CN112029742B - 植物als突变型蛋白及其应用 - Google Patents

植物als突变型蛋白及其应用 Download PDF

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CN112029742B
CN112029742B CN202010889258.7A CN202010889258A CN112029742B CN 112029742 B CN112029742 B CN 112029742B CN 202010889258 A CN202010889258 A CN 202010889258A CN 112029742 B CN112029742 B CN 112029742B
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刘学军
张融雪
孙林静
孙玥
苏京平
王胜军
郭彦丽
李军玲
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Jiangsu Province Golden Seed Technology Co ltd
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Abstract

本发明涉及植物遗传育种技术领域，具体涉及植物ALS突变型蛋白及其应用。本发明提供171位单点突变以及171位和627位双点突变的ALS突变型蛋白，该ALS突变型蛋白不仅使植物对多种ALS抑制剂类除草剂产生抗性，而且能显著提高植物对所述除草剂的抗性阈值。本发明提供的ALS突变型蛋白可用于除草剂抗性植物品种的培育，对于田间杂草防除以及降低除草剂对植物的药害具有重要的应用价值。

Description

植物ALS突变型蛋白及其应用
技术领域
本发明涉及植物遗传育种技术领域，具体涉及产生ALS抑制剂类除草剂抗性的ALS突变型蛋白及其应用。
背景技术
农田中杂草是影响农作物产量的最重要因素之一，而人工除草所需要消耗的人力资源和成本很高，且不便于农业集约化生产。种植具有抗除草剂性状的作物品种不仅利于杂草管理，能够有效降低种植成本，提高作物品质，而且播种更加方便(例如：具有抗除草剂性状的水稻适合直播，可避免插秧)。因此培育具有抗除草剂特性的作物品种是提高作物生产效率的有效手段。
在植物和微生物中，支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)的生物合成需要4种酶。乙酰乳酸合成酶(acetolactate synthase，ALS)、酮醇还原异构酶(ketolacidreductoisomerase)、二羟基酸脱水酶(dihydroxyavid dehydratase)和支链氨基酸转氨酶(branched-chain amino acidtransaminase)。其中，乙酰乳酸合成酶是支链氨基酸生物合成过程中第一阶段的关键酶：其在缬氨酸和亮氨酸的合成中催化2分子丙酮酸生成乙酰乳酸和二氧化碳，在异亮氨酸的合成中催化1分子丙酮酸与1分子α-丁酮酸生成2-乙醛基-2-羟基丁酸和二氧化碳。
ALS抑制剂类除草剂通过抑制植物体内的ALS酶活性，从而阻止支链氨基酸的合成，导致蛋白质的合成受到破坏，阻碍细胞分裂期的DNA合成，从而使植物细胞的有丝分裂停止在Gl阶段的S期(DNA合成期)和G2阶段的M期，细胞因此不能完成有丝分裂，进而使植物停止生长，最终导致植物个体死亡。目前，ALS抑制剂类除草剂主要包括磺酰脲类(Sulfonylureas,SU)、咪唑啉酮类(Imidazolinones,IMI)、三唑嘧啶类(Triazolopyrimidines,TP)、嘧啶氧(硫)苯甲酸类[Pyrimidinylthio(or oxy)-benzoates,PTB；pyrimidinyl-carboxyherbi cides；PCs]和磺酰胺基羰基三唑啉酮类(Sulfonylamino-carbonyltriazolinones,SCT)这5大类，共包括56种化合物。以水稻种植为例，其中部分ALS抑制剂类除草剂(如苄嘧磺隆、吡嘧磺隆、氯吡嘧磺隆)已经用于稻田除草，但这些除草剂如果剂量控制不当则会对一般不具有抗(耐)除草剂特性的水稻也产生药害；还有部分ALS抑制剂类除草剂(如咪唑乙烟酸、甲氧咪草烟、灭草烟等)，虽然对杂草灭杀效果很好，但完全不能用于不具有抗(耐)除草剂特性的水稻，必需要在农作物播种前一段时间使用除草剂才能避免农作物遭受药害。培育抗(耐)除草剂的作物品种可减少作物药害、扩大除草剂的使用范围。
ALS抑制剂类除草剂在旱作地区广泛使用，国际除草剂抗性行动委员会HRAC统计了不同地区收集的ALS抑制剂抗性杂草的ALS突变位点：Ala122、Pro197、Ala205、Asp 376、Arg 377、Trp 574、Ser 653、Gly 654处突变产生除草剂抗性。这些位点对应水稻ALS的位点分别是Ala96、Pro171、Ala179、Asp 350、Arg 351、Trp 548、Ser 627、Gly 628。但目前这些位点中只有部分位点在水稻中已经证明可产生除草剂抗性(如Ala96、Trp 548、Ser 627、Gly 628等位点突变对咪唑啉酮类的除草剂产生抗性)。获得能够使作物具有较高的除草剂抗性的ALS突变型蛋白，对于作物种植和杂草管理具有重要意义。
以往产生点突变植物材料多通过随机诱变的方法实现，该方法效率低，经济成本和时间成本都比较高，并且一般获得单点突变，无法同时获得多点突变。而针对特定农艺性状的改良，基因编辑育种效率显著高于传统诱变育种，不仅大大缩短育种周期，而且精确到具体性状，不影响基本性状。结合分子标记能够快速区分出纯合和杂合单株，加快选育周期。
发明内容
为解决现有技术中的技术问题，本发明的目的在于提供使植物产生ALS抑制剂抗性的ALS突变型蛋白、编码该突变型蛋白的核酸及其应用。
为实现上述目的，本发明通过对已报道的ALS抑制剂抗性杂草的ALS突变位点：Ala122、Pro197、Ala205、Asp 376、Arg 377、Trp 574、Ser 653、Gly 654等在水稻中对应的ALS位点(Ala96、Pro171、Ala179、Asp 350、Arg 351、Trp 548、Ser 627、Gly 628)进行单点突变以及不同位点组合的多点突变，通过大量的基因编辑植株的构建、研究、筛选和对比实验发现，171位氨基酸的特异突变能够赋予植物对多种除草剂的较高抗性。进一步地，在171位氨基酸的特异突变的基础上，本发明开发了双位点组合突变。本发明在研发过程中发现，将171位突变与其它突变位点组合并不一定会产生更优ALS抑制剂抗性，有些位点的组合甚至会对ALS抑制剂抗性产生负作用。在众多的组合突变中，同时具有171位和627位氨基酸突变的ALS蛋白与单点突变和其它组合突变相比具有明显更强的ALS抑制剂抗性以及更多种类的ALS抑制剂类除草剂抗性。
具体地，本发明的技术方案如下：
本发明提供植物ALS突变型蛋白，所述ALS突变型蛋白相对于野生型ALS蛋白包含171位的氨基酸突变。
优选地，所述ALS突变型蛋白相对于野生型ALS蛋白还包含和627位的具有氨基酸突变。
本发明所述的ALS突变型蛋白中，171位氨基酸突变可为将野生型ALS蛋白的171位氨基酸突变为丙氨酸、丝氨酸、苯丙氨酸、酪氨酸、缬氨酸、半胱氨酸、甘氨酸、组氨酸、精氨酸、赖氨酸、异亮氨酸、亮氨酸、色氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸和苏氨酸中的任意一种，或者为缺失171位氨基酸。
本发明所述的ALS突变型蛋白中，627位氨基酸突变可为将野生型ALS蛋白的627位氨基酸突变为丙氨酸、丝氨酸、苯丙氨酸、酪氨酸、缬氨酸、半胱氨酸、甘氨酸、组氨酸、精氨酸、赖氨酸、异亮氨酸、亮氨酸、色氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸和苏氨酸中的任意一种，或者为缺失627位氨基酸。
以上所述的171位氨基酸突变优选突变为丙氨酸、丝氨酸、苯丙氨酸、酪氨酸、缬氨酸、半胱氨酸或甘氨酸。
以上所述的627位氨基酸突变优选突变为天冬酰胺、丝氨酸、苏氨酸、谷氨酰胺、缬氨酸、异亮氨酸、亮氨酸或半胱氨酸。
本发明所述的植物ALS突变型蛋白优选为水稻ALS突变型蛋白，其中，野生型ALS蛋白为水稻野生型ALS蛋白，其中，籼稻明恢63的ALS蛋白的氨基酸序列如SEQ ID NO.15所示，基因登录号为OsMH_02T0304200.1(http://rice.hzau.edu.cn/cgi-bin/rice2/search)，cDNA序列如SEQ ID NO.52所示，籼稻珍汕97的ALS蛋白的氨基酸序列如SEQ ID NO.16所示，基因登录号为OsZS_02T0322000.1(http://rice.hzau.edu.cn/cgi-bin/rice2/search)，粳稻日本晴的ALS蛋白的氨基酸序列如SEQ ID NO.17所示，基因登录号为LOC_Os02g30630.1(http://rice.plantbiology.msu.edu/analyses_search_locus.shtml)，粳稻Bengal的ALS蛋白的氨基酸序列如SEQ ID NO.18所示，Genebank登录号为AY885674.1。
