggcgtgaccg agaagcctgg cagcggcctg gctgccctga tcaccgacgg tccgggaggc 2760
agcaagtgga tgtacgtggg caagcagcac gctggcaagg tgttctacga cctgaccggc 2820
aacaggagcg acaccgtgac catcaacagc gacggctggg gcgagttcaa ggtgaacggt 2880
ggcagcgtga gcgtgtgggt gccgaggaag accaccgtga gcaccatcgc ctggccgatc 2940
accaccaggc cgtggaccgg cgagttcgtg aggtggaccg agccgaggct ggtggcctgg 3000
ccgcgacata tgaagatgaa gatgaaatat ttggtgtgtc aaataaaaag ctagtgtgct 3060
taagtttgtg tttttttctt ggcttgttgt gttatgaatt tgtggctttt tctaatatta 3120
aatgaatgta agatctcatt ataatgaata aacaaatgtt tctataatcc attgtgaatg 3180
ttttgttgga tctcttctgc agcatataac tactgtatgt gctatggtat ggactatgga 3240
atatgattaa agataagatg ggctcataga gtaaaacgag gcgagggacc tataaacctc 3300
ccttcatcat gctatttcat gatctatttt ataaaataaa gatgtagaaa aaagtaagcg 3360
taataaccgc aaaacaaatg atttaaaaca tggcacataa tgaggagatt aagttcggtt 3420
tacgtttatt ttagtactaa ttgtaacgtg agactacgta tcgggaatcg cctaattaaa 3480
gcattaatgc gaacctgatt agattcaccg accctcctat cgtgtcgacc tttctgtttc 3540
ttagaatttt ttggtagtct atgtactaat aatgtcagct tcgtatttat ttcataagca 3600
atttgcattt gcaatttgtt ttttactttt atttttattg tattgtggaa tgtggactcg 3660
taccaacatg aagttatata ccaccaaaaa aattacagtt agtcaaaaga ttcacgagtg 3720
agagctactt atgattgtct tttacgtata tgtctaattg tctatttgct caataatctt 3780
tgtactttct tttgtcgttg ataaaatcac aaagttccaa aagtaatcga atgatttgct 3840
tttaagaaaa gaacagctca ataattcaac atatatctgt acacacataa ttgaataagg 3900
gtacccgatt aaaaatccca attatatttg gtctaattta gtttggtatt gagtaaaaca 3960
aattcgaacc aaaccaaaat ataaatatat agtttttata tatatgcctt taagactttt 4020
tatagaattt tctttaaaaa atatctagaa atatttgcga ctcttctggc atgtaatatt 4080
tcgttaaata tgaagtgctc catttttatt aactttaaat aattggttgt acgatcactt 4140
tcttatcaag tgttactaaa atgcgtcaat ctctttgttc ttccatattc atatgtcaaa 4200
atctatcaaa attcttatat atctttttcg aatttgaagt gaaatttcga taatttaaaa 4260
ttaaatagaa catatcatta tttaggtatc atattgattt ttatacttaa ttactaaatt 4320
tggttaactt tgaaagtgta catcaacgaa aaattagtca aacgactaaa ataaataaat 4380
atcatgtgtt attaagaaaa ttctcctata agaatatttt aatagatcat atgtttgtaa 4440
aaaaaattaa tttttactaa cacatatatt tacttatcaa aaatttgaca aagtaagatt 4500
aaaataatat tcatctaaca aaaaaaaaac cagaaaatgc tgaaaacccg gcaaaaccga 4560
accaatccaa accgatatag ttggtttggt ttgattttga tataaaccga accaactcgg 4620
tccatttgca cccctaatca taatagcttt aatatttcaa gatattatta agttaacgtt 4680
gtcaatatcc tggaaatttt gcaaaatgaa tcaagcctat atggctgtaa tatgaattta 4740
aaagcagctc gatgtggtgg taatatgtaa tttacttgat tctaaaaaaa tatcccaagt 4800
attaataatt tctgctagga agaaggttag ctacgattta cagcaaagcc agaatacaaa 4860
gaaccataaa gtgattgaag ctcgaaatat acgaaggaac aaatattttt aaaaaaatac 4920
