CN117616128A - C-20萜醇的重组生产 - Google Patents

C-20萜醇的重组生产 Download PDF

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CN117616128A
CN117616128A CN202280047957.4A CN202280047957A CN117616128A CN 117616128 A CN117616128 A CN 117616128A CN 202280047957 A CN202280047957 A CN 202280047957A CN 117616128 A CN117616128 A CN 117616128A
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M·J·贝克威尔德
M·Q·斯泰尔斯
H·J·博施
A·M·沃斯
A·M·M·L·范豪韦林根
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Abstract

披露了一种生产至少一种C‑20萜醇的方法，该方法包括将香叶基香叶基焦磷酸转化为柯巴基二磷酸(CPP)或半日花‑13‑烯‑8‑醇二磷酸(LPP)并将CPP或LPP转化为至少一种C‑20萜醇的步骤，其中所述转化是通过表现出二萜醇合酶活性的多肽实施的，该二萜醇合酶活性能够将CPP转化为泪杉醇、将LPP转化为香紫苏醇和/或将LPP转化为冷杉醇，其中所述多肽包含如权利要求中指定的氨基酸序列。本发明进一步涉及上述表现出二萜醇合酶活性的多肽以及包含所述多肽的融合蛋白，编码其的多核苷酸，包含所述多核苷酸的载体或基因构建体，包含所述载体或基因构建体的宿主细胞，包含该多核苷酸、载体、基因构建体或宿主细胞的非人转基因生物体，以及其用于生产至少一种C‑20萜醇的用途。

Description

C-20萜醇的重组生产
本发明涉及C-20萜醇的重组生产领域。特别地，本发明涉及生产至少一种C-20萜醇的方法，该方法包括将香叶基香叶基焦磷酸转化为柯巴基二磷酸(CPP)或半日花-13-烯-8-醇(labda-13-en-8-ol)二磷酸(LPP)并将CPP或LPP转化为至少一种C-20萜醇的步骤，其中所述转化是通过表现出二萜醇合酶活性的多肽实施的，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇(manool)、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇(sclareol)和/或将LPP转化为冷杉醇(abienol)，其中所述多肽包含选自由以下组成的组的氨基酸序列：a)如SEQ ID NO:3至7或34中任一个所示的氨基酸序列；b)与如SEQID NO:3至7或34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；c)由如SEQ ID NO:1或2或35所示的核酸序列编码的氨基酸序列；d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或2或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。本发明进一步涉及上述表现出二萜醇合酶活性的多肽以及包含所述多肽的融合蛋白，编码其的多核苷酸，包含所述多核苷酸的载体或基因构建体，包含所述载体或基因构建体的宿主细胞，包含该多核苷酸、载体、基因构建体或宿主细胞的非人转基因生物体。然而，本发明考虑所述多肽、融合多肽、多核苷酸、载体或基因构建体、宿主细胞或非人转基因生物体用于生产至少一种C-20萜醇(优选地冷杉醇、泪杉醇和/或香紫苏醇)的用途。进一步，本发明涵盖用于生产至少一种C-20萜醇(优选地冷杉醇、泪杉醇和/或香紫苏醇)的试剂盒。
香紫苏醇((+)-香紫苏醇)、冷杉醇(Z-冷杉醇)和泪杉醇((+)-泪杉醇)是半日花烷型二萜成员。二萜是C-20类异戊二烯，并且天然存在于植物和微生物中。半日花烷型二萜分子具有商业价值，因为它们可以转化为琥珀香调(amber note)，应用于香精工业。琥珀香调的实例包括琥珀缩酮(amberketal)、泪杉醇酮(manool ketone)、降龙涎香醚(ambroxide)和香紫苏内酯(sclareolide)。为了将二萜分子转化为琥珀香调，已经披露了几种化学或生物催化途径。香紫苏醇可以被转化为降龙涎香醚(例如Barrero等人1993,tetrahedron[四面体]49,10405-10412；Farbood EP 0 204 009 B1)或转化为香紫苏内脂(Farbood EP 0419 026A1)。泪杉醇可以被转化为琥珀缩酮，例如US 7,294,492(隐球菌属)，或转化为泪杉醇酮(EP 1 688 501 B1)；冷杉醇可以被转化为降龙涎香醚(例如Barrero等人1993,tetrahedron[四面体]49,10405-10412)或转化为香紫苏内酯(US 5,525,728)。
这些化合物的植物来源包括南欧丹参(Salvia sclarea)和心叶烟(Nicotianaglutinosa)(香紫苏醇)；二型白袍杉(Halocarpus biformis)(粉红松或黄松)(泪杉醇)；香脂冷杉(Balsam fir)(香脂冷杉(Abies balsamea))(冷杉醇)。
编码用于产生二萜的萜类环化酶的基因已被广泛描述(Zerbe,TrendsBiotechnol[生物技术趋势]2015年7月；33(7):419-28.)，并且这些化合物的微生物生产已得到证实(例如Schalk J.Am.Chem.Soc.[美国化学学会杂志]2012,134,18900-18903)。二萜生物合成从香叶基香叶基焦磷酸(GGPP)开始。GGPP广泛存在于自然界中，因为它是类胡萝卜素、植物激素等的前体。GGPP合酶是众所周知的，包括例如来自聚球藻菌属物种(Synechococcus sp.)PCC 7002、酿酒酵母(Saccharomyces cerevisiae)、辣薄荷(Menthapiperita)、拟南芥(Arabidopsis thaliana)的crtE(Feng Front.Plant Sci[植物科学前沿],2020年5月25日及其中的参考文献)，以及来自谷氨酸棒杆菌(Corynebacteriumglutamicum)的idsA基因(Heider FEBS Journal[二月刊]281(2014)4906-4920)。
二萜生物合成从GGPP开始，通常由两个步骤介导：步骤1形成环化二磷酸盐(例如，半日花-13-烯-8-醇二磷酸或LPP、柯巴基-PP或CPP)，步骤2将这一底物转化为最终产物。步骤1通常是通过II型二萜合酶实施的，而步骤2是通过I型二萜合酶实施的。已知有II型合酶实施这两个步骤，例如来自香脂冷杉的冷杉醇合酶(Zerbe JOURNAL OF BIOLOGICALCHEMISTRY[生物化学杂志]第287卷,第15期,第12121-12131页,2012年4月6日)。步骤1酶通常是αβγ结构域蛋白，其特征在于γ结构域中存在DXDD基序。步骤2酶可以是αβγ结构域蛋白或αβ结构域蛋白，其特征在于β结构域中存在DDXXD基序。二萜合酶综述见Zerbe等人,Trends in Biotechnology[生物技术趋势],2015,33(7),419-428。
对于相关二萜的生物合成，已描述了以下基因：
对于香紫苏醇，来自南欧丹参的LPP合酶(LPPS)和香紫苏醇合酶(SS)(Caniard等人BMC Plant Biology[BMC植物生物学]2012,12:119；Schalk WO 2009/101126)，LPPS是一种αβγ蛋白(II型)，SS是一种αβ蛋白(I型)，Ignea等人(Metabolic Engineering[代谢工程]27(2015),65-75)已证明仅用LPPS和来自心叶烟的相似酶即可在酵母中合成香紫苏醇(Julien，WO 2014/022434 A1)。
对于冷杉醇，来自烟草(nicotiana tabacum)的LPPS和ABS(Salaud,The PlantJournal[植物杂志](2012)72,1-17；WO 2008/07031A1)、香脂冷杉ABS，其可实施两个步骤(Zerbe JOURNAL OF BIOLOGICAL CHEMISTRY VOL.[生物化学杂志]第287卷,第15期,第12121-12131页,2012年4月6日)，以及冷杉属ABS和烟草ABS或鼠尾草属SS(WO 2016/94178A1)。
对于泪杉醇，来自普通小麦(Triticum aestivum)或丹参(SalviaMiltiorrhiza)、或细疣篮状菌(Talaromyces verruculosus)或毛喉鞘蕊花(ColeusForskohlii)、欧夏至草(Marrubium vulgare)、迷迭香(Rosmarinus officinale)的步骤1CPPS；以及步骤2鼠尾草属SS(US2019/0352673)、来自毛喉鞘蕊花的步骤1CPPS；来自马郁兰(Origanum majorana)的步骤2OmTPS4(Johnson J.Biol.Chem.[生物化学杂志](2019)294(4)1349-1362；WO 2020/028795)。
已描述了用于生产香紫苏醇、泪杉醇或冷杉醇的工程微生物。这包括引入以下遗传元件：
例如从Feng Front.Plant Sci.[植物科学前沿],2020年5月25日中描述的GGPP合酶组中选择GGPP合酶。此外，还使用了CrtE型微生物酶来产生GGPP，例如来自成团泛菌(Pantoea agglomerans)的crtE(AAA24819)(Schalk J.Am.Chem.Soc.[美国化学会志]2012,134,18900-18903)。已证明棒杆菌属IdsA具有极高的催化效率(Heider FEBSJournal[二月刊]281(2014)4906-4920)。
在现有技术中，从不同来源选择生成LPP或(+)-CPP的步骤1基因。来自南欧丹参(Caniard等人.BMC Plant Biology[BMC植物生物学]2012,12:119；Schalk WO 2009/101126)、心叶烟(WO 2014/022434 Allylix)的LPPS，来自毛喉鞘蕊花的CfLPPS(PaterakiPlant Physiol.[植物生理学报],164,1222-1236；WO 2015/091943)，来自烟草的NtLPPS(Salaud,The Plant Journal[植物杂志](2012)72,1-17；WO 2008/07031A1)，来自胶草(Grindelia hirsutula)的GhLPPS，来自雷公藤(Tripterygium wilfordii)的TwLPPS，来自克里特岩蔷薇(Cistus creticus)的CcLPPS(Falara,Plant Physiology[植物生理学报],2010,第154卷,第301-310页)。还使用了来自普通小麦或丹参或细疣篮状菌或毛喉鞘蕊花、欧夏至草、迷迭香的CPPS(US2019/0352673)。
Ma和同事描述了台湾杉(Taiwania cryptomerioides)的二萜合酶的生物化学特性(Ma Li-Ting等人,The Plant Journal[植物杂志],第100卷,第6期,1254-1272)。特别地，表征了先前未在裸子植物中观察到的5种单官能diTPS功能，包括形成半日花-13-烯-8-醇二磷酸(LPP，TcCPS2)和(+)-柯巴基二磷酸(CPP，TcCPS4)的单官能II类酶，以及三种分别产生两形体烯(biformene)(TcKSL1)、左旋海松二烯(TcKSL3)和phyllocladanol(TcKSL5)的I类diTPS。然而，这些二萜合酶均未显示出二萜醇合酶活性，更不会产生香紫苏醇、泪杉醇或冷杉醇。
事实上，生成香紫苏醇、泪杉醇或冷杉醇的步骤2步基因是罕见的。已知南欧丹参香紫苏醇合酶与CPPS(US 2019/0352673)组合时可产生泪杉醇。来自马郁兰的OmTPS4是一种与CPPS组合的泪杉醇合酶，但与LPPS组合不会产生香紫苏醇，而会产生泪柏醚(manoyloxide)(Johnson 2019)。Jia(ACS Catal.[ACS催化]2018,8,3133-3137)披露了通过将残基N431突变为I、D或E可以将鼠尾草紫杉醇合酶转化为异冷杉醇合酶：通过突变N431Q可以将其从13R-香紫苏醇合酶改变为13S-香紫苏醇合酶。他们声称，香紫苏醇合酶的特殊之处在于在该残基周围的产物结果决定区中含有天冬酰胺(N431)，这是向labdanoyl-PP加水形成香紫苏醇的关键。烟草冷杉醇合酶与来自灌状鼠尾草(Salvia fruticosa)的CPPS组合产生Z-两形体烯。Jia等人将来自南欧丹参的香紫苏醇合酶与来自不同物种的许多步骤2二萜合酶进行了比对，其中包括来自毛喉鞘蕊花的泪柏醚合酶(GenBank登录号：KF444508)；1IrMS，来自碎米桠(Isodon rubescens)的次丹参酮二烯合酶(KX831652)；CfMS，来自毛喉鞘蕊花的次丹参酮二烯合酶(KF444509)；RoMS1，来自迷迭香(Rosemariusofficinalis)的次丹参酮二烯合酶1(KF805858)；SmMS，来自丹参的次丹参酮二烯合酶(ABV08817)；RoMS1，来自迷迭香的次丹参酮二烯合酶(KF805859)；SfMS，来自灌状鼠尾草的次丹参酮二烯合酶(KP091841)；MvELS，来自欧夏至草的9,13-环氧-半日花-14-烯合酶(KJ584454)。据报告，SsSS的残基N438决定了产生13-羟基化半日花烷型二萜(例如香紫苏醇或泪杉醇)的能力。
虽然在现有技术中已报告了多种编码步骤2酶的基因，但仍需要可以用于在C-20萜醇，以及特别是冷杉醇、香紫苏醇和/或泪杉醇的生产中催化步骤2反应的高效酶。此外，希望具有不限于仅生产一种C-20萜醇的酶。
本发明的基本技术问题应被视为提供符合上述需求的手段和方法。该技术问题通过权利要求书及本文下文中描述的实施例得到解决。
因此，本发明涉及生产至少一种C-20萜醇的方法，该方法包括以下步骤：
a)将香叶基香叶基焦磷酸转化为柯巴基二磷酸(CPP)或半日花-13-烯-8-醇二磷酸(LPP)；以及
b)将CPP或LPP转化为至少一种C-20萜醇，其中所述转化是通过表现出二萜醇合酶活性的多肽实施的，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，
其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或SEQ ID NO:35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或SEQ ID NO:35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
应理解，在说明书和权利要求书中，“一个”(“a”或“an”)可以意指以下提及的一个或多个项目，这取决于其所使用的上下文。因此，例如，提及“一个”项目可能意味着可以使用至少一个项目。
如以下所使用的，术语“具有”、“包含”或“包括”意指具有非限制性含义或限制性含义。因此，具有限制含义的这些术语可以指除了由这些术语引入的特征之外，在所描述的实施例中不存在其他特征的情况，即，术语具有“由……组成”或“基本上由……组成”的意义上的限制含义。具有非限制性含义的术语是指除了由这些术语引入的特征之外，在所描述的实施例中还存在一个或多个其他特征的情况。
进一步，如以下所使用的，术语“优选地”、“更优选地”、“最优选地”、“特别地”、“更特别地”、“典型地”和“更典型地”与特征结合使用，以指示这些特征是优选的特征，即，术语应指示根据本发明还可以设想替代特征。
进一步，应理解，本文所用的术语“至少一个”意指可根据本发明使用该术语之后提及的一个或多个项目。例如，如果术语表示应使用至少一个项目，则可将其理解为一个项目或一个以上项目，即两个、三个、四个、五个或任何其他数字。根据术语所指的项目，本领域技术人员理解该术语可以指代的上限(如有)。
根据本发明的方法可以由以上所提及的步骤(a)和(b)组成，或可包含额外的步骤。这样的额外的步骤可以是预处理步骤或生产C-20萜醇所需的步骤，例如纯化步骤。
如本文所用，术语“生产”是指从CPP或LPP(CAS号1000876-36-7)生成至少一种C-20萜醇，特别是环状C-20萜醇，更优选地泪杉醇、香紫苏醇和/或冷杉醇。该生产可产生所述至少一种C-20萜醇的任何纯度。设想的纯度越高，需要的额外的纯化将越多。该方法可以离体实施，例如在一个或多个反应瓶中实施。可替代地，方法可全部或部分在生物体中实施，例如包括本文其他地方提及的宿主细胞的微生物或包括植物的非人转基因生物体。
根据本发明使用的术语“C-20萜醇”涉及包含乙醇部分的C-20萜类化合物。萜类是聚合异戊二烯。萜类化合物可能具有另外的功能化学部分。C-20萜类化合物也称为二萜类化合物或二萜。优选地，根据本发明提及的所述至少一种C-20萜醇是环状C-20萜醇。更优选地，其是泪杉醇(CAS号596-85-0，分子式C20H34O)、香紫苏醇(CAS号515-03-7，分子式C20H36O2)或冷杉醇(CAS号17990-16-8，分子式C20H34O)。
根据本发明使用的术语“多肽”是指通过肽键彼此连接的氨基酸的连续序列。根据本发明的多肽典型地包含至少50个、至少100个或至少200个长度的氨基酸，使得氨基酸链可以形成发挥本文其他地方提及的一种或多种酶活性所需的三维空间结构。术语“蛋白质”在本文中可以互换使用。
如本文所用，术语“二萜醇合酶活性”是指允许将起始物料(例如LPP或CPP)转化为C-20萜醇的酶的活性。二萜合酶经历复杂的亲电循环形成和/或重排，生成不同的主链结构。二萜合酶可以分类为I类酶，其使用萜二磷酸盐作为底物，而萜二磷酸盐是由II类酶从香叶基香叶基磷酸生成的。以上所提及的具有二萜醇合酶活性的多肽典型地是I型酶。优选地，所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)(CAS号1000876-36-7)转化为香紫苏醇和/或将LPP(CAS号1000876-36-7)转化为冷杉醇。优选地，根据本发明的具有二萜醇合酶活性的多肽包含如SEQ ID NO:24所示的保守区域或与SEQ ID NO:24具有一个或几个氨基酸改变的序列，其中SEQ ID NO:24的位置4处的丝氨酸是保守的或被苏氨酸替换；优选地，这一位置处的丝氨酸是保守的。
此外，根据本发明的具有二萜醇合酶活性的多肽包含Pfam结构域PF01397.23(萜合酶，N-末端结构域)、PF03936.18(萜合酶家族，金属结合结构域)和PF19086.2(萜合酶家族2，C-末端金属结合)(PFAM 35.0版)；参见Pfam:The protein families database in2021[2021年Pfam蛋白家族数据库]:J.Mistry,S.Chuguransky,L.Williams,M.Qureshi,G.A.Salazar,E.L.L.Sonnhammer,S.C.E.Tosatto,L.Paladin,S.Raj,L.J.Richardson,R.D.Finn,A.Nucleic Acids Research[核酸研究](2020)doi:10.1093/nar/gkaa913。
根据本发明的表现出二萜醇合酶活性的多肽，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，该多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
优选地，所述表现出二萜醇合酶活性的多肽能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇。更优选地，所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:4、6、7、9、10或SEQ ID NO:34所示的氨基酸序列；
b)与如SEQ ID NO:4、6、7、9、10或SEQ ID NO:34所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:2、16、17或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:2、16、17或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇的二萜醇合酶活性的多肽。