具体地，本发明提供具有如下(1)或(2)中所列氨基酸序列中的ALS突变型蛋白：
(1)如SEQ ID NO.1～14任一所示的氨基酸序列；
(2)如SEQ ID NO.1～14任一所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的能够使所述植物具有ALS抑制剂抗性的ALS突变型蛋白的氨基酸序列。
如SEQ ID NO.1～14所示的氨基酸序列分别为将水稻的野生型ALS蛋白的171位和627位进行如下突变：
SEQ ID NO.1：P171A；SEQ ID NO.2：P171S；SEQ ID NO.3：P171F；SEQ ID NO.4：P171Y；SEQ ID NO.5：P171V；SEQ ID NO.6：P171C；SEQ ID NO.7：P171G；SEQ ID NO.8：P171A和S627N；SEQ ID NO.9：P171S和S627N；SEQ ID NO.10：P171F和S627N；SEQ ID NO.11：P171Y和S627N；SEQ ID NO.12：P171V和S627N；SEQ ID NO.13：P171C和S627N；SEQ ID NO.14：P171G和S627N。
本发明提供了水稻的ALS突变型蛋白，根据该序列以及其它植物中ALS蛋白的序列，本领域技术人员可获得其它植物中与171位和627位氨基酸相对应的氨基酸位点，并获得除水稻之外的本发明所述的ALS突变型蛋白的氨基酸序列。
本发明提供编码所述ALS突变型蛋白的核酸。
以上所述的核酸为DNA或RNA。
在已知ALS突变型蛋白序列的情况下，本领域技术人员可根据各氨基酸对应的密码子获得编码所述ALS突变型蛋白的核酸序列。所有能够编码本发明所述ALS突变型蛋白的核酸均在本发明的保护范围内。
具体地，SEQ ID NO.1(P171A)所示突变蛋白的cDNA序列如SEQ ID NO.19所示；SEQID NO.2(P171S)所示突变蛋白的cDNA序列如SEQ ID NO.20所示；SEQ ID NO.3(P171F)所示突变蛋白的cDNA序列如SEQ ID NO.21所示；SEQ ID NO.4(P171Y)所示突变蛋白的cDNA序列如SEQ ID NO.22所示；SEQ ID NO.5(P171V)所示突变蛋白的cDNA序列如SEQ ID NO.23所示；SEQ ID NO.6(P171C)所示突变蛋白的cDNA序列如SEQ ID NO.24所示；SEQ ID NO.7(P171G)所示突变蛋白的cDNA序列如SEQ ID NO.25所示；SEQ ID NO.8(P171A和S627N)所示突变蛋白的cDNA序列如SEQ ID NO.26所示；SEQ ID NO.9(P171S和S627N)所示突变蛋白的cDNA序列如SEQ ID NO.27所示；SEQ ID NO.10(P171F和S627N)所示突变蛋白的cDNA序列如SEQ ID NO.28所示；SEQ ID NO.11(P171Y和S627N)所示突变蛋白的cDNA序列如SEQ IDNO.29所示；SEQ ID NO.12(P171V和S627N)所示突变蛋白的cDNA序列如SEQ ID NO.30所示；SEQ ID NO.13(P171C和S627N)所示突变蛋白的cDNA序列如SEQ ID NO.31所示；SEQ IDNO.14(P171G和S627N)所示突变蛋白的cDNA序列如SEQ ID NO.32所示。
本发明提供包含编码所述ALS突变型蛋白的核酸的生物材料，所述生物材料为表达盒、载体、微生物细胞、植物细胞或植物细胞系。
具体地，所述表达盒可为包含所述ALS突变型蛋白的编码核酸以及用于其转录、翻译或基因编辑的分子元件的DNA或RNA片段。所述载体可为包含所述ALS突变型蛋白的编码核酸或所述表达盒的转座子、克隆载体、表达载体或基因编辑载体等。所述微生物细胞可为包含所述ALS突变型蛋白的编码核酸、所述表达盒的细菌、真菌或病毒等。
本发明提供所述ALS突变型蛋白、其编码核酸或含有其编码核酸的生物材料的如下任一应用：
(1)在使得植物具有ALS抑制剂类除草剂抗性或提高植物对ALS抑制剂类除草剂抗性中的应用；
(2)在降低ALS抑制剂类除草剂对植物的药害中的应用；
(3)在杂草防治中的应用；
(4)在植物遗传育种中的应用；
(5)在植物种质资源改良中的应用。
本发明提供一种抗除草剂植物的制备方法，其包括使所述植物含有编码ALS突变型蛋白的核酸或使植物表达所述ALS突变型蛋白的步骤。
具体地，可通过基因编辑、杂交、回交、自交或无性繁殖的方法使所述植物含有编码所述ALS突变型蛋白的核酸或使植物表达所述ALS突变型蛋白。
本发明提供一种利用CRISPR基因编辑技术制备抗除草剂植物的方法，包括如下步骤：
(1)重组CRISPR基因编辑载体的构建：将靶点sgRNA双链片段与CRISPR基因编辑载体连接，构建重组CRISPR基因编辑载体；
(2)重组农杆菌的构建：将步骤(1)获得的重组CRISPR基因编辑载体导入农杆菌，获得含有重组CRISPR基因编辑载体的重组农杆菌；
(3)农杆菌侵染水稻愈伤，获得T0代水稻植株，对获得的T0代转基因植株进行鉴定，鉴定得到的纯合或者杂合植株进行测序，对所获得纯合或者杂合突变植株进行繁种，在其后代中鉴定出无转基因成分突变植株。
本发明提供一种抗除草剂植物的鉴定方法，包括：鉴定所述植物是否含有编码所述ALS突变型蛋白的核酸或是否表达所述ALS突变型蛋白。
上述鉴定方法可为利用PCR技术、杂交技术、芯片技术或高通量测序技术鉴定所述植物是否含有编码ALS突变型蛋白的核酸或是否表达所述ALS突变型蛋白。
本发明提供用于鉴定所述ALS突变型蛋白或编码所述ALS突变型蛋白的核酸的分子标记，其为由序列如SEQ ID NO.33-34所示的引物扩增得到的DNA片段，或者为由序列如SEQ ID NO.35-36所示的引物扩增得到的DNA片段。
本发明还提供用于鉴定所述ALS突变型蛋白或编码所述ALS突变型蛋白的核酸的特异性引物，其包括序列如SEQ ID NO.33-34所示的引物和/或序列如SEQ ID NO.35-36所示的引物。
上述特异性引物为dCAPS标记引物，可配合限制性内切酶进行酶切鉴定。其中，如SEQ ID NO.33-34所示的引物用于鉴定171位氨基酸突变，配合Kpn1或其同工酶进行酶切鉴定，如SEQ ID NO.35-36所示的引物用于鉴定627位氨基酸突变，配合Dde1或其同工酶进行酶切鉴定，具体地，对于如SEQ ID NO.33-34所示的特异性引物，其扩增产物为155bp，如果171位氨基酸未发生突变，该扩增产物可被Kpn1或其同工酶酶切为128bp和27bp，如果171位氨基酸发生突变，则扩增产物不能被Kpn1或其同工酶酶切。对于如SEQ ID NO.35-36所示的特异性引物，其扩增产物为186bp，如果627位氨基酸未发生突变，该扩增产物可被Dde1或其同工酶酶切为157bp和29bp，如果627位氨基酸发生突变，则扩增产物不能被Dde1或其同工酶酶切。
本发明还提供包含上述特异性引物的试剂或试剂盒。
本发明提供一种抗除草剂植物，其含有编码所述ALS突变型蛋白的核酸或表达所述ALS突变型蛋白。
具体地，本发明提供抗除草剂水稻，其含有编码所述ALS突变型蛋白的核酸或表达所述ALS突变型蛋白。
本发明通过使水稻含有编码所述ALS突变型蛋白的核酸或表达所述ALS突变型蛋白获得的水稻植株对ALS抑制剂类除草剂的抗性显著提高，表现为对原有除草剂的抗性阈值显著提高或产生新的除草剂抗性。
本发明提供一种防治杂草或降低除草剂对植物药害的方法，在植物种植过程中施用有效剂量的除草剂；所述植物含有编码所述ALS突变型蛋白的核酸或表达所述ALS突变型蛋白。
优选地，所述除草剂为咪唑啉酮类、磺酰脲类、嘧啶水杨酸类、三唑嘧啶类和三唑啉酮类除草剂中的一种或多种。
具体地，所述咪唑啉酮类除草剂可选自甲氧咪草烟、咪唑乙烟酸、咪唑烟酸、甲咪唑烟酸、咪唑喹啉酸和咪草酯中的一种或多种。所述磺酰脲类除草剂可选自烟嘧磺隆、砜嘧磺隆、甲基二磺隆、苯磺隆、四唑嘧磺隆、苄嘧磺隆、氯嘧磺隆、氯磺隆、醚磺隆、三氟啶磺隆、环丙嘧磺隆、胺苯磺隆、乙氧磺隆、啶嘧磺隆、氟啶嘧磺隆钠盐、氯吡嘧磺隆、唑吡嘧磺隆、甲基碘磺隆钠盐、甲磺隆、甲酰胺磺隆、环氧嘧磺隆、氟嘧磺隆、氟磺隆、吡嘧磺隆、甲嘧磺隆、磺酰磺隆、三氟甲磺隆、噻吩磺隆、醚苯磺隆、氟胺磺隆、氟吡磺隆、嘧苯胺磺隆、酰嘧磺隆、丙嗪嘧磺隆中的一种或多种。所述嘧啶水杨酸类除草剂可选自双草醚、嘧啶肟草醚、嘧草硫醚、环酯草醚、嘧草醚、Pyrimisulfan中的一种或多种。所述三唑嘧啶类除草剂可选自氯酯磺草胺、双氯磺草胺、双氟磺草胺、唑嘧磺草胺、磺草唑胺、五氟磺草胺、啶磺草胺中的一种或多种。所述三唑啉酮类除草剂可选自氟唑磺隆、丙苯磺隆、噻酮磺隆中的一种或多种。