gcaatgactt ggaacaaaag aaagtgatat attttttgtt cttaaacaag catcccctct 4980
aaagaatggc agttttcctt tgcatgtaac tattatgctc ccttcgttac aaaaattttg 5040
gactactatt gggaacttct tctgaaaata gtggtaccgt aatcatggtc atagctgttt 5100
cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 5160
tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 5220
cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 5280
gggagaggcg gtttgcgtat tggctagagc agcttgccaa catggtggag cacgacactc 5340
tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt 5400
ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact 5460
tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat tgcgataaag 5520
gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 5580
cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 5640
gtgaacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca 5700
gaagaccaaa gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga 5760
ttccattgcc cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc 5820
tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt 5880
ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 5940
acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa 6000
tcccactatc cttcgcaaga ccttcctcta tataaggaag ttcatttcat ttggagagga 6060
cacgctgaaa tcaccagtct ctctctacaa atctatctct ctcgagtcaa cacaacatat 6120
acaaaacaaa cgaatctcaa gcaatcaagc attctacttc tattgcagca atttaaatca 6180
tttcttttaa agcaaaagca attttctgaa aattttcacc atttacgaac gatagccaga 6240
tctaacaatg gataaggcct acgtggccct cctctccttc gcctccctct tcttgctcca 6300
ctacctcgtt tcccgccgca atggcaccgg gaagggcagc aaggccaagg gcgcgctgcc 6360
gccaagccct ccatccgttc cgttcctggg ccacctccac cttgtcaaga cgccattcca 6420
cgctgcgctg gcacgcctcg cggactgcca cggcccggtc ttctccctgc ggatgggagc 6480
ccgccccgca gttgtggtgt cctcgccgga gcacgccaag gagtgcttca cggagcacga 6540
cgtggccttc gccaaccggc cgcgctttcc ctcgcagcag ctcgcctcct tcaacggtgc 6600
cgcgctgggt tccgccagct acggcccgta ctggcgcaac ctccgccgcg tcgccaccgt 6660
ccacctcctg tccgcgcacc gcgtcgcgtg catgacgggg actatcgcgg ccgaggtgcg 6720
ggccatggtg cgacggatga accgcgccgc gcaggtggca tcaggcggcg cggcgcgcat 6780
cgagctcaag cggaggctat ttgaggtctc gctcagcgtg cttatggaga ccatcgcgcg 6840
gaccaagacg tcacgtacgg aggcggacga cgacacggac atgtcgcctg aggcccggga 6900
gttcaagcag atcgtggatg agctcctgcc tcacctcggc acggctaact tgtgggacta 6960
catgccggtg ttgcggtggt tcgacgtgtt cggcgtgagg aagaagatcg tgtccgcggt 7020
gaggagaagg gacgcgttcc tgcggcatct tgtcgacgca gagaggacga ggctggacga 7080
cggcaacgat gcgggcgaga agaagagcat cattgctatg ctgctcactc tgcagaagtc 7140
agagccggac gtctactcgg ataccatgat catggctcta tgtgggaact tgtttggggc 7200
cggcacagag accacgtcga cgaccaccga atgggccatg tctctcctcc tcaaccaccc 7260