还优选地，所述表现出二萜醇合酶活性的多肽能够将LPP转化为冷杉醇。更优选地，所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3、5或8所示的氨基酸序列；
b)与如SEQ ID NO:3、5或8所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1或18所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或18所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
上文所提及的序列同一性定义了氨基酸序列或核酸序列之间的关系，并且可通过比较这些序列来确定。通常，通过在序列的整个长度上比较两个序列确定序列同一性，但还可以仅比较与彼此比对的序列的一部分。优选地，本文中在序列的整个长度上比较序列同一性。序列同一性是指多肽序列或核酸序列之间的关联程度。它将以彼此比较的两个序列中相同氨基酸或核苷酸的百分比来表示。因此，在比对两个序列时，通常确定这些序列之间匹配的氨基酸或核苷酸的数量，并将其与比对序列或序列部分中的氨基酸或核苷酸的总数相关联。例如，当与亲本序列(即，如SEQ ID No:3至7或SEQ ID NO:34中任一个所示的氨基酸序列，或如SEQ ID NO:1或2或35所示的核酸序列)比较时，变体序列可通过其序列同一性来定义。为了在第一步中确定两个序列之间的同一性百分比，在这两个序列之间生成成对序列比对，其中这两个序列在它们的完整、全部或全长上比对(即，成对全局比对)。比对是用本文所述的程序或软件生成的。用于本发明目的的优选比对是可以从中确定最高序列同一性的比对。
序列比对可以用许多软件工具生成，例如Needleman和Wunsch算法-Needleman,Saul B.和Wunsch,Christian D.(1970).“A general method applicable to the searchfor similarities in the amino acid sequence of two proteins[适用于寻找两种蛋白质氨基酸序列相似性的通用方法]”.Journal of Molecular Biology[分子生物学杂志]48(3):443-453。例如，该算法被实现到“NEEDLE”程序中，该程序执行两个序列的全局比对。NEEDLE程序包含在例如欧洲分子生物学开放软件套件(European Molecular BiologyOpen Software Suite，EMBOSS)中。EMBOSS-acollection of various programs:TheEuropean Molecular Biology Open Software Suite(EMBOSS)[EMBOSS-各种程序的集合：欧洲分子生物学开放软件套件(EMBOSS)],Trends in Genetics[遗传学趋势]16(6),276(2000)。BLOSUM(BLOcks替代矩阵)-典型地基于保守区域(例如，蛋白质结构域)的比对而生成(Henikoff S,Henikoff JG:Amino acid substitution matrices from proteinblocks.[来自蛋白质嵌段的氨基酸取代矩阵]Proceedings of the National Academy ofSciences of the USA.[美国国家科学院院刊]1992年11月15日；89(22):10915-9)。许多BLOSUM中的一个是“BLOSUM62”，其通常是许多程序在比对蛋白质序列时的“默认”设置。BLAST(基本局部比对搜索工具)-由几个单独的程序(BlastP、BlastN)组成，主要用于在大型序列数据库中搜索相似的序列。BLAST程序还创建局部比对。通常使用的是由NCBI(美国国家生物技术信息中心(National Centre for Biotechnology Information))提供的“BLAST”界面，它是改进版本(“BLAST2”)。“原始”BLAST：Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.和Lipman,D.J.(1990)“Basic local alignment search tool.[基本局部比对搜索工具]”J.Mol.Biol.[分子生物学杂志]215:403-410；BLAST2：Altschul,StephenF.,Thomas L.Madden,Alejandro A.Schaffer,Jinghui Zhang,Zheng Zhang,WebbMiller,和David J.Lipman(1997),“Gapped BLAST and PSI-BLAST:a new generation ofprotein database search programs[空位型BLAST和PSI-BLAST：新一代蛋白质数据库搜索程序]”,Nucleic Acids Res.[核酸研究]25:3389-3402。
如本文所用，序列同一性优选地是通过EMBOSS成对比对算法“Needle”确定的值。特别地，可以使用EMBOSS包中的NEEDLE程序(版本2.8.0或更高版本，EMBOSS：欧洲分子生物学开放软件套件-Rice,P.,等人Trends in Genetics[遗传学趋势](2000)16:276-277；http://emboss.bioinformatics.nl)，使用NOBRIEF选项(与NO的‘简化同一性和相似性’)计算“最高同一性(longest-identity)”。在如下这样的情况下，计算两个比对序列之间的同一性：在比对中显示两个序列中相同氨基酸的对应位置的数量除以减去比对中空位总数后的比对总长度。对于氨基酸序列的比对，默认参数是：矩阵＝Blosum62；开放空位罚分＝10.0；空位延伸罚分＝0.5。对于核酸序列的比对，默认参数是：矩阵＝DNAfull；开放空位罚分＝10.0；空位延伸罚分＝0.5。
本文所提及的变体氨基酸或核酸序列可以是天然存在的变体，例如等位基因变体或直系同源、旁系同源或同源变体。可替代地，这样的序列可以是人工生成的，例如通过本领域技术人员已知的生物技术(比如，例如分子进化或合理设计)或通过使用本领域已知以及本文其他地方描述的诱变技术(随机诱变、位点定向诱变、定向进化、基因重组等)来试图改善酶或核酸的特性(例如，改善酶的表达或增加酶的酶活性)。
编码由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列，或由与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的核酸序列编码的氨基酸序列的变体核酸序列可能出于本文其他地方列出的原因由于至少一个核苷酸置换、添加和/或缺失而与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列不同。应理解，包含本文所提及的这样的变体核酸序列的多核苷酸优选地能够在严格杂交条件下彼此杂交。本文所提及的严格杂交条件优选地是在约45℃下的6x氯化钠/柠檬酸钠(SSC)，然后是在50℃至65℃下在0.2x SSC、0.1％ SDS中进行的一个或多个洗涤步骤。本领域技术人员知道，这些杂交条件根据核酸的类型以及例如当存在有机溶剂时的缓冲液的温度和浓度而不同。例如，在“标准杂交条件”下，在浓度为0.1至5x SSC(pH 7.2)的水性缓冲液中，温度根据核酸类型而不同，温度在42℃至58℃之间。如果上述缓冲液中存在有机溶剂，例如50％甲酰胺，则标准条件下的温度约为42℃。DNA:DNA杂合体的杂交条件优选地是0.1x SSC和20℃至45℃，优选地30℃至45℃之间。DNA:RNA杂合体的杂交条件优选地是0.1x SSC和30℃至55℃，优选地45℃至55℃之间。上述杂交温度是例如在不存在甲酰胺的情况下针对长度约为100bp(＝碱基对)且G+C含量为50％的核酸确定的。本领域技术人员知道如何通过参考教科书(例如上述教科书或以下教科书)来确定所需的杂交条件：Sambrook等人,“Molecular Cloning”[分子克隆],Cold Spring Harbor Laboratory[冷泉港实验室],1989；Hames和Higgins(编辑)1985,“Nucleic Acids Hybridization:A Practical Approach”[核酸杂交：一种实用方法],IRLPress at Oxford University Press[牛津大学出版社IRL出版社],牛津；Brown(编辑)1991,“Essential Molecular Biology:APractical Approach”[基本分子生物学：一种实用方法],IRL Press at Oxford University Press[牛津大学出版社IRL出版社],牛津。因此，变体核酸序列可以来源于多核苷酸，这些多核苷酸能够在严格杂交条件下与以下核酸序列杂交，这些核酸序列编码由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列，或与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的核酸序列编码的氨基酸序列。
在另一实施例中，本发明的多肽包含图5或图6所示位置处的保守氨基酸，并且优选地图6中给定位置处的保守氨基酸。保守氨基酸位置在图5和图6中用黑色背景上白色字体的字母表示。
发现本发明的表现出二萜醇合酶活性的多肽典型地在N-末端区域中包含一系列氨基酸，其单字母编码是EKKSFGSMCI(SEQ ID NO:56)或ENKSFGSMCI(SEQ ID NO:58)或ENNSFGSMCI(SEQ ID NO:55)或EKNSFGSMCI(SEQ ID NO:57)。优选地，本发明的多肽包含如SEQ ID NO:56或58所示的序列。如本文所提及的，分别用天冬酰胺替换这一序列段中的第一个赖氨酸，或用赖氨酸替换该序列段中的天冬酰胺，对生产至少一种C-20萜醇的酶的性能没有显著影响。
本发明的表现出二萜醇合酶活性的多肽片段可以是包含上述序列和具有足够长度以表现出上文指定的二萜醇合酶活性的序列变体的任何氨基酸序列的多肽。在此上下文中，已根据本发明鉴定出以上所提及的多肽的保守区域。这一区域(在SEQ ID NO:24中示出或与SEQ ID NO:24具有一个或几个氨基酸改变的序列，其中SEQ ID NO:24的位置4处的丝氨酸是保守的或被苏氨酸替换-优选地所述丝氨酸是保守的)位于SEQ ID NO:3中的氨基酸486至氨基酸497处或SEQ ID NO:4中的氨基酸486至氨基酸497处。根据本发明的表现出二萜醇合酶活性的多肽中的这一区域不同于其他合酶中的同源产物决定区，并且特别地不同于已知的鼠尾草香紫苏醇合酶。因此，优选地设想具有上述多肽的生物活性的片段包含上文指定的保守产物决定区的氨基酸序列。典型地，片段包含或由至少20、至少30、至少40、至少50、至少100、至少150或至少200个长度的连续氨基酸组成，这些连续氨基酸来自本发明的上述序列或序列变体并且可提供二萜醇合酶活性。
上述表现出二萜醇合酶活性的上述多肽(其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇)还可以包含在融合多肽中。除了表现出二萜醇合酶活性的多肽的氨基酸序列外，这样的融合多肽还包含一个或多个额外的氨基酸序列。所述额外的氨基酸序列可以是例如具有其他酶活性(例如用于催化步骤1的II型二萜合酶活性)的多肽、对表现出二萜合酶活性的多肽的功能具有支持功能的多肽、或具有用于例如监测正确表达或用于纯化目的的标记或标签功能(例如标签(例如，MYC标签、FLAG标签、His标签等)或荧光蛋白(例如，GFP、BFP、YFP或CFP))的多肽或肽。
进一步，本披露涉及用于制备C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇的方法，该方法包括在存在酶的情况下分别将柯巴基二磷酸(CPP)和/或半日花-13-烯-8-醇二磷酸(LPP)转化为C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇，该酶包含含有标签肽的第一区段和含有根据本发明的二萜醇合酶的第二区段。包含所述第一和所述第二区段的酶在本文中可称为“标记的酶”。
标签肽优选地选自下组：氮利用蛋白(NusA)、硫氧还蛋白(Trx)、麦芽糖结合蛋白(MBP)、谷胱甘肽S-转移酶(GST)、小泛素样修饰物(SUMO)或钙结合蛋白(Fh8)及其功能同源物。如本文所用，标签肽的功能同源物是与未标记的酶相比，对标记的酶的溶解度具有至少约相同影响的标签肽。典型地，同源物的不同之处在于，一个或多个氨基酸从作为其同源物的肽中插入、取代、缺失，或延伸至其同源物的肽上。特别地，同源物可以包含亲水性氨基酸对另一种亲水性氨基酸的一个或多个取代，或疏水性氨基酸对另一种疏水性氨基酸的一个或多个取代。特别地，同源物可以与NusA、Trx、MBP、GST、SUMO或Fh8的序列具有至少40％，更特别地至少50％，优选地至少55％，更优选地至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％序列同一性。
特别适合的是来自大肠杆菌(Escherichia coli)的麦芽糖结合蛋白或其功能同源物。
特别地，根据本发明的标记的酶的用途是有利的，因为它可以有助于增加萜类化合物或萜(例如C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇)的产生，尤其是增加细胞产生。
为了改善标记的酶的溶解度(与没有标签的酶相比)，酶的第一区段优选地在其C-末端与第二区段的N-末端结合。可替代地，标记的酶的第一区段在其N-末端与第二区段的C-末端结合。
进一步，本发明涉及包含编码多肽的核苷酸序列的核酸，该多肽包含含有标签肽(优选地MBP、NusA、Trx、GST、SUMO或Fh8-标签或这些中任一种的功能同源物)的第一区段，以及含有任何二萜醇合酶的第二区段。第二区段可以例如包含如SEQ ID NO:3至7、28至30、34或40至54中任一个所示的氨基酸序列或其功能类似物。
进一步，本发明涉及包含编码所述标记的二萜醇合酶的所述核酸的宿主细胞。根据本发明的编码标记的酶的特异性核酸在SEQ ID NO:8至SEQ ID NO:10和SEQ ID NO:28至30的任一个中示出。宿主细胞特别地可以包含含有这些序列中的任一个或其功能类似物的基因。
进一步，本发明涉及一种酶，该酶包含含有标签肽的第一区段和含有具有用于将聚戊二烯基二磷酸转化为萜，特别是二萜醇合酶的酶活性的多肽的第二区段，该标签肽优选地选自MBP、NusA、Trx或SET的组。包含根据本发明的标记的酶的特异性酶在SEQ ID NO:8至SEQ ID NO:10和SEQ ID NO:28至30的任一个中示出。
优选地，融合蛋白应进一步包含表现出II型二萜合酶的酶活性的多肽。步骤a)中的转化是通过另外的多肽实施的，该另外的多肽表现出将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP的II型二萜合酶的酶活性。因此，表现出二萜合酶活性的多肽优选地包含在融合多肽中，该融合多肽包含至少一种另外的多肽，该另外的多肽表现出II型二萜合酶的酶活性，优选地，将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP，该另外的多肽具有麦芽糖结合特性，或者该另外的多肽是硫氧还蛋白或硫氧还蛋白融合蛋白。更优选地，所述另外的多肽选自由以下组成的组：LPP合酶(优选地来自毛喉鞘蕊花(CfLPPS)(Pateraki,PlantPhysiol.[植物生理学报],164,1222-1236(2014)；WO 2015/091943)或烟草(NtLPPS)(Salaud,The Plant Journal[植物杂志](2012)72,1-17；WO 200807031A1))、CPP合酶(优选地来自毛喉鞘蕊花(CfCPPS)(Johnson,J.Biol.Chem.[生物化学杂志](2019)294(4)1349-1362；WO 2020028795))、硫氧还蛋白和麦芽糖结合蛋白(MBP)。
在本发明的方法的步骤a)中，将香叶基香叶基焦磷酸盐转化为柯巴基二磷酸(CPP)或半日花-13-烯-8-醇二磷酸(LPP)。典型地，所述转化是通过酶促实施的。能够将香叶基香叶基磷酸转化为CPP或LPP的酶是本领域熟知的。优选地，转化是通过多肽实施的，该多肽表现出将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP的II型二萜合酶，更优选地LPP合酶(优选地来自毛喉鞘蕊花(CfLPPS)或烟草(NtLPPS))、CPP合酶(优选地来自毛喉鞘蕊花(CfCPPS))的酶活性。应理解，表现出II型二萜合酶活性的多肽与本发明的表现出二萜醇合酶活性的多肽一起包含在融合多肽中，如本文其他地方更详细描述的。
上述步骤a)可以在体外实施，即在包含上述转化所需的所有组分的合适的反应瓶中实施。本领域技术人员非常了解如何调节反应条件，以便有效地实施反应。例如，合适的缓冲液可用于在具有合适pH和合适盐浓度的环境中提供组分。在这样的情况中也可应用合适的温度，无需赘述。
可替代地，步骤a)可以在宿主细胞中实施，如本文其他地方所述。应理解，宿主细胞应能够产生上文指定的GGP和II型转化酶。如有必要，需要对宿主细胞进行遗传修饰，以表达这样的II型酶或GGP合成所需的其他酶或蛋白。宿主细胞的培养条件和时间应足以允许表达上述酶并将GGP转化为CPP和/或LPP。在下面的随附实例中也描述了特别优选的条件。
然而，本发明的方法的步骤a)还可以在生物体中实施，典型地多细胞生物体，例如本文其他地方提及的转基因非人生物体。典型地，所述生物体经遗传修饰以表达将GGP转化为CPP和/或LPP所需的II型酶。
在本发明的方法的步骤b)中，将CPP或LPP转化为至少一种C-20萜醇的步骤，其中所述转化是通过表现出二萜醇合酶活性的多肽实施的，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，优选地通过本发明的二萜醇合酶。
本发明的方法的步骤b)还可在体外或在宿主细胞或生物体中实施，如上文对于步骤a)所指定。在下面的随附实例中描述了特别优选的条件。
优选地，所述步骤b)或所述步骤a)和b)在宿主细胞或非人转基因生物体中实施。更优选地，所述宿主细胞或非人转基因生物体是本发明的宿主细胞或非人转基因生物体，如本文其他地方更详细描述的。应理解，在宿主细胞或非人转基因生物体中实施步骤b)或步骤a)和b)需要应用的条件取决于所述宿主细胞或非人转基因生物体。然而，本领域技术人员非常了解取决于所给定宿主细胞或非人转基因生物体的选择需要应用哪些条件。
优选地，本发明的方法包括获得所述生产的至少一种C-20萜醇的步骤。
如本文所用，术语“获得”是指在步骤b)之后以任何纯度提供至少一种C-20萜醇。因此，该至少一种C-20萜醇可以基本纯的形式或作为包含额外的组分的组合物提供。因此，本发明的方法可以在步骤b)完成后涵盖一个或多个纯化步骤。需要应用的纯化技术取决于如何实施本发明的方法的步骤a)和/或b)。例如，如果这些步骤已在体外实施，即在使用分离的组分(例如分离的酶)、加合物和辅助组分(例如反应缓冲液)的反应小瓶中实施，应理解，为了获得例如基本上纯的至少一种C-20萜醇，需要较少的纯化。然而，如果步骤a)和b)在体内实施，即在本文其他地方定义的宿主细胞中实施，则可能需要进一步纯化和预处理步骤。典型地，需要收获宿主细胞并且可能必须裂解收获的细胞以从所述细胞中释放C-20萜醇。后续纯化步骤应去除细胞碎片，并旨在从剩余组分中纯化C-20萜醇。此外，如果步骤在动物或植物体内实施，则甚至可能需要另外的预处理和/或纯化步骤以获得至少一种C-20萜醇。本领域技术人员非常了解取决于实施步骤a)和b)的给定情况，合适的预处理和/或纯化步骤。所设想的纯化技术可以是萃取技术、层析(例如LC、GC或HPLC)、尺寸排阻层析、亲和层析、蒸馏、离心、过滤等。所设想的预处理步骤可以是收获、热处理、超声波处理、用化学品和/或酶处理等。在下面的随附实例中描述了特别优选的措施。