以上所述的植物为单子叶植物或双子叶植物。所述植物可选自水稻、小麦、玉米、大豆、棉花、大麦、燕麦、黑麦、谷子、高粱、烟草、青稞、向日葵、油菜、苜蓿、粟、甘蔗、番茄、木薯、马铃薯、白菜、甘蓝、黄瓜、西瓜、甜瓜、花椰菜、西蓝花、拟南芥等。本发明的有益效果在于：
本发明通过对ALS突变位点进行单点突变以及不同位点组合的多点突变，通过大量的基因编辑植株的构建、研究、筛选和对比实验获得了能够使植物对ALS抑制剂类除草剂的抗性显著提高、除草剂抗性种类显著增加的ALS突变型蛋白。在171位发生突变的ALS突变型蛋白能够赋予植物对多种除草剂具有较高的抗性，在171位和627位同时突变的ALS突变型蛋白与单点突变的ALS突变型蛋白相比具有明显更强的ALS抑制剂抗性，能够显著提高植物对ALS抑制剂类除草剂的抗性阈值，同时使植物产生新的除草剂抗性，显著增加了植物具有抗性的ALS抑制剂类除草剂的种类。本发明提供的ALS突变蛋白可用于除草剂抗性植物品种的培育，对于田间杂草防除以及降低除草剂对植物的药害具有重要的应用价值。
附图说明
图1为本发明实施例1中单碱基编辑载体hAID-nCas9的结构示意图。
图2为本发明实施例1中hAID-nCas9连接sgRNA靶序列的PCR鉴定结果图；其中，marker为DNA marker，1是P171位点单碱基编辑载体用171sgRNA和OsU3T R引物检测的PCR产物，该条带大小符合预期，证明sgRNA片段已经连入基因编辑载体，2是hAID-nCas9空载体作为对照，无条带，说明引物没有非特异扩增。
图3为本发明实施例1中利用dCAPS标记鉴定171位突变和627位突变的结果图:其中R是基因编辑材料ALS基因171位纯合突变材料带型，S是ALS基因171位未发生突变材料的带型，H是ALS基因171位发生杂合突变材料的带型，R0是金粳818的ALS基因627位点带型，S0是627位点恢复为野生型的带型，H0是627位点杂合材料的带型。
图4为本发明实施例2中TadA-nCas9载体的图谱。
图5为本发明实施例2TadA-nCas9连接sgRNA靶序列的PCR鉴定结果图；其中，Marker为DNAmarker，1是TadA-nCas9空载体作为对照，无条带，说明引物没有非特异扩增，2是N627位点单碱基编辑载体用627sgRNA F和OsU3T R引物检测PCR产物，该条带大小符合预期，证明sgRNA片段已经连入基因编辑载体。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1ALS蛋白171位和627位双位点突变的水稻植株的获得
本发明利用基因编辑技术构建171位和627位双突变的ALS突变水稻材料，常规基因编辑方法是对野生型水稻进行编辑，但是对同一基因的两个位点进行同时编辑时效率较低。因此，本发明利用携带ALS S627N(627位丝氨酸突变为天冬酰胺)突变的水稻材料金粳818作为受体材料，再利用单碱基编辑方法对金粳818的ALS基因进行171位氨基酸突变，创制了ALS双点突变水稻材料，该方法的效率是常规单碱基编辑创制双点突变效率的2倍。
金粳818是申请人前期选育得到的粳型常规水稻品种，生育期155.4天，具有ALSS627N突变，对咪唑啉酮类除草剂有抗性，适宜直播，该水稻品种已经在生产上大面积推广应用。
1、金粳818水稻材料的ALS基因克隆及sgRNA设计
采用CTAB法提供金粳818基因组DNA，利用引物001F：CCCAAACCCAGAAACCCTCG(SEQID NO.37)和2019R:GCTGGTGCTTTGCCAACATA(SEQ ID NO.38)扩增ALS基因，PCR扩增体系如表1所示。
表1 ALS基因的PCR扩增体系
组分 体积 浓度
金粳818DNA 0.5μl 100ng/μl
001F 0.5μl 10μM
2019R 0.5μl 10μM
2×Q5(NEB) 5μl —
DMSO 0.5μl 分析纯99％
H<sub>2</sub>O 3μl —
总计 10μl —
在BIO-RAD C1000运行以下PCR程序：98℃、30s；98℃、7s，59℃、20s，72℃、90s，35Cycles；72℃5min。
将PCR产物在1％琼脂糖胶中进行水平电泳，目的条带约为2000bp，切胶回收后送华大基因测序，获得ALS基因序列如SEQ ID NO.16所示，将此序列在NCBI(https://blast.ncbi.nlm.nih.gov/Blast.cgi)比对，发现金粳818ALS基因1880位为A，而常规野生型水稻品种为G。金粳818的ALS蛋白序列如SEQ ID NO.21所示。
根据金粳818的ALS基因序列(如SEQ ID NO.39所示)，在http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR网站上设计进行171位氨基酸突变的sgRNA，选择GAGGTCCCCCGCCGCATGAT(SEQ ID NO.40)为编辑的靶向序列，其中加粗三字母是171位氨基酸(脯氨酸，P)的密码子，利用胞嘧啶脱氨酶融合nCas9的单碱基编辑工具能将C突变成T或者G，导致171位点突变为丝氨酸(S)、丙氨酸(A)、苯丙氨酸(F)等氨基酸。
2、单碱基编辑载体的构建
(1)C-T单碱基编辑载体hAID-nCas9构建
hAID-nCas9载体的构建过程如下：以pCAMBIA1300为基础载体，在RB附近插入U3启动子以及pretRNA、双BsaI和sgRNA(Xie K,Minkenberg B,Yang Y.Boosting CRISPR/Cas9multiplex editing capability with the endogenous tRNA-processing system[J].Proc Natl Acad,U S A,2015,112(11):3570-3575.)，接着插入NLS-hAID-nCas9-UGI-NLS融合蛋白表达盒(Ma Y,Zhang J,Yin W,et al.Targeted AID-mediated mutagenesis(TAM)enables efficient genomic diversification in mammalian cells[J].NatureMethods,2016.，Komor A C,Zhao K T,Packer M S,et al.Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity[J].Science Advances,2017,3(8):eaao4774.)，构建得到C-T单碱基编辑载体hAID-nCas9。
(2)靶向片段制备
合成sgRNA 171F:AGACGAGGTCCCCCGCCGCATGA(SEQ ID NO.41)T和sgRNA171R:ACAAATCATGCGGCGGGGGACCTC(SEQ ID NO.42)，分别用超纯水溶解配制100μM的溶液，各取20μl，煮沸5min，移至室温自然冷却，完成退火，获得带有BsaI粘性末端的靶位点双链片段；
(3)单碱基编辑载体连接
连接反应体系如下：将hAID-nCas9载体(质粒图谱如图1所示)、步骤(1)得到的靶标片段，BsaI、10×T4连接酶缓冲液、T4连接酶混合，将10×buffer稀释至1×。
反应程序如下：放入PCR仪，设置37℃、5min，16℃、10min，循环30次，最后80℃、20min。
(4)采用热击法将步骤(2)得到的连接产物转化大肠杆菌DH5α获得重组菌，菌液涂布于含有50mg/L卡那霉素的LB平板上，培养约12h；挑取平板上长出的单菌落，摇菌扩繁；以菌液为模板进行PCR鉴定，鉴定引物序列如下：171sgRNA F:GAGGTCCCCCGCCGCATGAT(SEQ IDNO.43)和OsU3T R:CTCGGGATAGTTGGCACAAA(SEQ ID NO.44)。PCR鉴定的反应体系如表2所示。
表2PCR鉴定的反应体系
组分 体积 浓度
菌液 2μl —
171sgRNA F 0.5μl 10μM
OsU3T R 0.5μl 10μM
2x Mix(TIANGEN) 5μl —
H<sub>2</sub>O 2μl —
总计 10μl —
在BIO-RAD C1000运行以下PCR程序：94℃、3min；94℃、30s，55℃、30s，72℃、40s，35Cycles；72℃、5min。
PCR鉴定的部分结果如图2所示。将PCR鉴定得到的阳性菌送华大基因测序，将测序正确的阳性菌扩繁提取质粒，所得质粒即为单碱基编辑载体。
3、转化农杆菌及侵染金粳818愈伤组织
将上述步骤2构建的单碱基编辑载体转化至EHA105中，得到携带单碱基编辑载体的农杆菌。