ggagaagctc aggaaggcgc aggctgagat cgatgctgtc gtgggcacat cccgccttct 7320
taccgccgac gacatgcctc gtctcaccta cctccgctgc atcatcgacg agaccatgcg 7380
cctgtacccg gccgcaccac ttctgctgcc acacgagtcc tcgacacact gcaaggtcgg 7440
cggctacgac gtgcccgccg gcacgatgct gctcgtcaac gtgtacgcca tccacaggga 7500
ccccgcggtg tgggacgggc cgaccgagtt cgtgccggag cggttcgagg atggcaaggc 7560
agaaggccgg ctgctgatgc cgttcgggat gggacggcgc aagtgtcccg gcgagacgct 7620
cgcgctgcgg acgatcgggc tggtgctcgg cacgctgatc cagtgtttcg actgggaccg 7680
ggttgatggt cttgaggtcg acatgactga aagtggtggg ctcacgatcc ccagggctgt 7740
cccgttggag gccatgtgca ggcctcgtgc gacgatgcgt gaggttttgc aggagctctg 7800
agaattcttc gagtttctcc ataataatgt gtgagtagtt cccagataag ggaattaggg 7860
ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat 7920
ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc agtactaaaa 7980
tccagatccc ccgaattaat tcggcgttaa ttcagtacat taaaaacgtc cgcaatgtgt 8040
tattaagttg tctaagcgtc aatttgttta caccacaata tatcctgcca 8090
<210> 18
<211> 131
<212> DNA
<213> 未知(Unknown)
<400> 18
tcaacacaac atatacaaaa caaacgaatc tcaagcaatc aagcattcta cttctattgc 60
agcaatttaa atcatttctt ttaaagcaaa agcaattttc tgaaaatttt caccatttac 120
gaacgatagc c 131
<210> 19
<211> 63
<212> DNA
<213> 未知(Unknown)
<400> 19
atggctaaca agctcttcct tgtttgcgct actttcgctc tttgcttcct tcttactaac 60
gct 63
<210> 20
<211> 1167
<212> DNA
<213> 未知(Unknown)
<400> 20
cgattaaaaa tcccaattat atttggtcta atttagtttg gtattgagta aaacaaattc 60
gaaccaaacc aaaatataaa tatatagttt ttatatatat gcctttaaga ctttttatag 120
aattttcttt aaaaaatatc tagaaatatt tgcgactctt ctggcatgta atatttcgtt 180
aaatatgaag tgctccattt ttattaactt taaataattg gttgtacgat cactttctta 240
tcaagtgtta ctaaaatgcg tcaatctctt tgttcttcca tattcatatg tcaaaatcta 300
tcaaaattct tatatatctt tttcgaattt gaagtgaaat ttcgataatt taaaattaaa 360
tagaacatat cattatttag gtatcatatt gatttttata cttaattact aaatttggtt 420
aactttgaaa gtgtacatca acgaaaaatt agtcaaacga ctaaaataaa taaatatcat 480
gtgttattaa gaaaattctc ctataagaat attttaatag atcatatgtt tgtaaaaaaa 540
attaattttt actaacacat atatttactt atcaaaaatt tgacaaagta agattaaaat 600
aatattcatc taacaaaaaa aaaaccagaa aatgctgaaa acccggcaaa accgaaccaa 660
tccaaaccga tatagttggt ttggtttgat tttgatataa accgaaccaa ctcggtccat 720
ttgcacccct aatcataata gctttaatat ttcaagatat tattaagtta acgttgtcaa 780
tatcctggaa attttgcaaa atgaatcaag cctatatggc tgtaatatga atttaaaagc 840
agctcgatgt ggtggtaata tgtaatttac ttgattctaa aaaaatatcc caagtattaa 900
taatttctgc taggaagaag gttagctacg atttacagca aagccagaat acaaagaacc 960
ataaagtgat tgaagctcga aatatacgaa ggaacaaata tttttaaaaa aatacgcaat 1020
gacttggaac aaaagaaagt gatatatttt ttgttcttaa acaagcatcc cctctaaaga 1080
atggcagttt tcctttgcat gtaactatta tgctcccttc gttacaaaaa ttttggacta 1140