有利地，本发明的基础研究揭示，来自巨柏(Cupressa gigantea)的步骤2酶的家族(即Cup2v1和Cup2v2b)能够有效地将CPP和LPP转化为C-20萜醇泪杉醇、香紫苏醇和/或冷杉醇。特别地，发现当在例如红杆菌属(Rhodobacter)中表达时，Cup2v1和Cup2v2b酶在C-20萜醇的重组生产中特别有效，如以下随附实例中所述。此外，发现Cup2v2a和Cup2v2b酶，即具有如SEQ ID NO:4、6、7、9、10或34中任一个所示的氨基酸序列或如本文其他地方指定的其变体的多肽，能够产生两种C-20萜醇，即泪杉醇和香紫苏醇。Cup2v1，即具有如SEQ IDNO:3、5或8所示的氨基酸序列或本文其他地方指定的其变体的多肽，在冷杉醇的生产中是有效的。
由于本发明，可以更有效地生产C-20萜醇，特别是在重组生产方法中。
在一个实施例中，如果酶优先地产生一种或多种C-20萜醇，则认为该酶在本发明的方法中有用。在另一实施例中，优先地产生一种或多种C-20萜醇应理解为，当在适合酶具有活性的条件下酶与大量底物一起提供时，在该酶产生的产物中，该一种或多种C-20萜醇占优势。例如，从酶产生的所有分子中，50％以上的分子是C-20萜醇。
在另一个实施例中，本发明的表现出二萜醇合酶活性的多肽的特征在于，其优先地从CPP产生泪杉醇、和/或从LPP产生香紫苏醇和/或从LPP产生冷杉醇。
在另一实施例中，优先地产生泪杉醇、香紫苏醇和/或冷杉醇应理解为，当在适合酶具有活性的条件下酶与合适的底物(例如LPP或CPP)一起提供时，在该酶产生的产物中，泪杉醇、香紫苏醇和/或冷杉醇占优势。例如，从酶产生的所有分子中，50％以上的分子是以下任一种分子：泪杉醇、香紫苏醇或冷杉醇。
本发明进一步涉及用于生产芳香组合物的方法，该方法包括以下步骤：
a)根据本发明的方法，优选地根据权利要求1-5中任一项所述的方法，生产一种或多种C-20萜醇，优选地冷杉醇、泪杉醇和/或香紫苏醇，
b)任选地纯化所述一种或多种C-20萜醇，以及
c)用所述一种或多种C-20萜醇制备或配制芳香组合物。
本文所用的芳香组合物可以是例如香料、香精或香水；参见例如Chemistry andTechnology of Flavors and Fragrances[香料和香精的化学和技术],编辑:DavidJ.Rowe,首版出版日期:2004年10月26日,印刷ISBN:9781405114509|在线ISBN:9781444305517|DOI:10.1002/9781444305517,Blackwell Publishing Ltd[布莱克威尔出版有限公司]。
除非另有指定，否则本文之前对术语的定义和解释经必要修改后适用于本发明的以下实施例。
本发明还提供组合物或芳香组合物，该组合物包含通过本发明的方法获得的所述至少一种C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇。
此外，本发明涉及一种组合物，该组合物包含宿主细胞或非人转基因生物体，以及通过本发明的方法，优选地根据权利要求1-5中任一项所述的方法获得的所述至少一种C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇，其中宿主细胞或非人转基因生物体包含适于重组的至少一种表现出二萜醇合酶活性的多肽，该多肽具有以下氨基酸序列
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
然而，本发明还涉及表现出二萜醇合酶活性的多肽，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，所述多肽具有选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
优选地，所述二萜醇合酶活性能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇。更优选地，所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列；
b)与如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:2、16、17或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:2、16、17或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇的二萜醇合酶活性的多肽。
还优选地，所述二萜醇合酶活性能够将LPP转化为冷杉醇。更优选地，所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3、5或8所示的氨基酸序列；
b)与如SEQ ID NO:3、5或8所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1或18所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或18所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
本发明还考虑一种融合多肽，该融合多肽包含本发明的多肽和至少一种另外的多肽，该另外的多肽(i)表现出II型二萜合酶的酶活性，优选地，将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP，(ii)具有麦芽糖结合特性，或者(iii)是硫氧还蛋白或硫氧还蛋白融合蛋白。更优选地，所述另外的多肽选自由以下组成的组：LPP合酶(优选地来自毛喉鞘蕊花(CfLPPS)或烟草(NtLPPS))、CPP合酶(优选地来自毛喉鞘蕊花(CfCPPS))、硫氧还蛋白和麦芽糖结合蛋白(MBP)。
本发明还涉及用于生产本发明的具有二萜醇合酶活性的多肽的方法，该方法包括：
(a)用本发明的核酸序列转化宿主细胞或单细胞生物体以表达具有二萜醇合酶活性的多肽；
(b)从步骤(a)的宿主细胞中获得或分离所述具有二萜醇合酶活性的多肽；以及
(c)任选地，纯化所述具有二萜醇合酶活性的多肽。
本发明进一步涉及用于制备具有二萜醇合酶活性的变体多肽的方法，该方法包括以下步骤：
a)选择本发明的核酸或编码本发明的多肽的核酸；
b)修饰所选核酸以获得至少一种突变体核酸；
c)用突变体核酸序列转化宿主细胞或单细胞生物体以表达由该突变体核酸序列编码的多肽；
d)筛选具有至少一种修饰的特性以及二萜醇合酶活性的多肽；以及，
e)任选地，如果多肽不具有所需变体二萜醇合酶活性，则重复工艺步骤(a)至(d)，直到获得具有所需变体二萜醇合酶活性的多肽；
f)任选地，如果在步骤(d)中鉴定出具有所需变体二萜醇合酶活性的多肽，则分离在步骤(c)中获得的相应突变体核酸。
本发明涉及编码本发明的多肽或本发明的融合多肽或其反向互补或互补序列的多核苷酸。
根据本发明所用的术语“多核苷酸”是指处于单链或双链形式的脱氧核糖核苷酸或核糖核苷酸聚合物，并且除非另外限制，否则涵盖在以下方面具有天然核苷酸的基本性质的已知类似物：其以与天然存在的核苷酸(例如，肽核酸)相似的方式杂交至单链核酸。本文所用的术语涵盖本文指定的序列及其互补或反向互补序列。因此，该术语涵盖因稳定性或其他原因具有修饰的主链的DNA或RNA。此外，仅举两个例子，包含稀有碱基(例如肌苷)或修饰碱基(例如三苯甲基化的碱基)的DNA或RNA还涵盖在多核苷酸内。应认识到，已对DNA和RNA进行了很多种修饰，这些修饰起到本领域技术人员已知的许多有用目的。本文中编码本发明的某个多肽的每一个核酸序列都可能由于遗传密码的简并而具有沉默变异。遗传密码的简并产生大量编码相同多肽的功能相同的多核苷酸。例如，密码子GCA、GCC、GCG和GCU都编码氨基酸丙氨酸。因此，在密码子指定丙氨酸的每个位置，该密码子可以改变为任何所述的相应密码子而不改变编码的多肽。这样的核酸变异是沉默变异。
本发明的多核苷酸应编码本发明的多肽，即其应包含编码本发明的所述多肽的核酸序列。此外，本发明的多核苷酸可以包含额外的核酸序列。优选地，除可读框外，本发明的多核苷酸可在编码基因区的3’和5’末端包含另外的非翻译序列：编码区5’末端序列上游的至少500个，优选地200个，更优选地100个核苷酸，以及编码基因区3’末端序列下游的至少100个，优选地50个，更优选地20个核苷酸。
本发明的多核苷酸应优选地以分离的多核苷酸(即纯化或至少从其天然背景(例如其天然基因位点)分离)或以遗传修饰或外源性(即人工)操纵的形式提供。分离的多核苷酸可以例如包含小于约5kb、4kb、3kb、2kb、1kb、0.5kb、或0.1kb的核苷酸序列，该核苷酸序列在该核酸所来源的细胞的基因组DNA中天然地位于该核酸分子的侧翼。优选地，多核苷酸以双链或单链分子的形式提供。应理解，本发明通过提及任何本发明的上述多核苷酸，也指之前提及的特定序列或变体的互补或反向互补链。多核苷酸涵盖DNA(包括cDNA和基因组DNA)或RNA多核苷酸。
然而，本发明还涉及来源于本发明的多核苷酸且能够干扰本发明的多核苷酸的转录或翻译的多核苷酸变体。这样的变体多核苷酸包括反义核酸、核酶、siRNA分子、吗啉代核酸(磷酸二酰胺吗啉代寡聚物)、三螺旋形成寡核苷酸、抑制性寡核苷酸或小RNA分子，由于存在互补或基本上互补的序列，所有这些多核苷酸均能特异性识别本发明的多核苷酸。这些技术是本领域技术人员熟知的。基于本发明的多核苷酸的结构，可以容易地设计上述类型的合适变体多核苷酸。
此外，还包括化学修饰的多核苷酸(包括天然存在的修饰多核苷酸，例如糖基化或甲基化多核苷酸)或人工修饰的多核苷酸(例如生物素化的多核苷酸)。
本发明还涉及包含本发明的多核苷酸的载体或基因构建体。
术语“载体”优选地涵盖噬菌体、质粒、粘粒、病毒载体以及人工染色体，例如细菌或酵母人工染色体(YAC)。涵盖本发明的多核苷酸的载体优选地进一步包含用于在宿主中繁殖和/或选择的选择性标记。可以通过本领域熟知的多种技术将载体整合到宿主细胞中。如果被引入宿主细胞，载体可能存在于细胞质中，或者可能被整合到基因组中。在后一种情况下，应理解载体可以进一步包含允许同源重组或异源插入的核酸序列。可以通过常规转化或转染技术将载体引入原核或真核细胞。在本发明上下文中使用的术语“转化”和“转染”、接合和转导旨在包括将外来核酸(例如DNA)引入宿主细胞的多种现有方法，包括磷酸钙、氯化铷或氯化钙共沉淀、DEAE-右旋糖酐介导的转染、脂质体转染、f-交配、自然感受态、碳基团簇、化学介导的转移、电穿孔或粒子轰击。用于转化或转染宿主细胞(包括植物细胞)的合适方法可以在Sambrook等人(Molecular Cloning:A Laboratory Manual[分子克隆：实验室手册],第2版,Cold Spring Harbor Laboratory[冷泉港实验室],Cold SpringHarbor Laboratory Press[冷泉港实验室出版社],冷泉港,纽约州,1989)和其他实验室手册(例如Methods in Molecular Biology[分子生物学方法],1995,第44卷、Agrobacteriumprotocols[土壤杆菌实验方案],编辑:Gartland和Davey,Humana Press[胡马纳出版社],托托瓦,新泽西州)中找到。可替代地，可以通过热激或电穿孔技术引入质粒载体。如果载体是病毒，可在应用于宿主细胞之前使用适当的包装细胞系对其进行体外包装。
优选地，本文所提及的载体适合作为克隆载体，即在微生物系统中可复制。这样的载体可确保在细菌和优选地酵母或真菌中高效克隆，并使植物的稳定转化成为可能。其中必须提及的是，特别是适合T DNA介导的转化的多种二元和共整合载体系统。这样的载体系统通常具有以下特征：它们至少包含土壤杆菌介导的转化所需的vir基因，以及定界T-DNA的序列(T-DNA边界)。这些载体系统优选地还包含另外的顺式调节区，例如启动子和终止子和/或选择标记，选择标记可用于鉴定合适的转化的宿主细胞或生物体。虽然共整合载体系统将vir基因和T DNA序列排列在相同载体上，但二元系统基于至少两个载体，其中一个携带vir基因，但不携带T-DNA，而另一个携带T DNA，但不携带vir基因。因此，最后提到的载体相对较小，易于操作，并且可在大肠杆菌和土壤杆菌中复制。这些二元载体包括来自pBIB-HYG、pPZP、pBecks、pGreen系列的载体。优选地，根据本发明使用的是Bin19、pBI101、pBinAR、pGPTV和pCAMBIA。二元载体及其使用的概述可在Hellens等人,Trends in PlantScience[植物科学趋势](2000)5,446-451中找到。此外，通过使用适当的克隆载体，可以将多核苷酸引入宿主细胞或生物体，例如植物或动物，并且因此用于植物的转化，例如在以下中发表和引用的那些：Plant Molecular Biology and Biotechnology[植物分子生物学和生物技术](CRC Press[CRC出版社],博卡拉顿,佛罗里达州),第6/7章,第71-119页(1993)；F.F.White,Vectors for Gene Transfer in Higher Plants；in:Transgenic Plants[转基因植物：用于高等植物基因转移的载体],第1卷,Engineering and Utilization[工程化和利用],编辑:Kung和R.Wu,Academic Press[学术出版社],1993,15-38；B.Jenes等人,Techniques for Gene Transfer,in:Transgenic Plants[转基因植物：用于基因转移的技术],第1卷,Engineering and Utilization[工程化和利用],编辑:Kung和R.Wu,AcademicPress[学术出版社](1993),128-143；Potrykus 1991,Annu.Rev.Plant Physiol.PlantMolec.Biol[植物生理学和植物分子生物学年评].42,205 225。
更优选地，本发明的载体是表达载体。在这样的表达载体，即包含本发明的多核苷酸的载体中，核酸序列可操作地连接至表达控制序列(也称为“表达盒”)，允许在原核或真核细胞或其分离的组分中表达。合适的表达载体是本领域已知的，例如Okayama-Berg cDNA表达载体pcDV1(法玛西亚公司(Pharmacia))、pCDM8、pRc/CMV、pcDNA1、pcDNA3(英杰公司(Invitrogen))或pSPORT1(吉布科BRL公司(GIBCO BRL))。典型的融合表达载体的另外的实例是pGEX(法玛西亚生物技术公司(Pharmacia Biotech Inc)；Smith 1988,Gene[基因]67:31-40)、pMAL(新英格兰生物实验室公司(New England Biolabs)，贝弗利，马萨诸塞州)和pRIT5(法玛西亚公司，皮斯卡特维，新泽西州)，其中谷胱甘肽S转移酶(GST)、麦芽糖E结合蛋白和蛋白A分别与重组靶蛋白融合。合适的诱导性非融合大肠杆菌表达载体的实例尤其是pTrc(Amann 1988,Gene[基因]69:301-315)和pET 11d(Studier 1990,Methods inEnzymology[酶学方法]185,60-89)。pTrc载体的tar-get基因表达基于通过宿主RNA聚合酶从杂合trp-lac融合启动子的转录。从pET 11d载体的靶基因表达基于T7-gn10-lac融合启动子的转录，该启动子由共表达的病毒RNA聚合酶(T7 gn1)介导。这种病毒聚合酶由宿主菌株BL21(DE3)或HMS174(DE3)提供，来自携带在lacUV 5启动子转录控制下的T7 gn1基因的常驻λ原噬菌体。本领域技术人员熟悉适用于原核生物的其他载体；这些载体是例如在大肠杆菌中，pLG338、pACYC184、pBR系列(例如pBR322)、pUC系列(例如pUC18或pUC19)、M113mp系列、pKC30、pRep4、pHS1、pHS2、pPLc236、pMBL24、pLG200、pUR290、pIN-III113-B1、lambdagt11或pBdCl；在链霉菌属中，plJ101、plJ364、plJ702或plJ361；在芽孢杆菌属中，pUB110、pC194或pBD214；在棒杆菌属中，pSA77或pAJ667。用于在酵母酿酒酵母中表达的载体的实例包括pYep Sec1(Baldari 1987,Embo J.[欧洲分子生物学学会杂志]6:229-234)、pMFa(Kurjan 1982,Cell[细胞]30:933-943)、pJRY88(Schultz1987,Gene[基因]54:113-123)和pYES2(英杰公司，圣地亚哥，加利福尼亚州)。用于构建适用于其他真菌(例如丝状真菌)的载体的载体和过程包括在以下中详述的那些：在Applied Molecular Genetics offungi[真菌应用分子遗传学],J.F.Peberdy等人,编辑,第1-28页,Cambridge UniversityPress[剑桥大学出版社]:Cambridge[剑桥]中或在More Gene Manipulations in Fungi[真菌中的更多基因操作](J.W.Bennett&L.L.Lasure,编辑,第396-428页:Academic Press[学术出版社]:圣地亚哥)中的van den Hondel,C.A.M.J.J.,&Punt,P.J.(1991)“Genetransfer systems and vector development for filamentous fungi”[丝状真菌的基因转移系统和载体开发]。另外的合适的酵母载体是例如pAG-1、YEp6、YEp13或pEMBLYe23。作为替代，本发明的多核苷酸还可以使用杆状病毒表达载体在昆虫细胞中表达。可用于在培养的昆虫细胞(例如Sf9细胞)中表达蛋白质的杆状病毒载体包含pAc系列(Smith 1983,Mol.Cell Biol[分子细胞生物学].3:2156-2165)和pVL系列(Lucklow 1989,Virology[病毒学]170:31-39)。
然而，载体可以是整合载体。整合载体是指线性或环状的DNA分子，其可以整合到例如微生物的基因组(如细菌的基因组)中，并提供编码目的多肽(例如本发明的醇酰基转移酶)的基因的稳定遗传。整合载体通常包含一个或多个区段，该一个或多个区段包含编码目的多肽的基因序列，该基因序列在提供其转录的额外核酸区段的控制下(即可操作地连接)。
这样的额外的区段可以包括启动子和终止子序列，以及通常通过同源重组过程驱动目的基因掺入靶细胞基因组中的一个或多个区段。典型地，整合载体将是一种可以被转移到靶细胞中的载体，但其具有在该生物体中无功能的复制子。如果包含目的基因的区段中包含适当的标记，则可以选择该区段的整合。编码不与本发明宿主细胞中待表达的多肽天然相关的适当信号肽的一个或多个核酸序列可整合到(表达)载体中。例如，信号肽前导序列的DNA序列可以框内融合至本发明的核酸，使得本发明的醇酰基转移酶初始翻译为包含信号肽的融合蛋白。根据信号肽的性质，表达的多肽将被不同地靶向。例如，在预期宿主细胞中起作用的分泌信号肽增强表达的多肽的细胞外分泌。其他信号肽将表达的多肽引导至某些细胞器，如叶绿体、线粒体和过氧化物酶体。信号肽可在转运至预期的细胞器后从多肽中或从细胞中裂解出来。在多肽的氨基或羧基末端提供额外的肽序列的融合物是可能的。
如本文所用，术语“基因构建体”是指包含本发明的多核苷酸和额外的功能核酸序列的多核苷酸。根据本发明的基因构建体优选地是线性DNA分子。典型地，根据本发明的基因构建体可以是靶向构建体，其允许靶向构建体随机或定点整合到基因组DNA中。这样的靶构建体优选地包含用于同源或异源重组的足够长度的DNA，如下文所详述的。在这两种情况下，构建体必须优选地是完美的，具有控制基因表达的结构，例如启动子、转录起始位点、多聚腺苷酸化位点和转录终止位点。
然而，本发明涉及包含本发明的载体或基因构建体的宿主细胞。
本发明的宿主细胞能够表达包含在本发明的载体或基因构建体中的本发明的多肽。宿主细胞典型地用所述载体或基因构建体转化，使得本发明的多肽可以从载体或基因构建体中表达。转化的载体或基因构建体可维持作为非整合载体，例如质粒，或可替代地，可整合到宿主细胞基因组中，如本文其他地方更详细指定的。