利用携带单碱基编辑载体的农杆菌侵染金粳818愈伤组织，经过潮霉素筛选，分化诱导再生，最终获得337棵T0转基因苗。
4、T0转基因苗ALS基因171位点和627位点突变检测针对野生型ALS基因171位氨基酸的密码子序列CCC，设计dCAPS标记。
dCAPS标记引物序列如下：
ALS171Kpn1F:CGATGGTCGCCATCACGGGCCAGGTA(SEQ ID NO.33)；
ALS171R:CGGTGCCGATCATGCGGC(SEQ ID NO.34)。
利用该dCAPS标记引物得到的扩增产物为155bp，如果该位点未发生突变，扩增片段可被Kpn1酶切为128bp和27bp两个片段，如果CCC中前两个C单独或者同时突变，扩增片段不能被Kpn1酶切，仍为155bp(如图3所示)。
针对野生型ALS基因627位氨基酸密码子AGT，设计dCAPS标记。
dCAPS标记引物序列如下：
ALS627Dde1F:CACCAGGAGCATGTGCTGCCTATGATCCTAA(SEQ ID NO.35)；
ALS627R:GATAGAGCACATACAAACATCATAG(SEQ ID NO.36)。
利用该dCAPS标记引物得到的扩增产物长度是186bp，进行Dde I酶切，野生型可以被切成157bp和29bp，而627位发生突变的金粳818因为没有酶切位点，仍是186bp(如图3所示)。
对步骤3所获得的T0转基因苗提取DNA，用以上dCAPS标记进行检测，共获得了56株突变株，进一步对这些突变株进行测序，分别得到了在171位和627位发生双位点突变的突变体：P171A+S627N(ALS突变型蛋白的序列如SEQ ID NO.8所示)、P171S+S627N(ALS突变型蛋白的序列如SEQ ID NO.9所示)、P171F+S627N(ALS突变型蛋白的序列如SEQ ID NO.10所示)、P171Y+S627N(ALS突变型蛋白的序列如SEQ ID NO.11所示)、P171V+S627N(ALS突变型蛋白的序列如SEQ ID NO.12所示)、P171C+S627N(ALS突变型蛋白的序列如SEQ ID NO.13所示)、P171G+S627N(ALS突变型蛋白的序列如SEQ ID NO.14所示)，将这些突变体进行大田种植繁种。
实施例2ALS蛋白171位单位点突变的水稻植株的获得
在实施例1得到的P171A+S627N、P171S+S627N、P171F+S627N、P171Y+S627N、P171V+S627N、P171C+S627N、P171G+S627N水稻突变体植株的基础上，本实施例对这些双位点突变的水稻植株的627位进行基因编辑，使其恢复为野生型位点(Ser)，从而获得一系列171位点单点突变的水稻突变体植株。具体方法如下：
1、ALS N627靶位点sgRNA设计
根据金粳818的ALS基因序列(如SEQ ID NO.39所示)，在http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR网站上设计进行627位氨基酸突变的sgRNA，选择GCCAAATGGGGGCGCATTCA(SEQ ID NO.45)为编辑的靶向序列，其中加粗三字母是金粳818ALS 627位氨基酸(天冬酰胺，Asn,N)的密码子，利用腺嘌呤脱氨酶融合nCas9的单碱基编辑工具能将A突变成G，导致627位点由AAT突变为AGT，即将上述系列双突变体的ALSN627恢复为野生的S627，从而得到ALS的P171A、P171S、P171F、P171Y、P171V、P171C、P171G单点突变体(序列分别如SEQ ID NO.1～7所示)。
2、单碱基编辑载体的构建
(1)A-G单碱基编辑载体TadA-nCas9构建
TadA-nCas9载体的构建过程如下：以pCAMBIA1300为基础载体，在RB附近插入OsU3启动子以及pretRNA、双BsaI和sgRNA(Xie K,Minkenberg B,Yang Y.Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[J].Proc Natl Acad,U S A,2015,112(11):3570-3575.)，接着插入NLS-TadA-nCas9-NLS融合蛋白表达盒(Gaudelli N M,Komor A C,Rees H A,et al.Programmable baseediting of A.T to G.C in genomic DNA without DNAcleavage[J].Nature,2017,551(7681):464-471..)，构建得到A-G单碱基编辑载体TadA-nCas9。
(2)靶向片段制备
合成sgRNA 627F:AGACGCCAAATGGGGGCGCATTCA(SEQ ID NO.46)和sgRNA 627RACAATGAATGCGCCCCCATTTGGC(SEQ ID NO.47)，分别用超纯水溶解配制100μM的溶液，各取20μl，煮沸5min，移至室温自然冷却，完成退火，获得带有BsaI粘性末端的靶位点双链片段；
(3)单碱基编辑载体连接
连接反应体系如下：将TadA-nCas9载体(质粒图谱如图4所示)、步骤(1)得到的靶标片段，BsaI、10×T4连接酶缓冲液、T4连接酶混合，将10×buffer稀释至1×。反应程序如下：放入PCR仪，设置37℃、5min，16℃、10min，循环30次，最后80℃、20min。
(4)采用热击法将步骤(2)得到的连接产物转化大肠杆菌DH5α获得重组菌，菌液涂布于含有50mg/L卡那霉素的LB平板上，培养约12h；挑取平板上长出的单菌落，摇菌扩繁；以菌液为模板进行PCR鉴定，鉴定引物序列如下：627sgRNAF:GCCAAATGGGGGCGCATTCA(SEQ IDNO.48)和OsU3T R:CTCGGGATAGTTGGCACAAA(SEQ ID NO.49)。PCR鉴定的反应体系如表3所示。
表3PCR鉴定的反应体系
组分 体积 浓度
菌液 2μl —
627sgRNA F 0.5μl 10μM
OsU3T R 0.5μl 10μM
2x Mix(TIANGEN) 5μl —
H<sub>2</sub>O 2μl —
总计 10μl —
在BIO-RAD C1000运行以下PCR程序：94℃、3min；94℃、30s，55℃、30s，72℃、40s，35Cycles；72℃、5min。
PCR鉴定的部分结果如图5所示。将PCR鉴定得到的阳性菌送华大基因测序，将测序正确的阳性菌扩繁提取质粒，所得质粒即为单碱基编辑载体。
3、转化农杆菌及侵染P171A+S627N、P171S+S627N、P171F+S627N、P171Y+S627N、P171V+S627N、P171C+S627N、P171G+S627N愈伤组织。
将上述步骤2构建的单碱基编辑载体转化至EHA105中，得到携带单碱基编辑载体的农杆菌。利用携带单碱基编辑载体的农杆菌侵染P171A+S627N、P171S+S627N、P171F+S627N、P171Y+S627N、P171V+S627N、P171C+S627N、P171G+S627N愈伤组织，经过潮霉素筛选，分化诱导再生，每个双突变体最终得到约100株再生苗。
4、T0转基因苗ALS P171位点和N627位点突变检测
利用ALS171位点检测标记引物(SEQ ID NO.33-34)和ALS 627位点检测标记引物(SEQ ID NO.35-36)进行8％PAGE胶电泳后银染显色检测。SEQ ID NO.33-34引物的扩增产物为155bp，如果该位点未发生突变，扩增片段可被Kpn1酶切为128bp和27bp两个片段(27bp太短，电泳后迁移出PAGE胶，胶图中只有128bp长片段)，如图3中S泳道所示。如果CCC中前两个C单独或者同时突变，扩增片段不能被Kpn1酶切，仍为155bp(如图3中R泳道所示)，如果是杂合突变则155bp和128bp都存在，如图3中H泳道所示。SEQ ID NO.35-36引物的扩增产物长度是186bp，进行Dde I酶切，如果上述双突变体的627位点被恢复为野生型，扩增片段可以被Dde1酶切为157bp和29bp(29bp太短，电泳后迁移出PAGE胶，胶图中只有157bp长片段)，如图3中S0泳道所示，如果没有恢复，则仍是186bp，如图3中R0泳道所示，如果是杂合突变，则如图3中H0泳道所示。
对步骤3所获得的T0转基因苗提取DNA，用以上dCAPS标记进行检测，共获得了63株突变株，进一步对这些突变株进行测序，分别得到了在ALS 171位仍为突变而627位恢复为野生型位点的单点突变体：P171A、P171S、P171F、P171Y、P171V、P171C、P171G，将这些突变体进行大田种植繁种。
实施例3水稻突变体后代种子的除草剂抗性鉴定