ctattgggaa cttcttctga aaatagt 1167
<210> 21
<211> 1551
<212> DNA
<213> 未知(Unknown)
<400> 21
atggataagg cctacgtggc cctcctctcc ttcgcctccc tcttcttgct ccactacctc 60
gtttcccgcc gcaatggcac cgggaagggc agcaaggcca agggcgcgct gccgccaagc 120
cctccatccg ttccgttcct gggccacctc caccttgtca agacgccatt ccacgctgcg 180
ctggcacgcc tcgcggactg ccacggcccg gtcttctccc tgcggatggg agcccgcccc 240
gcagttgtgg tgtcctcgcc ggagcacgcc aaggagtgct tcacggagca cgacgtggcc 300
ttcgccaacc ggccgcgctt tccctcgcag cagctcgcct ccttcaacgg tgccgcgctg 360
ggttccgcca gctacggccc gtactggcgc aacctccgcc gcgtcgccac cgtccacctc 420
ctgtccgcgc accgcgtcgc gtgcatgacg gggactatcg cggccgaggt gcgggccatg 480
gtgcgacgga tgaaccgcgc cgcgcaggtg gcatcaggcg gcgcggcgcg catcgagctc 540
aagcggaggc tatttgaggt ctcgctcagc gtgcttatgg agaccatcgc gcggaccaag 600
acgtcacgta cggaggcgga cgacgacacg gacatgtcgc ctgaggcccg ggagttcaag 660
cagatcgtgg atgagctcct gcctcacctc ggcacggcta acttgtggga ctacatgccg 720
gtgttgcggt ggttcgacgt gttcggcgtg aggaagaaga tcgtgtccgc ggtgaggaga 780
agggacgcgt tcctgcggca tcttgtcgac gcagagagga cgaggctgga cgacggcaac 840
gatgcgggcg agaagaagag catcattgct atgctgctca ctctgcagaa gtcagagccg 900
gacgtctact cggataccat gatcatggct ctatgtggga acttgtttgg ggccggcaca 960
gagaccacgt cgacgaccac cgaatgggcc atgtctctcc tcctcaacca cccggagaag 1020
ctcaggaagg cgcaggctga gatcgatgct gtcgtgggca catcccgcct tcttaccgcc 1080
gacgacatgc ctcgtctcac ctacctccgc tgcatcatcg acgagaccat gcgcctgtac 1140
ccggccgcac cacttctgct gccacacgag tcctcgacac actgcaaggt cggcggctac 1200
gacgtgcccg ccggcacgat gctgctcgtc aacgtgtacg ccatccacag ggaccccgcg 1260
gtgtgggacg ggccgaccga gttcgtgccg gagcggttcg aggatggcaa ggcagaaggc 1320
cggctgctga tgccgttcgg gatgggacgg cgcaagtgtc ccggcgagac gctcgcgctg 1380
cggacgatcg ggctggtgct cggcacgctg atccagtgtt tcgactggga ccgggttgat 1440
ggtcttgagg tcgacatgac tgaaagtggt gggctcacga tccccagggc tgtcccgttg 1500
gaggccatgt gcaggcctcg tgcgacgatg cgtgaggttt tgcaggagct c 1551

Claims (5)

1.一种利用转基因大豆生产α-淀粉酶的方法，其特征在于所述方法是将含α-淀粉酶表达框的表达载体导入大豆基因组中，获得能够在大豆种子中表达α-淀粉酶的转基因大豆；所述α-淀粉酶表达框包括启动子、α-淀粉酶编码基因、终止子；
所述启动子为核苷酸序列为SEQ ID NO: 1、SEQ ID NO: 2、SEQ ID NO: 3或SEQ IDNO: 4中的一种；
所述α-淀粉酶氨基酸序列为下列之一所示：SEQ ID NO: 11、SEQ ID NO: 12、SEQ IDNO: 13、SEQ ID NO: 14、SEQ ID NO: 5、SEQ ID NO: 16。
2.如权利要求1所述的方法，其特征在于所述终止子为花椰菜花叶病毒35s终止子或拟南芥heat shock protein 18.2基因终止子。
3.如权利要求1所述的方法，其特征在于所述表达载体还包括筛选标记基因表达框，所述筛选标记基因表达框为耐草甘膦表达框、耐草铵膦表达框或耐啶嘧磺隆表达框。
4.如权利要求3所述的方法，其特征在于所述耐啶嘧磺隆表达框中啶嘧磺隆抗性基因CdP450的核苷酸序列如SEQ ID NO:21所示。
5.如权利要求1所述的方法，其特征在于使用核苷酸序列如SEQ ID NO: 18所示的烟草蚀纹病毒5'非翻译区TEV 5'UTR编码基因、核苷酸序列如SEQ ID NO: 19所示的拟南芥2S白蛋白信号肽编码基因和核苷酸序列如SEQ ID NO: 20所示的烟草Tobacco RB7核基质结合区调控序列来增强α-淀粉酶基因的表达。
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