根据本发明的宿主细胞可以基于本领域公知的标准遗传和分子生物学技术来生产，例如如以下中所述：Sambrook,J.,和Russell,D.W.“Molecular Cloning:A LaboratoryManual[分子克隆：实验室手册]”第3版,Cold Spring Harbor Laboratory Press[冷泉港实验室出版社],冷泉港,纽约州,(2001)；和F.M.Ausubel等人,编辑,“Current protocolsin molecular biology[当前分子生物学方案]”,John Wiley and Sons,Inc.[约翰威利父子公司],纽约(1987)以及后者的增刊。
优选地，所述宿主细胞选自由以下组成的组：细菌细胞、酵母细胞、真菌细胞、藻类细胞或蓝细菌细胞、非人动物细胞或非人哺乳动物细胞以及植物细胞。更优选地，宿主细胞可选自以下生物体中的任何一种：
细菌：
细菌宿主细胞可以例如选自由以下组成的组：埃希氏菌属、克雷伯氏菌属、螺杆菌属、芽孢杆菌属、乳杆菌属、链球菌属、拟无枝酸菌属、红杆菌属、假单胞菌属、副球菌属、乳球菌属或泛菌属。
革兰氏阳性：芽孢杆菌属、链霉菌属：有用的革兰氏阳性菌宿主细胞包括但不限于芽孢杆菌属细胞，例如，嗜碱芽孢杆菌(Bacillus alkalophius)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、短芽孢杆菌(Bacillus brevis)、环状芽孢杆菌(Bacillus circulans)、克劳氏芽孢杆菌(Bacillus clausii)、凝结芽孢杆菌(Bacilluscoagulans)、坚强芽孢杆菌(Bacillus firmus)、Jautus芽孢杆菌(Bacillus Jautus)、迟缓芽孢杆菌(Bacillus lentus)、地衣芽孢杆菌(Bacillus licheniformis)、巨大芽孢杆菌(Bacillus megaterium)、短小芽孢杆菌(Bacillus pumilus)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)、枯草芽孢杆菌(Bacillus subtilis)和苏云金芽孢杆菌(Bacillus thuringiensis)。最优选的原核生物是芽孢杆菌属细胞，优选地枯草芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌或迟缓芽孢杆菌的芽孢杆菌属细胞。
一些其他优选的细菌包括放线菌目的菌株，优选地链霉菌属，优选地类球形链霉菌(Streptomyces spheroides)(ATTC 23965)、热紫链霉菌(Streptomycesthermoviolaceus)(IFO 12382)、浅青紫链霉菌(Streptomyces lividans)或鼠灰链霉菌(Streptomyces murinus)或轮丝链轮丝菌物种轮枝菌(Streptoverticillumverticillium ssp.verticillium)。其他优选的细菌包括类球红杆菌(Rhodobactersphaeroides)、沼泽红单胞菌(Rhodomonas palustri)、乳链球菌(Streptococcuslactis)。进一步优选的细菌包括属于粘球菌属(Myxococcus)的菌株，例如变绿粘球菌(M.virescens)。
革兰氏阴性：大肠杆菌、假单胞菌属、红杆菌属、副球菌属：优选的革兰氏阴性菌是大肠杆菌、假单胞菌属物种，优选地，吡咯菌素假单胞菌(Pseudomonas purrocinia)(ATCC15958)或荧光假单胞菌(Pseudomonas fluorescens)(NRRL B-11)、荚膜红杆菌(Rhodobacter capsulatus)或类球红杆菌、产类胡萝卜素副球菌(Paracoccuscarotinifaciens)或玉米黄质副球菌(Paracoccus zeaxanthinifaciens)或菠萝泛菌(Pantoea ananatis)。
真菌：
曲霉属、镰刀菌属、木霉属：宿主细胞可以是真菌细胞。如本文所用，“真菌”包括子囊菌门、担子菌门、壶菌门和接合菌门、以及卵菌门和半知菌亚门、和所有的丝分孢子(mitosporic)真菌。子囊菌门的代表性群体包括例如脉孢菌属、正青霉属(＝青霉属)、翘孢霉属(＝曲霉属)、散囊菌属(＝曲霉属)和下面列出的真酵母。担子菌门的实例包括蘑菇、锈菌和黑粉菌。壶菌门的代表性群体包括例如异水霉属(Allomyces)、芽枝霉属(Blastocladiella)、雕蚀菌属(Coelomomyces)和水生真菌。卵菌门的代表性群体包括例如水霉属(Saprolegniomycetous)水生真菌(水霉菌(water molds))，如绵霉属(Achlya)。丝分孢子真菌的实例包括曲霉属、青霉属、念珠菌属和链格孢属。接合菌门的代表性群体包括例如根霉属和毛霉属。
一些优选的真菌包括属于半知菌亚门，丝孢纲的菌株，例如镰刀菌属、腐质霉属(Humicola)、木霉属(Tricoderma)、漆斑菌属(Myrothecium)、轮枝孢属(Verticillum)、Arthromyces、卡尔黑霉属(Caldariomyces)、单格孢属(Ulocladium)、埃里砖格孢属(Embellisia)、枝孢属(Cladosporium)或德氏霉属(Dreschlera)，特别是尖孢镰刀菌(Fusarium oxysporum)(DSM 2672)、特异腐质霉(Humicola insolens)、里氏木霉(Trichoderma resii)、疣孢漆斑菌(Myrothecium verrucana)(IFO 6113)、黑白轮枝孢(Verticillum alboatrum)、大丽轮枝孢(Verticillum dahlie)、Arthromyces ramosus(FERM P-7754)、煤卡尔黑霉(Caldariomyces fumago)、纸单格孢(Ulocladiumchartarum)、大蒜埃里砖格孢(Embellisia alli)或Dreschlera halodes。其他优选的真菌包括属于担子菌亚门，担子菌纲的菌株，例如鬼伞属、平革菌属(Phanerochaete)、革盖菌属(Coriolus)或栓菌属，特别是灰盖鬼伞微孢变型(Coprinus cinereus f.microsporus)(IFO 8371)、长根鬼伞(Coprinus macrorhizus)、黄孢原毛平革菌(Phanerochaetechrysosporium)(例如，NA-12)或栓菌属(先前称为多孔菌属)，例如变色栓菌(T.versicolor)(例如，PR4 28-A)。
进一步优选的真菌包括属于接合菌亚门，毛霉(Mycoraceae)纲的菌株，例如根霉属或毛霉属，特别是冻土毛霉(Mucor hiemalis)。
酵母、毕赤酵母属、酵母属：真菌宿主细胞可以是酵母细胞。如本文所用，酵母包括产子囊酵母(ascosporogenous yeast)(内孢霉目(Endomycetales))、产担子酵母(basidiosporogenous yeast)和属于半知菌类(Fungi lmperfecti)的酵母(芽生菌目(Blastomycetes))。产子囊酵母分为蚀精霉科(Spermophthoraceae)和酵母科(Saccharomycetaceae)。后者由四个亚科组成：裂殖酵母亚科(Schizosaccharomycoideae)(例如，裂殖酵母属)、拿逊酵母亚科(Nadsonioideae)、油脂酵母亚科(Lipomycoideae)和类酵母亚科(Saccharomycoideae)(例如，克鲁维酵母属、毕赤酵母属和酵母属)。产担子酵母包括白冬孢酵母属、红冬孢酵母属、锁掷酵母属(Sporidiobolus)、线黑粉酵母属(Filobasidium)和线黑粉菌属(Filobasidiella)。属于半知菌类的酵母分为两个科：掷孢酵母科(例如，掷孢酵母属和布勒掷孢酵母属(Bullera))和隐球酵母科(例如，念珠菌属)。
真核生物：
真核宿主细胞进一步包括但不限于非人动物细胞、非人哺乳动物细胞、禽类细胞、爬行动物细胞、昆虫细胞或植物细胞。
最优选地，宿主细胞是细菌宿主细胞，特别是红杆菌属宿主细胞。
本发明涉及转基因非人生物体，其包含本发明的多核苷酸、本发明的载体或基因构建体或本发明的宿主细胞。
如本文所用，术语“转基因非人生物体”是指经遗传修饰以包含本发明的多核苷酸、载体或基因构建体的生物体。所述遗传修饰可能是任何类型的同源或异源重组事件、诱变或基因编辑过程的结果。因此，转基因非人生物体与其非转基因对应物的不同之处在于，其在其基因组中包含非天然存在的(即异源)多核苷酸、载体或基因构建体。根据本发明设想作为转基因非人生物体的非人生物体优选地是多细胞生物体。此外，非人生物体优选地是动物或植物。优选的动物是哺乳动物，特别是实验动物，例如啮齿动物，如小鼠、大鼠、兔等，或农场动物，如绵羊、山羊、奶牛、马等。优选的植物是农作物植物或蔬菜，特别是选自由以下组成的组：拟南芥属(Arabidopsis spp.)、烟草属(Nicotiana spp)、菊苣(Cichorumintybus)、莴苣(Lactuca sativa)、薄荷属(Mentha spp)、黄花蒿(Artemisiaannua)、块茎形成植物、油料作物(如芸薹属(Brassica spp.)或甘蓝型油菜(Brassicanapus))、产生果实的开花植物(被子植物)和树。
在一个实施例中，非人转基因生物体是转基因本发明的多肽、包含所述多肽的融合蛋白、编码其的多核苷酸、包含所述多核苷酸的载体或基因构建体的非人转基因生物体。
在一个实施例中，宿主细胞是体外(例如，在细胞培养物中)的非人细胞。
在另一个实施例中，术语“非人”应理解为指除人以外的非动物生物体(例如，植物、真菌或微生物)或除哺乳动物以外的动物，优选地非脊椎动物的动物。
用于生产转基因非人生物体的方法是本领域众所周知的；参见例如Lee-Yoon Low等人,Transgenic Plants:Gene constructs,vector and transformation method.[转基因植物：基因构建体、载体和转化方法]2018.DOI.10.5772/intechopen.79369；Pinkert,C.A.(编辑)1994.Transgenic animal technology:A laboratory handbook.[转基因动物技术：实验室手册]Academic Press,Inc.[学术出版社公司],圣地亚哥,加利福尼亚州；Monastersky G.M.和Robl,J.M.(编辑)(1995)Strategies in Transgenic AnimalScience.[转基因动物科学策略]ASM Press.[ASM出版社]华盛顿特区；Sambrook,上述引文,Ausubel,上述引文。
一般而言，本发明考虑使用本发明的多肽或本发明的融合多肽、本发明的多核苷酸、本发明的载体或基因构建体、本发明的宿主细胞或本发明的非人转基因生物体用于生产至少一种C-20萜醇，优选地冷杉醇、泪杉醇和/或香紫苏醇。
根据本发明生产的C-20萜醇可以在不同的工业领域中具有多种用途。特别地，所述C-20萜醇用于生产香料、农用化学品、香精、药物组合物、化妆品或化学合成砌块。
此外，本发明还涉及用于生产至少一种C-20萜醇，优选地冷杉醇、泪杉醇和/或香紫苏醇的试剂盒，该试剂盒包含本发明的多肽或本发明的融合多肽、本发明的多核苷酸、本发明的载体或基因构建体、本发明的宿主细胞或本发明的非人转基因生物体。
如本文所用，术语“试剂盒”是指实施本发明的方法来生产至少一种C-20萜醇所需的组分的集合。试剂盒应包括作为单个组分或其任何组合的任何上述组分。典型地，试剂盒的组分在单独的容器中或在单个容器内提供。容器还典型地包含用于实施本发明的方法来生产至少一种C-20萜醇的说明。此外，试剂盒可优选地包含实施本发明方法所需的另外的组分，例如孵育试剂、培养介质、洗涤溶液、溶剂和/或纯化至少一种C-20萜醇所需的试剂或手段。
以下实施例是根据本发明设想的特定优选实施例。上述术语的所有定义和解释经必要修改后适用。
实施例1：一种用于生产至少一种C-20萜醇的方法，该方法包括以下步骤：
a)将香叶基香叶基焦磷酸转化为柯巴基二磷酸(CPP)或半日花-13-烯-8-醇二磷酸(LPP)；以及
b)将CPP或LPP转化为至少一种C-20萜醇，其中所述转化是通过表现出二萜醇合酶活性的多肽实施的，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，
其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
实施例2：根据权利要求1所述的方法，其中所述多肽包含如SEQ IDNO:24所示的保守区域的氨基酸序列。
实施例3：根据实施例1或2所述的方法，其中所述至少一种C-20萜醇是环状C-20萜醇。
实施例4：根据实施例1至3中任一项所述的方法，其中所述至少一种C-20萜醇是泪杉醇、香紫苏醇或冷杉醇。
实施例5：根据实施例1至4中任一项所述的方法，其中所述表现出二萜醇合酶活性的多肽能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇。
实施例6：根据实施例5所述的方法，其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列；
b)与如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:2、16、17或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:2、16、17或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇的二萜醇合酶活性的多肽。
实施例7：根据实施例1至4中任一项所述的方法，其中所述表现出二萜醇合酶活性的多肽能够将LPP转化为冷杉醇。
实施例8：根据实施例7所述的方法，其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3、5或8所示的氨基酸序列；
b)与如SEQ ID NO:3、5或8所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1或18所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或18所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
实施例9：根据实施例1至8中任一项所述的方法，其中步骤a)中的所述转化是通过另外的多肽实施的，该另外的多肽表现出将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP的II型二萜合酶的酶活性。
实施例10：根据实施例1至9中任一项所述的方法，其中所述表现出二萜合酶活性的多肽包含在融合多肽中，该融合多肽包含至少一种另外的多肽，该另外的多肽表现出II型二萜合酶的酶活性，优选地，将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP，该另外的多肽具有麦芽糖结合特性，或者该另外的多肽是硫氧还蛋白或硫氧还蛋白融合蛋白。
实施例11：根据实施例10所述的方法，其中所述另外的多肽选自由以下组成的组：LPP合酶(优选地来自毛喉鞘蕊花(CfLPPS)或烟草(NtLPPS))、CPP合酶(优选地来自毛喉鞘蕊花(CfCPPS))、硫氧还蛋白和麦芽糖结合蛋白(MBP)。
实施例12：根据实施例1至12中任一项所述的方法，其中所述步骤b)或所述步骤a)和b)在宿主细胞或非人转基因生物体中实施。
实施例13：根据实施例1至12中任一项所述的方法，其进一步包括获得所述生产的至少一种C-20萜醇的步骤。
实施例14：一种组合物，该组合物包含通过根据实施例1至14中任一项所述的方法可获得的所述至少一种C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇。
实施例15：一种表现出二萜醇合酶活性的多肽，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，所述多肽具有选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
实施例16：根据实施例15所述的多肽，其中所述多肽包含如SEQ ID NO:24所示的保守区域的氨基酸序列。
实施例17：根据实施例15或16所述的多肽，其中所述二萜醇合酶活性能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇。
实施例18：根据实施例17所述的多肽，其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列；
b)与如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:2、16、17或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:2、16、17或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇的二萜醇合酶活性的多肽。
实施例19：根据实施例15或16所述的多肽，其中所述二萜醇合酶活性能够将LPP转化为冷杉醇。
实施例20：根据实施例19所述的多肽，其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3、5或8所示的氨基酸序列；
b)与如SEQ ID NO:3、5或8所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1或18所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或18所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
实施例21：一种融合多肽，该融合多肽包含根据实施例15至20中任一项所述的多肽和至少一种另外的多肽，该另外的多肽(i)表现出II型二萜合酶的酶活性，优选地，将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP，(ii)具有麦芽糖结合特性，或者(iii)是硫氧还蛋白或硫氧还蛋白融合蛋白。
实施例22：根据实施例21所述的融合多肽，其中所述另外的多肽选自由以下组成的组：LPP合酶(优选地来自毛喉鞘蕊花(CfLPPS)或烟草(NtLPPS))、CPP合酶(优选地来自毛喉鞘蕊花(CfCPPS))、硫氧还蛋白和麦芽糖结合蛋白(MBP)。
实施例23：一种多核苷酸，该多核苷酸编码根据实施例15至20中任一项所述的多肽或根据实施例21或22所述的融合多肽或其反向互补或互补序列。
实施例24：一种载体或基因构建体，该载体或基因构建体包含根据实施例23所述的多核苷酸。
实施例25：一种宿主细胞，该宿主细胞包含根据实施例24所述的载体或基因构建体。
实施例26：根据实施例25所述的宿主细胞，其中所述宿主细胞选自由以下组成的组：细菌细胞、酵母细胞、真菌细胞、藻类细胞或蓝细菌细胞、非人动物细胞或非人哺乳动物细胞以及植物细胞。
实施例27：一种转基因非人生物体，该转基因非人生物体包含根据实施例23所述的多核苷酸、根据实施例24所述的载体或基因构建体、或根据实施例25或26所述的宿主细胞。
实施例28：根据实施例15至20中任一项所述的多肽或根据实施例21或22所述的融合多肽、根据实施例23所述的多核苷酸、根据实施例24所述的载体或基因构建体、根据实施例25或26所述的宿主细胞或根据实施例27所述的非人转基因生物体用于生产至少一种C-20萜醇，优选地冷杉醇、泪杉醇和/或香紫苏醇的用途。
实施例29：根据实施例28所述的用途，其中所述C-20萜醇用于生产香料、农用化学品、香精、药物、化妆品或化学合成砌块。
实施例30：一种用于生产至少一种C-20萜醇，优选地冷杉醇、泪杉醇和/或香紫苏醇的试剂盒，该试剂盒包含根据实施例15至20中任一项所述的多肽或根据实施例21或22所述的融合多肽、根据实施例23所述的多核苷酸、根据实施例24所述的载体或基因构建体、根据实施例25或26所述的宿主细胞或根据实施例27所述的非人转基因生物体。
在整个说明书中引用的所有参考文献均通过引用以其全文或针对具体提及的披露内容并入本文。