选择ALS抑制剂类除草剂：咪唑啉酮类的甲氧咪草烟、咪唑乙烟酸、咪唑烟酸、甲咪唑烟酸、咪唑喹啉酸和咪草酯；磺酰脲类的烟嘧磺隆、砜嘧磺隆、甲基二磺隆，嘧啶水杨酸类的双草醚、嘧啶肟草醚，三唑嘧啶类的啶磺草胺和三唑啉酮的噻酮磺隆对实施例1和实施例2构建的ALS单点突变和ALS双位点突变的水稻突变体进行除草剂抗性测试，具体方法如下：
1、对鉴定出的纯合的突变体进行田间小区扩繁，每个小区400个单株，正常田间管理，每个小区挑选10株优良单株留种，其余单株混收脱粒待用。
2、田间除草剂抗性实验设计：选择除草剂为咪唑啉酮类的甲氧咪草烟、咪唑乙烟酸、咪唑烟酸、甲咪唑烟酸、咪唑喹啉酸和咪草酯；嘧啶水杨酸类的双草醚、嘧啶肟草醚；磺酰脲类的砜嘧磺隆、烟嘧磺隆、甲基二磺隆；三唑嘧啶类的啶磺草胺和三唑啉酮的噻酮磺隆。测试材料为7个ALS P171和S627双点突变体和7个ALS P717单点突变体，以常规粳稻品种苏秀867以及除草剂抗性品种金粳818为对照。合计材料数为16份，每份播种面积1平方米，每种除草剂设置1个正常生长对照(不喷施除草剂)和8个浓度梯度，具体剂量如表4所示。
表4除草剂种类及喷施剂量设置
Figure BDA0002656436000000081
注：表4中的除草剂单位为有效成分克/亩(每亩兑水量30公斤)，其中，1×是田间推荐用量。
3、将每个水稻材料用塑料网袋盛取90份样品，每份70克，分做10组，每组9份，常温浸种2天，32℃催芽1天，播种于秧田，每份播种面积1平方米，播种完毕后覆盖无纺布，1周后揭开无纺布，田间水面与秧田面齐，继续生长1周后进行除草剂喷施实验。
4、喷施除草剂1周后拍照，统计存活情况和苗长，评价抗性效果。
各种除草剂的喷施后效果如下：
(1)甲氧咪草烟喷施后效果：结果如表5所示，常规对照品种苏秀867在最低剂量下就已经死亡，ALS P171系列单突变体在最低剂量下也全部死亡，金粳818在甲氧咪草烟(有效成分)100克/亩死亡，而ALS P171+S627系列双突变体在250克/亩才死亡。
表5苗后喷施甲氧咪草烟材料存活统计
Figure BDA0002656436000000091
进一步对金粳818和ALS P171+S627系列双突变体都能存活下的75克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表6所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表6甲氧咪草烟75克/亩处理材料相对苗长
Figure BDA0002656436000000092
以上结果均说明：ALS P171+S627系列双突变体对甲氧咪草烟抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。
(2)咪唑烟酸喷施后效果：结果如表7所示，常规对照品种苏秀867在最低剂量下就已经死亡，ALS P171系列单突变体在最低剂量下也全部死亡，金粳818在咪唑烟酸(有效成分)100克/亩死亡，而ALS P171+S627系列双突变体在250克/亩才死亡。
表7苗后喷施咪唑烟酸材料存活统计
Figure BDA0002656436000000093
Figure BDA0002656436000000101
进一步对金粳818和ALS P171+S627系列双突变体都能存活下的75克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表8所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表8咪唑烟酸75克/亩处理材料相对苗长
Figure BDA0002656436000000102
以上结果均说明ALS P171+S627系列双突变体对咪唑烟酸抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。
(3)咪唑乙烟酸喷施后效果：结果如表9所示，常规对照品种苏秀867在最低剂量下就已经死亡，ALS P171系列单突变体在最低剂量下也全部死亡，金粳818在咪唑乙烟酸(有效成分)100克/亩死亡，而ALS P171+S627系列双突变体在250克/亩才死亡。
表9苗后喷施咪唑乙烟酸材料存活统计
Figure BDA0002656436000000103
Figure BDA0002656436000000111
进一步对金粳818和ALS P171+S627系列双突变体都能存活下的75克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表10所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强.
表10咪唑乙烟酸75克/亩处理材料相对苗长
Figure BDA0002656436000000112
以上结果均说明ALS P171+S627系列双突变体对咪唑乙烟酸抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。
(4)甲咪唑烟酸喷施后效果：结果如表11所示，常规对照品种苏秀867在最低剂量下就已经死亡，ALS P171系列单突变体在最低剂量下也全部死亡，金粳818在甲咪唑烟酸(有效成分)100克/亩死亡，而ALS P171+S627系列双突变体在250克/亩才死亡。
表11苗后喷施甲咪唑烟酸材料存活统计
Figure BDA0002656436000000113
进一步对金粳818和ALS P171+S627系列双突变体都能存活下的75克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表12所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表12甲咪唑烟酸75克/亩处理材料相对苗长
Figure BDA0002656436000000121
以上结果均说明：ALS P171+S627系列双突变体对甲咪唑烟酸抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。
(5)咪唑喹啉酸喷施后效果：结果如表13所示，常规对照品种苏秀867在最低剂量下就已经死亡，ALS P171系列单突变体在最低剂量下也全部死亡，金粳818在咪唑喹啉酸(有效成分)100克/亩死亡，而ALS P171+S627系列双突变体在250克/亩才死亡。
表13苗后喷施咪唑喹啉酸材料存活统计
Figure BDA0002656436000000122
进一步对金粳818和ALS P171+S627系列双突变体都能存活下的75克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表14所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表14咪唑喹啉酸75克/亩处理材料相对苗长
Figure BDA0002656436000000123
Figure BDA0002656436000000131
以上结果均说明：ALS P171+S627系列双突变体对咪唑喹啉酸抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。
(6)咪草酯喷施后效果：结果如表15所示，常规对照品种苏秀867在最低剂量下就已经死亡，ALS P171系列单突变体在最低剂量下也全部死亡，金粳818在咪草酯(有效成分)100克/亩死亡，而ALS P171+S627系列双突变体在250克/亩才死亡。
表15苗后喷施咪草酯材料存活统计
Figure BDA0002656436000000132
进一步对金粳818和ALS P171+S627系列双突变体都能存活下的75克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表16所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表16咪草酯75克/亩处理材料相对苗长
Figure BDA0002656436000000133
以上结果均说明：ALS P171+S627系列双突变体对咪草酯抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。
(7)双草醚喷施后效果：结果如表17所示，双草醚本就是水稻用除草剂，常规品种对其有一定抗性。对照品种苏秀867在双草醚(有效成分)60克/亩剂量下死亡，金粳818、ALSP171系列单突变体和ALS P171+S627系列双突变体在最高剂量100克/亩条件下均未死亡，但有不同程度矮化。
表17苗后喷施双草醚材料存活统计
Figure BDA0002656436000000141