附图说明
图1：巨柏的二氯甲烷萃取物的GC MS分析。在19.7min处观察到一个清晰的泪杉醇峰，对应于泪杉醇标准品的Rt。
图2：菌株pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA和pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA的GC分析。A)和b)；分析显示一种化合物在13.61min处洗脱(随后被鉴定为冷杉醇)；c，d，e，来自表达Cup2v2a和Cup2v2b与LPP合酶组合的构建体的结果；分析显示在14.03min处洗脱的一种新化合物(随后被鉴定为香紫苏醇)，f)菌株pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA的GC分析显示产生一种化合物，其在13.29min处洗脱(随后被鉴定为泪杉醇)。
图3：菌株的GC MS分析。a)pBBR-MEV-PcrtE-TrxCfLPPS-mbpCupr2v1-Prplm-CgIsdA-GC MS分析证实这种化合物对应于冷杉醇；b)pBBR-MEV-PcrtE-TrxCfLPPS-mbpCupr2v2b-Prplm-CgIsdA-GC MS分析证实这种化合物对应于香紫苏醇；c)pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA GC MS分析表明这种化合物对应于泪杉醇。
图4：产物决定区的比对。CfMOS，来自毛喉鞘蕊花的泪柏醚合酶(GenBank登录号：KF444508)；IrMS，来自碎米桠的次丹参酮二烯合酶(KX831652)；CfMS，来自毛喉鞘蕊花的次丹参酮二烯合酶(KF444509)；RoMS1，来自迷迭香(Rosemarius officinalis)的次丹参酮二烯合酶1(KF805858)；SmMS，来自丹参的次丹参酮二烯合酶(ABV08817)；RoMS1，来自迷迭香(Rosemarius officinalis)的次丹参酮二烯合酶(KF805859)；SfMS，来自灌状鼠尾草的次丹参酮二烯合酶(KP091841)；MvELS，来自欧夏至草的9,13-环氧-半日花-14-烯合酶(KJ584454)；SsSS南欧丹参香紫苏醇合酶(JN133922)；SsSS的SsSS-iAS变体，其是异冷杉醇合酶(Jia等人ACS Catal.[ACS催化]2018,8,3133-3137)。
图5：蛋白Cup2v2b(SEQ ID NO:4)、Cup2v2a(SEQ ID NO:34)、Cup2v1(SEQ ID NO:3)与TcKSL1、TcKSL2和TcKSL8的比对，这些蛋白如分别在美国国家生物技术信息中心(National Center for Biotechnology Information，NCBI)数据库中登录号KT588484、KT588485和KT588489下所发现的；另外的ScSS的序列，如在国际专利申请WO 2009101126的SEQ ID NO:3中所发现的。
图6：蛋白Cup2v2b(SEQ ID NO:4)、Cup2v2a(SEQ ID NO:34)、Cup2v1(SEQ ID NO:3)与TcKSL1、TcKSL2和TcKSL8的比对，这些蛋白如分别在美国国家生物技术信息中心(NCBI)数据库中登录号KT588484、KT588485和KT588489下所发现的。
在整个说明书和随附的序列方案中提及了以下序列：
SEQ ID NO:1：Cup2v1 cDNA序列
SEQ ID NO:2：Cup2v2b cDNA序列
SEQ ID NO:3：Cup2v1蛋白
SEQ ID NO:4：Cup2v2b蛋白
SEQ ID NO:5：截短Cup2v1蛋白
SEQ ID NO:6：截短Cup2v2a蛋白
SEQ ID NO:7：截短Cup2v2b蛋白
SEQ ID NO:8：MBP-截短Cup2v1蛋白
SEQ ID NO:9：MBP-截短Cup2v2a蛋白
SEQ ID NO:10：MBP截短cup2v2b蛋白
SEQ ID NO:11：SsSS截短蛋白
SEQ ID NO:12：Trx-CfCPS蛋白
SEQ ID NO:13：Trx-CfLPPS蛋白
SEQ ID NO:14：Trx-NtLPPS蛋白
SEQ ID NO:15：CgIdsA蛋白
SEQ ID NO:16：MBP-Cup2v2b DNA
SEQ ID NO:17：MBP-Cup2v2a DNA
SEQ ID NO:18：MBP-Cup2v1 DNA
SEQ ID NO:19：SsSS cDNA
SEQ ID NO:20：Trx-CfLPPSDNA
SEQ ID NO:21：Trx-CfCPSDNA
SEQ ID NO:22：CgidsA cDNA
SEQ ID NO:23：Trx-NtLPPSDNA
SEQ ID NO:24：Cup2v1、Cup2v2a和Cup2v2b蛋白的保守区域
SEQ ID NO:25：CfMOS蛋白的产物决定区；IrMS蛋白的产物决定区；CfMS蛋白的产物决定区；CRoMS1蛋白的产物决定区；SmMS蛋白的产物决定区；RoMS2蛋白的产物决定区；SfMS蛋白的产物决定区；MvELS蛋白的产物决定区
SEQ ID NO:26：SsSS蛋白的产物决定区
SEQ ID NO:27：SsSS-iAS蛋白的产物决定区
SEQ ID NO:28：MBP-Cupr2v2b-2多肽
SEQ ID NO:29MBP-Cupr2v2b-3多肽
SEQ ID NO:30MBP-Cupr2v2b-4多肽
SEQ ID NO:31MBP-Cupr2v2b-2DNA
SEQ ID NO:32MBP-Cupr2v2b-3DNA
SEQ ID NO:33MBP-Cupr2v2b-4DNA
SEQ ID NO:34Cup2v2a蛋白
SEQ ID NO:35Cup2v2a DNA
SEQ ID NO:36截短Cup2v1 DNA
SEQ ID NO:37截短Cup2v2b DNA
SEQ ID NO:38截短Cup2v2a DNA
SEQ ID NO:39 C-和N-末端双截短的DNA Cup2v1
SEQ ID NO:40 C-和N-末端双截短的蛋白Cup2v1
SEQ ID NO:41变体1蛋白
SEQ ID NO:42变体2蛋白
SEQ ID NO:43变体3蛋白
SEQ ID NO:44变体4蛋白
SEQ ID NO:45变体5蛋白
SEQ ID NO:46变体6蛋白
SEQ ID NO:47变体7蛋白
SEQ ID NO:48变体8蛋白
SEQ ID NO:49变体9蛋白
SEQ ID NO:50变体10蛋白
SEQ ID NO:51变体11蛋白
SEQ ID NO:52变体12蛋白
SEQ ID NO:53变体13蛋白
SEQ ID NO:54变体14蛋白
SEQ ID NO:55 Cup基序ENNSFGSMCI
SEQ ID NO:56 Cup基序EKKSFGSMCI
SEQ ID NO:57 Cup基序EKNSFGSMCI
SEQ ID NO:58 Cup基序ENKSFGSMCI
进一步，具有给定单氨基酸取代的以下多肽也是根据本发明的多肽：
在SEQ ID NO:4中，在位置84处，Lys可被Asn替换。
在SEQ ID NO:6中，在位置3处，Asn可被Lys替换。
在SEQ ID NO:7中，在位置3处，Lys可被Asn替换。
在SEQ ID NO:9中，在位置375处，Asn可被Lys替换。
在SEQ ID NO:10中，在位置375处，Asn可被Lys替换。
在SEQ ID NO:3中，在位置398处，填充有Ile或Thr。
在SEQ ID NO:5中，在位置317处，填充有Ile或Thr。
实例
实例仅用于说明本发明。无论如何，它们不应被理解为对范围进行限制。
实例1：Cup2v1、Cup2v2a和Cup2v2b的克隆
巨柏萜的分析。
从埃斯维尔德(Esveld)(博斯科普(Boskoop))获得巨柏树。通过在液氮下将皮层材料研磨成细粉末，并用1ml二氯甲烷萃取100mg该粉末，从茎的皮层制备萃取物。在GC MS上分析二氯甲烷相。在19.7min处观察到一个清晰的泪杉醇峰，对应于泪杉醇标准品的Rt。
进行RNA提取并从柏木属组织的cDNA进行测序
将约15mL萃取缓冲液(2％十六烷基-三甲基溴化铵、2％聚乙烯吡咯烷酮K 30、100mM Tris-HCl(pH 8.0)、25mM EDTA、2.0M NaCl、0.5g/L亚精胺和2％β-巯基乙醇)加热至65℃，然后添加3g磨碎的皮层组织并混合。用等体积的氯仿:异戊醇(1:24)萃取混合物两次，并将四分之一体积的10M LiCl添加至上清液中并混合。RNA在4℃下沉淀过夜，并通过以10 000g离心20min进行收获。将颗粒溶于500μLSSTE[1.0M NaCl，0.5％ SDS，10mM Tris-HCl(pH 8.0)，1mM EDTA(pH 8.0)]中，并用等体积的氯仿:异戊醇萃取一次。向上清液中添加二体积的乙醇，在-20℃下孵育至少2h，以13 000g离心并除去上清液。将颗粒风干并重悬于水中。将总RNA(60μg)运输至Vertis生物技术公司(Vertis Biotechnology AG)(德国弗赖辛(Freising,Germany))。分离出PolyA+RNA，使用随机N6接头引物和M-MLV H-逆转录酶合成随机引发的cDNA。对cDNA进行剪切和分级分离，使用500bp大小的片段进行进一步分析。在cDNA的5′和3′端连接由因美纳公司(Illumina)指定的接头序列A和B。随后在Illumina MiSeq测序装置上对材料进行了分析。MiSeq总共读取了19,608,859个序列。Trimmomatic-0.32用于修剪来自Illumina测序接头的序列，Seqprep用于重叠双端序列，bowtie2(2.2.1版)用于去除phiX污染(phiX DNA用作加标(spike-in)对照，通常<1％)。在Trinity组装(trinityrnaseq-2.0.2)中使用双端读段和单读段。通过Trinity组装了共88667个重叠群。
为了鉴定倍半萜合酶，使用巨柏重叠群创建cDNA序列数据库。在这个数据库中，利用TBLASTN程序鉴定了编码显示与倍半萜合酶蛋白序列具有同一性的蛋白的cDNA序列，这些蛋白包括来自拟南芥的贝壳杉烯合酶(Q9SAK2)、来自南欧丹参的香紫苏醇合酶(AET21246.1)、来自胶冷杉(Abies balsamifera)的冷杉醇合酶(H8ZM73.1)、来自南欧丹参的13-半日花烯-8,15-二醇(13-labden-8,15-diol)焦磷酸盐合酶(AET21248.1)。在巨柏cDNA数据库中共鉴定出184个与倍半萜合酶具有显著同源性的重叠群。根据其序列中的重叠，将重叠群分为68组。通过使用BLASTX程序将这68个重叠群与UniProt数据库(2015年8月28日下载)中存在的蛋白序列进行比对分析，对这些重叠群进行了进一步表征，并且发明人根据它们与UniProt中存在的萜合酶序列的同源性及其特征，手动鉴定出其中12个重叠群是推定的二萜合酶序列。
Cup2v1、Cup2v2a和Cup2v2b的鉴定
基于对其特征的熟练分析，发明人选择了其中三个cDNA序列作为最有前景的候选基因。SEQ ID No:1和2中所示的cDNA序列分别被鉴定为Cup2v1和Cup2v2b。Cup2v1蛋白在SEQ ID NO:3中示出，Cup2v2b蛋白在SEQ ID NO:4中示出。Cup2v1和Cup2v2b蛋白在氨基酸水平上彼此有93.8％的同一性。
第三个cDNA序列与Cup2v2b相似，并且被命名为Cup2v2a。
发明人生成了序列的人工缩短版本，从而去除了质体靶向信号并改变了N-末端。这些截短的氨基酸序列(命名为trcup2v1、trcup2v2a和trcup2v2b)分别在SEQ ID NO:5至7中给出。全长Cup2v2a蛋白在SEQ ID NO:34中示出，cDNA序列在SEQ ID NO:35中绘出。
在已知的鼠尾草香紫苏醇合酶(SsSS)中，创建了截短版本(trSsSS)作为对照。
在NCBI nr蛋白质数据库中进行BLAST，发现这些蛋白的最接近的同源物是来自台湾杉的二萜合酶(AOG18231.1)，其产物特异性未知，具有67.6％的氨基酸同一性。在uniprot数据库中对表征的蛋白进行BLAST，发现来自穗花牡荊(Vitex agnuscastus)的对映贝壳杉烯合酶，其具有39.1％的氨基酸同一性。
#TOOL:needle
#GAPMETHOD:NOGAPS
#GAPOPEN:10,GAPEXTEND:0.5,MATRIX:EBLOSUM62
发明人已鉴定出Cup2v1、Cup2v2a和Cup2v2b蛋白是用于生成冷杉醇、泪杉醇和/或香紫苏醇的步骤2二萜醇合酶的候选蛋白。发明人在Cup2v1、Cup2v2a和Cup2v2b之间鉴定出一个基本保守的区域(参见比对图4)。合酶中的这一区域位于与其他合酶的产物决定区相对应的位置，但不同于所述其他合酶的产物决定区，包括已知的鼠尾草香紫苏醇合酶的产物决定区。尽管Cup2v1、Cup2v2a和Cup2v2b具有不同的产物特异性(参见下文)，但在已知的其他二萜合酶中典型地负责决定产物特异性的区域在所述Cup蛋白之间的差异很大，但却是保守的。
实例2：用于在红杆菌属中表达步骤1和步骤2基因的质粒的构建
对于在红杆菌属中的表达，设计了Cup2v1、Cup2v2a、Cup2v2b的截短版本与麦芽糖结合蛋白的融合蛋白(命名为mbpCup2v1、mbpCup2v2a和mbpCup2v2b，分别参见SEQ ID NO:8至10)，以及许多步骤1基因CfLPPS、CfCPPS和NtLPPS与硫氧还蛋白Trx的融合蛋白(参见SEQID No:12至14)。为了进行比较，还制备了表达CfLPPS与南欧丹参香紫苏醇合酶(SsSS)的截短版本组合的构建体。这一截短版本对应于在Schalk J.Am.Chem.Soc.[美国化学会志]2012,134,18900-18903中发表的SsSS。
制备了一种构建体，其中如EP 2 336 310 A1中所述用其天然启动子表达的来自玉米黄质副球菌的甲羟戊酸操纵子，连同如WO 2018/160066A1中所述通过Lppa启动子表达的CgIdsA，以及包含crtE启动子的操纵子，然后是trx-步骤1基因、核糖体结合位点和mbp-步骤2基因。
制备了以下构建体集：
a.pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA
b.pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA
c.pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v2a-Prplm-CgIsdA
d.pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2a-Prplm-CgIsdA
e.pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2b-Prplm-CgIsdA
f.pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA
g.pBBR-MEV-PcrtE-TrxCfLPPS-SsSS-Prplm-CgIsdA
这些构建体被引入大肠杆菌S17-1，并使用标准程序将得到的菌株与类球红杆菌Rs265-9c接合。所得菌株以其质粒命名。
实例3：C-20萜醇的小规模重组生产
每个菌株都用于小规模生产试验，基本上如US2020/0010822 A1中所述。为此，在无挡板的100ml摇瓶中，用含有100mg/L新霉素的20ml RS102培养基和一圈甘油储备液进行种子培养。在110rpm、轨道为50mm的振荡培养箱中，在30℃下培养种子培养瓶72小时。
在72小时结束时，评估培养物的OD600，以计算转移至较大培养瓶的准确培养物体积。
在具有2个底部挡板的300ml摇瓶中进行摇瓶实验。将20ml RS102培养基和最终浓度为100mg/L的新霉素与2ml无菌正十二烷一起添加至培养瓶中。调整接种物的体积，以在20ml培养基中获得0.05的最终OD600值。
在110rpm、轨道为50mm的振荡培养箱中，在30℃下保持培养瓶72小时。随后，将培养物收集到预称重的50ml PP管中，然后以4500xg将其离心20分钟。将正十二烷层转移至微量离心管中，用于后续的GC分析。
称取10微升月桂酸乙酯，置于10ml玻璃小瓶中，向其中添加800μl分离的十二烷溶液并称重。随后，向小瓶中添加8ml丙酮以稀释十二烷浓度，用于更准确的GC分析。将丙酮溶液中的约1.5ml含萜十二烷转移至色谱小瓶中。通过气相色谱法对每份样品进行分析，如US2020/0010822A1中所述。对于化合物鉴定，使用气相色谱仪通过GC/MS对约2μL进行分析，如Cankar等人(2015)所详述的。通过将保留时间和质谱与香紫苏醇、泪杉醇和冷杉醇(西格玛奥德里奇公司(Sigma-Aldrich))的标准品进行比较，对产品进行鉴定。
对菌株pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA和pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA的GC分析显示在13.61min处洗脱的一种化合物(图2a，图2b)。GC MS分析证实这种化合物对应于冷杉醇(图3a)。对于冷杉醇，测得构建体的以下滴度(g/kg正十二烷)：pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA为1.9，pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA为3.5。
菌株pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA的GC分析(图2f)显示产生一种化合物，其在13.29min处洗脱。GC MS分析表明这种化合物对应于泪杉醇(图3c)。对于泪杉醇，测得构建体的以下滴度(g/kg正十二烷)：pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA为1.5
菌株pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v2a-Prplm-CgIsdA、pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2a-Prplm-CgIsdA、pBBR-MEV-PcrtE-TrxCfLPPS-SsSS-Prplm-CgIsdA和pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2b-Prplm-CgIsdA的GC分析显示在14.03min处洗脱的一种新化合物(图2c，图2d，图2e)。GC MS分析证实这种化合物对应于香紫苏醇(图3b)。使用不同构建体的香紫苏醇的定量分析在下表中示出：
表1：香紫苏醇相对量
其中，以g/kg正十二烷表示的滴度通过对照获得的滴度进行归一化。
与SEQ ID NO:7相比在N末端具有额外的序列的Cup2v2b的另外的序列变体也作为具有N-末端MBP的融合蛋白(SEQ ID NO:28至30)在相似的设置中进行了试验。所有三种蛋白均显示出相似的香紫苏醇生产水平，如上文表1第4行中的pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2b-Prplm-CgIsdA所示。
序列表
<110> 艾瑟拜奥尼克斯公司（Isobionics B.V.）
<120> C-20萜醇的重组生产
<130> B210230PC
<150> 21184067.3
<151> 2021-07-06
<160> 58
<170> BiSSAP 1.3.6版
<210> 1
<211> 1893
<212> DNA
<213> 巨柏（Cupressus gigantea）
<400> 1
atggcagcac ttagtttttc taccatctcc accactgcca actatgcaat tcctaagctg 60
ccatcttcaa atcttcaagg gcggtccaga gatacaaaat accatgtctt gataagatct 120
gcaggggcct ctaatttcaa cttcaatgga agaaagaagt tcaatccaag aacacatcag 180
aaagtcaaat ctttgagtgt tgaagattcc tccaatgtac ttattaaggc tgagaaagag 240
aacagagaga aaaaatcatt tggttcgatg tgcatagatg atgatattac aaagataaaa 300
atgttgttct cggaaattaa aagaagatat tcatctcttt ctgcttatga tacagtgcac 360
ggtgcttatc ctatcaattt gattcttggc ctctcaatgg taaaccactt ggagagcttg 420
ggtatagcaa gacatttcaa acttgagata aaagaggttc tagatgatgt ctatagattt 480
tggatagaga atgaaatggt tgaaggaatg ccatcagcat cagacataat gaccagttct 540
atcagtttca ggatcttgcg gtggaatgga tatgacgtgt ctccagatgt ctttttcagt 600
tacctgaggg atagtgactt cctactattt ttagaaaata gtgaccaagc tactatagca 660
ttgctgaatt tatacaaggc atctcagatg atgttcccag gcgaaagaat tcttgaagaa 720
gcaaaattgt tctcccaaaa ttatcttgaa aatatcaaga tgggcaatca aagtggagct 780
ctcaaagaca ttgaagcaga ggttaagcat gcactggatg tcccttggat ggctaatttg 840
gagagaatag aacatctgcg atacatgaaa gcatttgctt ttgacaatat ttggattgga 900
gagacctctt ctaggatccc atttacagga aaggatttat ttttatcttt tgccaaacaa 960
gacttcaata tctgccaagc agctcaacaa gaggatttga aggcgttgga aaaatggagt 1020
gtagactcta aacttggtga cctacacttt gctagacaaa agcttgtagc ctgctatttc 1080
tctgccgcct caatactttt tagccctgaa atgtctgcta ctcggattgt gtggactaaa 1140
aatgctgttc tcattacact aatggatgac ttttatgatg tggagggttc catygaagat 1200
attcaatcct ttgttgaagc ggttaagaga tgggacccaa tagttttatg taattgttca 1260
gctaatgtta aaattttatt ctctgctatt tataacactg tgaatgatat tgctcaagat 1320
atatggattt tccagggctg ggacattagc actcagttaa gagaaatttg gtacgaactg 1380
gcaatctcaa tgatgaagga ggctgaatgg accaaaactg gatttatacc atccttggaa 1440
gaatacatgg aaaatgggag aacatcaatt gccttggaac caattatttt tacatcacta 1500
ttttttattg gccctaagct ttccgagcaa atgatccacc atcacgagta taagaggctt 1560
ttgcaactca tgaatacttg tgggcgcctt ttgaatgaca ttcagagcga caagagggag 1620
atgaagcaag gaaagctcac ctctgtatca ttgttcgtga aagaataccc aattgaggat 1680
gctactaagt ggattagact cactgtaagt gaaaacacac aaaatatact caaaaaaata 1740
ttgcaaccaa gtgagatccc tcgtgaatgc aagcagatat tttggaatat ggtcaaaatt 1800
atccaactat tctgcctcca cagtgatgaa ttcacatctc ctaccacaat gcttgaacat 1860
atgaaggcag ttctttttta tcctgtttta taa 1893
<210> 2
<211> 1896
<212> DNA
<213> 巨柏（Cupressus gigantea）
<400> 2
atggcagcac ttagtttttc taccatctcc accactgcca actatgcaat tcctaagctg 60
ccatcttcaa atcttcaagg gcggtccaga gatacaaaat accatgtctt gataagatct 120
gcaggggcct ctaatttcaa cttcaatgga agaaagaagt tcaatccaag aacacatcag 180
aaagtcaaat ctttgagtgt tgaagattcc tccaatgtac ttattaaggc tgagaaagag 240
aacagagaga aaaaatcatt tggttcgatg tgcatagacg ataatattac aaggataaat 300
atgttgttct tggaaattaa gagaagatat tcatctcttt ctgcttatga tacagtgcac 360
ggtgcttatc ctatcaattt gattcttggc ctctcaatgg taaatcattt ggagagcttg 420
ggtatagcaa gacatttcaa acttgagata aaagaggttc tagatgatgt ctatagattt 480
tggatagaga atgaaatggt taaaggaatg ccatcagcat cagacataat gaccagttct 540
atcagtttca agatcttgcg gtggaatgga tatgacgtgt ctccagatgt ctttttcagt 600
tacctgaggg atagtgactt cctaccattt ctagaaaata gtggccaagc tactatggca 660
ttgctgaatt tatacaaggc atctcagatg atgttcccag gggaaagaat tcttgaagaa 720
gcaaaatcgt tctcccaaaa ttatcttgaa aatatcaagg tgggcgatca aagtggagct 780
ctcaaagata ttgaagcaga ggttaagcat gcactggatg ttccttggat ggctggtttg 840
gagagaatag aacatctgcg atacatgaaa gcatttgctt ttgatgatat ttggattgga 900
gagacctctt ctaggatccc atttacagga aaggatttat ttttatcttt agccaaacaa 960
gacttcaata tctgccaaga agctcaacaa gaggatttga aagcgttgga aaaatggagt 1020
gtagactcta aacttggtga cctacacttt gctagacaaa agcttgtagc ctgctatttc 1080
tctgccgcct caacactttt tagccctgaa atgtctacta ctcggattgt gtggactaaa 1140
aatgctgttc tcactactct aatggatgac ttttatgatg tggagggttc cattgaagat 1200
attcaatcct ttgttgaagc agttaagaga tgggacccaa tagttttatg taattgttca 1260
gctaatgtga aaaaattatt ctctgctatt tataacactg tgaatgatat tgctcaagat 1320
atatggattt tccagggctg ggacattagc actcagttaa gagaaatttg gtacgaactg 1380
gcaatctcaa tgatgaagga ggctgaatgg accaaaactg gatttatacc atcgctggaa 1440
gaatacatgg aaaatgggag aacgtcaatt gccttggaac caattatttt tacatcacta 1500
ttttttcttg gccctaagct ttccgagcaa atgatccacg agtataagag acttatgcaa 1560
ctcctgaata cttgtgggcg ccttttgaat gacagtcaga gctacaagag ggagataaag 1620
caaggaaagc tcaactcttt atcattgttc gtgaaagaat atcccgcaac atctattgag 1680
gatgctactg agtggattag actcactgta agtgaaagca gacaaaatat actcagaaat 1740
ttattgcaac caagtgagat ccctcgtgaa tgcaagcaga tattttggaa tatggtcaaa 1800
attatccaac tattctacct ccacagtgat gaattcacat ctcctactgc aatgcttgaa 1860
catatgcaag cagttctttt taatcctgtt ttataa 1896
<210> 3
<211> 630
<212> PRT
<213> 巨柏（Cupressus gigantea）
<220>
<221> 不确定
<222> 398
<400> 3
Met Ala Ala Leu Ser Phe Ser Thr Ile Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Lys Leu Pro Ser Ser Asn Leu Gln Gly Arg Ser Arg Asp Thr
20 25 30
Lys Tyr His Val Leu Ile Arg Ser Ala Gly Ala Ser Asn Phe Asn Phe
35 40 45
Asn Gly Arg Lys Lys Phe Asn Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asp Ile
85 90 95
Thr Lys Ile Lys Met Leu Phe Ser Glu Ile Lys Arg Arg Tyr Ser Ser
100 105 110
Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Val Glu Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Arg Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu
195 200 205
Leu Phe Leu Glu Asn Ser Asp Gln Ala Thr Ile Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Leu Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Met Gly Asn
245 250 255
Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Asn Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asn Ile Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Phe Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Ala Ala Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Ile Leu Phe Ser
355 360 365
Pro Glu Met Ser Ala Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Ile Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Ile Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Phe Ile Gly Pro Lys Leu Ser Glu Gln Met Ile
500 505 510
His His His Glu Tyr Lys Arg Leu Leu Gln Leu Met Asn Thr Cys Gly
515 520 525
Arg Leu Leu Asn Asp Ile Gln Ser Asp Lys Arg Glu Met Lys Gln Gly
530 535 540
Lys Leu Thr Ser Val Ser Leu Phe Val Lys Glu Tyr Pro Ile Glu Asp
545 550 555 560
Ala Thr Lys Trp Ile Arg Leu Thr Val Ser Glu Asn Thr Gln Asn Ile
565 570 575
Leu Lys Lys Ile Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln
580 585 590
Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Cys Leu His Ser
595 600 605
Asp Glu Phe Thr Ser Pro Thr Thr Met Leu Glu His Met Lys Ala Val
610 615 620
Leu Phe Tyr Pro Val Leu
625 630
<210> 4
<211> 631
<212> PRT
<213> 巨柏（Cupressus gigantea）
<400> 4
Met Ala Ala Leu Ser Phe Ser Thr Ile Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Lys Leu Pro Ser Ser Asn Leu Gln Gly Arg Ser Arg Asp Thr
20 25 30
Lys Tyr His Val Leu Ile Arg Ser Ala Gly Ala Ser Asn Phe Asn Phe
35 40 45
Asn Gly Arg Lys Lys Phe Asn Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Arg Ile Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser
100 105 110
Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu
195 200 205
Pro Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Ile
500 505 510
His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln Gly Lys Leu
530 535 540
Asn Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala Thr Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys
580 585 590
Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala
610 615 620
Val Leu Phe Asn Pro Val Leu
625 630
<210> 5
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 截短蛋白
<220>
<221> 不确定
<222> 317
<400> 5
Met Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asp Ile Thr
1 5 10 15
Lys Ile Lys Met Leu Phe Ser Glu Ile Lys Arg Arg Tyr Ser Ser Leu
20 25 30
Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile Leu
35 40 45
Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg His
50 55 60
Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe Trp
65 70 75 80
Ile Glu Asn Glu Met Val Glu Gly Met Pro Ser Ala Ser Asp Ile Met
85 90 95
Thr Ser Ser Ile Ser Phe Arg Ile Leu Arg Trp Asn Gly Tyr Asp Val
100 105 110
Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu Leu
115 120 125
Phe Leu Glu Asn Ser Asp Gln Ala Thr Ile Ala Leu Leu Asn Leu Tyr
130 135 140
Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu Ala
145 150 155 160
Lys Leu Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Met Gly Asn Gln
165 170 175
Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu Asp
180 185 190
Val Pro Trp Met Ala Asn Leu Glu Arg Ile Glu His Leu Arg Tyr Met
195 200 205
Lys Ala Phe Ala Phe Asp Asn Ile Trp Ile Gly Glu Thr Ser Ser Arg
210 215 220
Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Phe Ala Lys Gln Asp
225 230 235 240
Phe Asn Ile Cys Gln Ala Ala Gln Gln Glu Asp Leu Lys Ala Leu Glu
245 250 255
Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg Gln
260 265 270
Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Ile Leu Phe Ser Pro
275 280 285
Glu Met Ser Ala Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu Ile
290 295 300
Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp Ile