进一步测量了100克/亩剂量下的苗长，并计算了其相对未喷施对照组的相对苗长，结果如表18所示，金粳818相对苗长为0.3，ALS P171系列单突变体相对苗长在0.5左右，而ALS P171+S627系列双突变体相对苗长在0.8左右，表明该剂量下ALS P171+S627系列双突变体抗性最强。
表18双草醚100克/亩处理材料相对苗长
Figure BDA0002656436000000142
以上结果均说明ALS P171+S627系列双突变体对双草醚的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对双草醚的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALS P171V和ALS P171Y对双草醚抗性优于ALS P171S和ALS P171A。
(8)嘧啶肟草醚喷施后效果：结果如表19所示，嘧啶肟草醚本就是水稻用除草剂，常规品种对其有一定抗性。对照品种苏秀867在嘧啶肟草醚(有效成分)60克/亩剂量下死亡，金粳818、ALS P171系列单突变体和ALS P171+S627系列双突变体在最高剂量100克/亩条件下均未死亡，但有不同程度矮化。
表19苗后喷施嘧啶肟草醚材料存活统计
Figure BDA0002656436000000151
进一步测量了100克/亩剂量下的苗长，并计算了其相对未喷施对照组的相对苗长，结果如表20所示，金粳818相对苗长为0.3，ALS P171系列单突变体相对苗长在0.5左右，而ALS P171+S627系列双突变体相对苗长在0.8左右，表明该剂量下ALS P171+S627系列双突变体抗性最强。
表20嘧啶肟草醚100克/亩处理材料相对苗长
Figure BDA0002656436000000152
以上结果均说明ALS P171+S627系列双突变体对嘧啶肟草醚的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对嘧啶肟草醚的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALS P171V和ALS P171Y对嘧啶肟草醚抗性优于ALS P171S和ALS P171A。
(9)砜嘧磺隆喷施后效果：结果如表21所示，常规对照品种苏秀867在最低剂量下就已经死亡，金粳818在砜嘧磺隆(有效成分)3.75克/亩死亡，ALS P171系列单突变体在15克/亩全部死亡，而ALS P171+S627系列双突变体在22.5克/亩才全部死亡。
表21苗后喷施砜嘧磺隆材料存活统计
Figure BDA0002656436000000161
进一步对ALS P171单突变体和ALS P171+S627系列双突变体都能存活下的11.25克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表22所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表22砜嘧磺隆11.25克/亩处理材料相对苗长
Figure BDA0002656436000000162
以上结果均说明ALS P171+S627系列双突变体对砜嘧磺隆的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对砜嘧磺隆的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALSP171V和ALS P171Y对砜嘧磺隆抗性优于ALS P171S和ALS P171A。
(10)烟嘧磺隆喷施后效果：结果如表23所示，常规对照品种苏秀867在最低剂量下就已经死亡，金粳818在烟嘧磺隆(有效成分)7.5克/亩死亡，ALS P171系列单突变体在22.5克/亩全部死亡，而ALS P171+S627系列双突变体在45克/亩才全部死亡。
表23苗后喷施烟嘧磺隆材料存活统计
Figure BDA0002656436000000171
进一步对ALS P171单突变体和ALS P171+S627系列双突变体都能存活下的15克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表24所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表24烟嘧磺隆15克/亩处理材料相对苗长
Figure BDA0002656436000000172
以上结果均说明ALS P171+S627系列双突变体对烟嘧磺隆的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对烟嘧磺隆的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALSP171V和ALS P171Y对烟嘧磺隆抗性优于ALS P171S和ALS P171A。
(11)甲基二磺隆喷施后效果：结果如表25所示，常规对照品种苏秀867在最低剂量下就已经死亡，金粳818在甲基二磺隆(有效成分)5克/亩死亡，ALS P171系列单突变体在12.5克/亩全部死亡，而ALS P171+S627系列双突变体在20克/亩才全部死亡。
表25苗后喷施甲基二磺隆材料存活统计
Figure BDA0002656436000000181
进一步对ALS P171单突变体和ALS P171+S627系列双突变体都能存活下的7.5克/亩剂量下的苗长进行了统计，并计算了其相对未喷施对照组的相对苗长，结果如表26所示，结果表明该剂量下ALS P171+S627系列双突变体抗性最强。
表26甲基二磺隆7.5克/亩处理材料相对苗长
Figure BDA0002656436000000182
以上结果均说明ALS P171+S627系列双突变体对甲基二磺隆的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对甲基二磺隆的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALS P171V和ALS P171Y对甲基二磺隆抗性优于ALS P171S和ALS P171A。
(12)噻酮磺隆喷施后效果：结果如表27所示，常规对照品种苏秀867在最低剂量下就已经死亡，金粳818在噻酮磺隆(有效成分)5克/亩死亡，ALS P171系列单突变体在12.5克/亩全部死亡，而ALS P171+S627系列双突变体在20克/亩才全部死亡。
表27苗后喷施噻酮磺隆材料存活统计
Figure BDA0002656436000000191
进一步对ALS P171单突变体和ALS P171+S627系列双突变体都能存活下的7.5克/亩剂量下的苗长进行了统计，计算其相对未喷施对照组的相对苗长，结果如表28所示，该剂量下ALS P171+S627系列双突变体抗性最强。
表28噻酮磺隆7.5克/亩处理材料相对苗长
Figure BDA0002656436000000192
以上结果均说明ALS P171+S627系列双突变体对噻酮磺隆的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对噻酮磺隆的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALSP171V和ALS P171Y对噻酮磺隆抗性优于ALS P171S和ALS P171A。
(13)啶磺草胺喷施后效果：结果如表29所示，常规对照品种苏秀867在最低剂量下就已经死亡，金粳818在啶磺草胺(有效成分)5克/亩死亡，ALS P171系列单突变体在12.5克/亩全部死亡，而ALS P171+S627系列双突变体在20克/亩才全部死亡。
表29苗后喷施啶磺草胺材料存活统计
Figure BDA0002656436000000201
进一步对ALS P171单突变体和ALS P171+S627系列双突变体都能存活下的7.5克/亩剂量下的苗长进行统计，并计算其相对未喷施对照组的相对苗长，结果如表30所示，该剂量下ALS P171+S627系列双突变体抗性最强。
表30啶磺草胺7.5克/亩处理材料相对苗长
Figure BDA0002656436000000202
以上结果均说明ALS P171+S627系列双突变体对啶磺草胺的抗性不仅优于常规粳稻品种苏秀867，还优于金粳818和ALS P171系列单突变体。还说明ALS P171系列单突变体对啶磺草胺的抗性优于常规品种和金粳818，并且ALS P171F、ALS P171C、ALS P171G、ALSP171V和ALS P171Y对啶磺草胺抗性优于ALS P171S和ALS P171A。
(14)ALS P171单突变体和ALS P171+S627系列双突变体对其它已登记水稻ALS抑制剂类除草剂的抗性检测。