305 310 315 320
Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu Cys
325 330 335
Asn Cys Ser Ala Asn Val Lys Ile Leu Phe Ser Ala Ile Tyr Asn Thr
340 345 350
Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp Ile
355 360 365
Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met Met
370 375 380
Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu Glu
385 390 395 400
Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe
405 410 415
Thr Ser Leu Phe Phe Ile Gly Pro Lys Leu Ser Glu Gln Met Ile His
420 425 430
His His Glu Tyr Lys Arg Leu Leu Gln Leu Met Asn Thr Cys Gly Arg
435 440 445
Leu Leu Asn Asp Ile Gln Ser Asp Lys Arg Glu Met Lys Gln Gly Lys
450 455 460
Leu Thr Ser Val Ser Leu Phe Val Lys Glu Tyr Pro Ile Glu Asp Ala
465 470 475 480
Thr Lys Trp Ile Arg Leu Thr Val Ser Glu Asn Thr Gln Asn Ile Leu
485 490 495
Lys Lys Ile Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln Ile
500 505 510
Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Cys Leu His Ser Asp
515 520 525
Glu Phe Thr Ser Pro Thr Thr Met Leu Glu His Met Lys Ala Val Leu
530 535 540
Phe Tyr Pro Val Leu
545
<210> 6
<211> 550
<212> PRT
<213> 人工序列
<220>
<223> 截短蛋白
<400> 6
Met Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile Thr
1 5 10 15
Arg Ile Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser Leu
20 25 30
Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile Leu
35 40 45
Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg His
50 55 60
Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe Trp
65 70 75 80
Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile Met
85 90 95
Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp Val
100 105 110
Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu Pro
115 120 125
Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu Tyr
130 135 140
Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu Ala
145 150 155 160
Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp Gln
165 170 175
Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu Asp
180 185 190
Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr Met
195 200 205
Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser Arg
210 215 220
Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln Asp
225 230 235 240
Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu Glu
245 250 255
Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg Gln
260 265 270
Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser Pro
275 280 285
Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu Thr
290 295 300
Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp Ile
305 310 315 320
Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu Cys
325 330 335
Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn Thr
340 345 350
Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp Ile
355 360 365
Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met Met
370 375 380
Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu Glu
385 390 395 400
Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe
405 410 415
Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Ile His
420 425 430
Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu Leu
435 440 445
Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln Gly Lys Leu Thr
450 455 460
Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala His Leu Ile Glu Asp
465 470 475 480
Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn Ile
485 490 495
Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln
500 505 510
Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His Ser
515 520 525
Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala Val
530 535 540
Leu Phe Asn Pro Val Leu
545 550
<210> 7
<211> 550
<212> PRT
<213> 人工序列
<220>
<223> 截短蛋白
<400> 7
Met Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile Thr
1 5 10 15
Arg Ile Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser Leu
20 25 30
Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile Leu
35 40 45
Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg His
50 55 60
Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe Trp
65 70 75 80
Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile Met
85 90 95
Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp Val
100 105 110
Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu Pro
115 120 125
Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu Tyr
130 135 140
Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu Ala
145 150 155 160
Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp Gln
165 170 175
Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu Asp
180 185 190
Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr Met
195 200 205
Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser Arg
210 215 220
Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln Asp
225 230 235 240
Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu Glu
245 250 255
Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg Gln
260 265 270
Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser Pro
275 280 285
Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu Thr
290 295 300
Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp Ile
305 310 315 320
Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu Cys
325 330 335
Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn Thr
340 345 350
Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp Ile
355 360 365
Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met Met
370 375 380
Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu Glu
385 390 395 400
Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe
405 410 415
Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Ile His
420 425 430
Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu Leu
435 440 445
Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln Gly Lys Leu Asn
450 455 460
Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala Thr Ser Ile Glu Asp
465 470 475 480
Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn Ile
485 490 495
Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln
500 505 510
Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His Ser
515 520 525
Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala Val
530 535 540
Leu Phe Asn Pro Val Leu
545 550
<210> 8
<211> 921
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 8
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly
355 360 365
Gly Gly Ala Ala Met Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp
370 375 380
Asp Asp Ile Thr Lys Ile Lys Met Leu Phe Ser Glu Ile Lys Arg Arg
385 390 395 400
Tyr Ser Ser Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile
405 410 415
Asn Leu Ile Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly
420 425 430
Ile Ala Arg His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val
435 440 445
Tyr Arg Phe Trp Ile Glu Asn Glu Met Val Glu Gly Met Pro Ser Ala
450 455 460
Ser Asp Ile Met Thr Ser Ser Ile Ser Phe Arg Ile Leu Arg Trp Asn
465 470 475 480
Gly Tyr Asp Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser
485 490 495
Asp Phe Leu Leu Phe Leu Glu Asn Ser Asp Gln Ala Thr Ile Ala Leu
500 505 510
Leu Asn Leu Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile
515 520 525
Leu Glu Glu Ala Lys Leu Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys
530 535 540
Met Gly Asn Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys
545 550 555 560
His Ala Leu Asp Val Pro Trp Met Ala Asn Leu Glu Arg Ile Glu His
565 570 575
Leu Arg Tyr Met Lys Ala Phe Ala Phe Asp Asn Ile Trp Ile Gly Glu
580 585 590
Thr Ser Ser Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Phe
595 600 605
Ala Lys Gln Asp Phe Asn Ile Cys Gln Ala Ala Gln Gln Glu Asp Leu
610 615 620
Lys Ala Leu Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His
625 630 635 640
Phe Ala Arg Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Ile
645 650 655
Leu Phe Ser Pro Glu Met Ser Ala Thr Arg Ile Val Trp Thr Lys Asn
660 665 670
Ala Val Leu Ile Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser
675 680 685
Ile Glu Asp Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro
690 695 700
Ile Val Leu Cys Asn Cys Ser Ala Asn Val Lys Ile Leu Phe Ser Ala
705 710 715 720
Ile Tyr Asn Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln
725 730 735
Gly Trp Asp Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala
740 745 750
Ile Ser Met Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro
755 760 765
Ser Leu Glu Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu
770 775 780
Pro Ile Ile Phe Thr Ser Leu Phe Phe Ile Gly Pro Lys Leu Ser Glu
785 790 795 800
Gln Met Ile His His His Glu Tyr Lys Arg Leu Leu Gln Leu Met Asn
805 810 815
Thr Cys Gly Arg Leu Leu Asn Asp Ile Gln Ser Asp Lys Arg Glu Met
820 825 830
Lys Gln Gly Lys Leu Thr Ser Val Ser Leu Phe Val Lys Glu Tyr Pro
835 840 845
Ile Glu Asp Ala Thr Lys Trp Ile Arg Leu Thr Val Ser Glu Asn Thr
850 855 860