ALS抑制剂类除草剂除了双草醚和嘧啶肟草醚，还有一些登记于水稻的除草剂，包括苄嘧磺隆、吡嘧磺隆、氯吡嘧磺隆、五氟磺草胺、乙氧磺隆、嘧草醚、丙嗪嘧磺隆、嘧苯胺磺隆、醚磺隆、嗪吡嘧磺隆。本发明还利用这些除草剂对ALS P171单突变体和ALS P171+S627系列双突变体进行了抗性测试，结果如表31所示，在50倍田间推荐剂量下，常规品种苏秀867均死亡，金粳818在各除草剂田间推荐剂量下均表现出不同程度的矮化，而ALS P171单突变体和ALS P171+S627系列双突变体均能正常存活和生长，未出现矮化，说明这些突变材料对用于水稻的ALS抑制剂类除草剂也有很好的抗性。
表31苗后喷施50X田间推荐剂量除草剂材料存活统计
Figure BDA0002656436000000211
实施例4无转基因成分的水稻植株的获得
筛选实施例1和实施例2构建的ALS单位点和双位点突变的水稻材料后代中靶位点突变但是不含转基因成分的单株，具体方法如下：
(1)用国家标准农业部1782号公告-2-2012中的潮霉素引物HPT F:GAAGTGCTTGACATTGGGGAGT(SEQ ID NO.50)和HPT R:AGATGTTGGCGACCTCGTATT(SEQ IDNO.51)检测P171A+S627N和P171S+S627N突变体后代单株，鉴定出无转基因成分的单株。
(2)用dCAPS标记引物ALS171Kpn1 F:CGATGGTCGCCATCACGGGCCAGGTA和ALS171R:CGGTGCCGATCATGCGGC以及dCAPS标记ALS627Dde1F:CACCAGGAGCATGTGCTGCCTATG ATCCTAA和ALS627R:GATAGAGCACATACAAACATCATAG检测上述(1)中获得的无转基因成分单株，鉴定出突变单株，由此得到无转基因成分的ALS单位点和双位点突变的水稻材料，作为后续育种所用。
实施例5突变体水稻的农艺性状观察
对实施例4得到的无转基因成分的ALS单位点和双位点突变的水稻材料的农艺性状进行观察，结果如表32所示，鉴定出的无转基因成分的单株在株高、分蘖、生育期和千粒重方面，与受体材料金粳818相比均无显著差异。
表32突变体与金粳818农艺性状比较
Figure BDA0002656436000000221
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 天津市农业科学院
<120> 植物ALS突变型蛋白及其应用
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Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 5
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Val Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 6
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Cys Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 7
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 8
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Ala Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 9
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Ser Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 10
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Phe Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 11
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Tyr Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 12
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Val Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 13
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Cys Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 14
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 15
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 16
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Phe Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Asp Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 17
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Trp Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Gln Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Val Tyr
<210> 18
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Val Tyr
<210> 19
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc gcccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 20
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc tcccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 21
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ttccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 22
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc taccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 23
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc gtccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 24
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc tgccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 25
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ggccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 26
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc gcccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 27
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc tcccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 28
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ttccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 29
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc taccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 30
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc gtccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 31
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc tgccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 32
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ggccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 33
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
cgatggtcgc catcacgggc caggta 26
<210> 34
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
cggtgccgat catgcggc 18
<210> 35
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
caccaggagc atgtgctgcc tatgatccta a 31
<210> 36
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gatagagcac atacaaacat catag 25
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
cccaaaccca gaaaccctcg 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gctggtgctt tgccaacata 20
<210> 39
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ccccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaaa tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gaggtccccc gccgcatgat 20
<210> 41
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
agacgaggtc ccccgccgca tga 23
<210> 42
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
acaaatcatg cggcggggga cctc 24
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gaggtccccc gccgcatgat 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ctcgggatag ttggcacaaa 20
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gccaaatggg ggcgcattca 20
<210> 46
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
agacgccaaa tgggggcgca ttca 24
<210> 47
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
acaatgaatg cgcccccatt tggc 24
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
gccaaatggg ggcgcattca 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
ctcgggatag ttggcacaaa 20
<210> 50
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gaagtgcttg acattgggga gt 22
<210> 51
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
agatgttggc gacctcgtat t 21
<210> 52
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ccccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720
tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020
ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200
gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320
gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680
tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860
gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactatgt attaa 1935

Claims (8)

1.植物ALS突变型蛋白，其特征在于，其氨基酸序列如SEQ ID NO.8~14任一所示。
2.一种核酸，其特征在于，其编码权利要求1所述的ALS突变型蛋白。
3.一种生物材料，其特征在于，其为包含权利要求2所述核酸的表达盒、载体或微生物细胞。
4.权利要求1所述的ALS突变型蛋白或权利要求2所述的核酸或权利要求3所述的生物材料的如下任一应用：
（1）在使植物具有ALS抑制剂类除草剂抗性或提高植物对ALS抑制剂类除草剂抗性中的应用；
（2）在降低ALS抑制剂类除草剂对植物药害中的应用；
（3）在杂草防治中的应用；
（4）在植物遗传育种中的应用；
（5）在植物种质资源改良中的应用。
5.一种抗除草剂植物的制备方法，其特征在于，使所述植物含有权利要求2所述的核酸或使所述植物表达权利要求1所述的ALS突变型蛋白。
6.根据权利要求5所述的方法，其特征在于，通过基因编辑、杂交、回交、自交或无性繁殖的方法使所述植物含有权利要求2所述的核酸或使所述植物表达权利要求1所述的ALS突变型蛋白。
7.一种防治杂草或降低除草剂对植物药害的方法，其特征在于，在植物种植过程中施用有效剂量的除草剂；所述植物含有权利要求2所述的核酸或表达权利要求1所述的ALS突变型蛋白。
8.根据权利要求7所述的方法，其特征在于，所述除草剂为咪唑啉酮类、磺酰脲类、嘧啶水杨酸类、三唑嘧啶类和三唑啉酮类除草剂中的一种或多种。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107217044A (zh) * 2017-08-03 2017-09-29 江苏省农业科学院 使植物具有除草剂抗性的水稻als突变型蛋白及其应用
CN107964543A (zh) * 2017-12-25 2018-04-27 江苏省农业科学院 水稻除草剂抗性als突变型蛋白、核酸及其应用
CN111593031A (zh) * 2020-05-07 2020-08-28 海南波莲水稻基因科技有限公司 水稻ALS突变基因、含有该基因的植物转基因筛选载体pCALSm3及其应用

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CN107217044A (zh) * 2017-08-03 2017-09-29 江苏省农业科学院 使植物具有除草剂抗性的水稻als突变型蛋白及其应用
WO2019024534A1 (zh) * 2017-08-03 2019-02-07 江苏省农业科学院 使植物具有除草剂抗性的水稻als突变型蛋白及其应用
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CN111593031A (zh) * 2020-05-07 2020-08-28 海南波莲水稻基因科技有限公司 水稻ALS突变基因、含有该基因的植物转基因筛选载体pCALSm3及其应用

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