Gln Asn Ile Leu Lys Lys Ile Leu Gln Pro Ser Glu Ile Pro Arg Glu
865 870 875 880
Cys Lys Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Cys
885 890 895
Leu His Ser Asp Glu Phe Thr Ser Pro Thr Thr Met Leu Glu His Met
900 905 910
Lys Ala Val Leu Phe Tyr Pro Val Leu
915 920
<210> 9
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 9
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly
355 360 365
Gly Gly Ala Ala Met Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Asp
370 375 380
Asp Asn Ile Thr Arg Ile Asn Met Leu Phe Leu Glu Ile Lys Arg Arg
385 390 395 400
Tyr Ser Ser Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile
405 410 415
Asn Leu Ile Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly
420 425 430
Ile Ala Arg His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val
435 440 445
Tyr Arg Phe Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala
450 455 460
Ser Asp Ile Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn
465 470 475 480
Gly Tyr Asp Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser
485 490 495
Asp Phe Leu Pro Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu
500 505 510
Leu Asn Leu Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile
515 520 525
Leu Glu Glu Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys
530 535 540
Val Gly Asp Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys
545 550 555 560
His Ala Leu Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His
565 570 575
Leu Arg Tyr Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu
580 585 590
Thr Ser Ser Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu
595 600 605
Ala Lys Gln Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu
610 615 620
Lys Ala Leu Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His
625 630 635 640
Phe Ala Arg Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr
645 650 655
Leu Phe Ser Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn
660 665 670
Ala Val Leu Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser
675 680 685
Ile Glu Asp Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro
690 695 700
Ile Val Leu Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala
705 710 715 720
Ile Tyr Asn Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln
725 730 735
Gly Trp Asp Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala
740 745 750
Ile Ser Met Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro
755 760 765
Ser Leu Glu Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu
770 775 780
Pro Ile Ile Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu
785 790 795 800
Gln Met Ile His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys
805 810 815
Gly Arg Leu Leu Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln
820 825 830
Gly Lys Leu Thr Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala His
835 840 845
Leu Ile Glu Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Ser
850 855 860
Arg Gln Asn Ile Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg
865 870 875 880
Glu Cys Lys Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe
885 890 895
Tyr Leu His Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His
900 905 910
Met Gln Ala Val Leu Phe Asn Pro Val Leu
915 920
<210> 10
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 10
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly
355 360 365
Gly Gly Ala Ala Met Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Asp
370 375 380
Asp Asn Ile Thr Arg Ile Asn Met Leu Phe Leu Glu Ile Lys Arg Arg
385 390 395 400
Tyr Ser Ser Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile
405 410 415
Asn Leu Ile Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly
420 425 430
Ile Ala Arg His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val
435 440 445
Tyr Arg Phe Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala
450 455 460
Ser Asp Ile Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn
465 470 475 480
Gly Tyr Asp Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser
485 490 495
Asp Phe Leu Pro Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu
500 505 510
Leu Asn Leu Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile
515 520 525
Leu Glu Glu Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys
530 535 540
Val Gly Asp Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys
545 550 555 560
His Ala Leu Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His
565 570 575
Leu Arg Tyr Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu
580 585 590
Thr Ser Ser Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu
595 600 605
Ala Lys Gln Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu
610 615 620
Lys Ala Leu Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His
625 630 635 640
Phe Ala Arg Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr
645 650 655
Leu Phe Ser Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn
660 665 670
Ala Val Leu Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser
675 680 685
Ile Glu Asp Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro
690 695 700
Ile Val Leu Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala
705 710 715 720
Ile Tyr Asn Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln
725 730 735
Gly Trp Asp Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala
740 745 750
Ile Ser Met Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro
755 760 765
Ser Leu Glu Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu
770 775 780
Pro Ile Ile Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu
785 790 795 800
Gln Met Ile His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys
805 810 815
Gly Arg Leu Leu Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln
820 825 830
Gly Lys Leu Asn Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala Thr
835 840 845
Ser Ile Glu Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Ser
850 855 860
Arg Gln Asn Ile Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg
865 870 875 880
Glu Cys Lys Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe
885 890 895
Tyr Leu His Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His
900 905 910
Met Gln Ala Val Leu Phe Asn Pro Val Leu
915 920
<210> 11
<211> 525
<212> PRT
<213> 人工序列
<220>
<223> 截短蛋白
<400> 11
Met Ala Lys Met Lys Glu Asn Phe Lys Arg Glu Asp Asp Lys Phe Pro
1 5 10 15
Thr Thr Thr Thr Leu Arg Ser Glu Asp Ile Pro Ser Asn Leu Cys Ile
20 25 30
Ile Asp Thr Leu Gln Arg Leu Gly Val Asp Gln Phe Phe Gln Tyr Glu
35 40 45
Ile Asn Thr Ile Leu Asp Asn Thr Phe Arg Leu Trp Gln Glu Lys His
50 55 60
Lys Val Ile Tyr Gly Asn Val Thr Thr His Ala Met Ala Phe Arg Leu
65 70 75 80
Leu Arg Val Lys Gly Tyr Glu Val Ser Ser Glu Glu Leu Ala Pro Tyr
85 90 95
Gly Asn Gln Glu Ala Val Ser Gln Gln Thr Asn Asp Leu Pro Met Ile
100 105 110
Ile Glu Leu Tyr Arg Ala Ala Asn Glu Arg Ile Tyr Glu Glu Glu Arg
115 120 125
Ser Leu Glu Lys Ile Leu Ala Trp Thr Thr Ile Phe Leu Asn Lys Gln
130 135 140
Val Gln Asp Asn Ser Ile Pro Asp Lys Lys Leu His Lys Leu Val Glu
145 150 155 160
Phe Tyr Leu Arg Asn Tyr Lys Gly Ile Thr Ile Arg Leu Gly Ala Arg
165 170 175
Arg Asn Leu Glu Leu Tyr Asp Met Thr Tyr Tyr Gln Ala Leu Lys Ser
180 185 190
Thr Asn Arg Phe Ser Asn Leu Cys Asn Glu Asp Phe Leu Val Phe Ala
195 200 205
Lys Gln Asp Phe Asp Ile His Glu Ala Gln Asn Gln Lys Gly Leu Gln
210 215 220
Gln Leu Gln Arg Trp Tyr Ala Asp Cys Arg Leu Asp Thr Leu Asn Phe
225 230 235 240
Gly Arg Asp Val Val Ile Ile Ala Asn Tyr Leu Ala Ser Leu Ile Ile
245 250 255
Gly Asp His Ala Phe Asp Tyr Val Arg Leu Ala Phe Ala Lys Thr Ser
260 265 270
Val Leu Val Thr Ile Met Asp Asp Phe Phe Asp Cys His Gly Ser Ser
275 280 285
Gln Glu Cys Asp Lys Ile Ile Glu Leu Val Lys Glu Trp Lys Glu Asn
290 295 300
Pro Asp Ala Glu Tyr Gly Ser Glu Glu Leu Glu Ile Leu Phe Met Ala
305 310 315 320
Leu Tyr Asn Thr Val Asn Glu Leu Ala Glu Arg Ala Arg Val Glu Gln
325 330 335
Gly Arg Ser Val Lys Glu Phe Leu Val Lys Leu Trp Val Glu Ile Leu
340 345 350
Ser Ala Phe Lys Ile Glu Leu Asp Thr Trp Ser Asn Gly Thr Gln Gln
355 360 365
Ser Phe Asp Glu Tyr Ile Ser Ser Ser Trp Leu Ser Asn Gly Ser Arg
370 375 380
Leu Thr Gly Leu Leu Thr Met Gln Phe Val Gly Val Lys Leu Ser Asp
385 390 395 400
Glu Met Leu Met Ser Glu Glu Cys Thr Asp Leu Ala Arg His Val Cys
405 410 415
Met Val Gly Arg Leu Leu Asn Asp Val Cys Ser Ser Glu Arg Glu Arg
420 425 430
Glu Glu Asn Ile Ala Gly Lys Ser Tyr Ser Ile Leu Leu Ala Thr Glu
435 440 445
Lys Asp Gly Arg Lys Val Ser Glu Asp Glu Ala Ile Ala Glu Ile Asn
450 455 460
Glu Met Val Glu Tyr His Trp Arg Lys Val Leu Gln Ile Val Tyr Lys
465 470 475 480
Lys Glu Ser Ile Leu Pro Arg Arg Cys Lys Asp Val Phe Leu Glu Met
485 490 495
Ala Lys Gly Thr Phe Tyr Ala Tyr Gly Ile Asn Asp Glu Leu Thr Ser
500 505 510
Pro Gln Gln Ser Lys Glu Asp Met Lys Ser Phe Val Phe
515 520 525
<210> 12
<211> 864
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 12
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Gly Asp
100 105 110
Asp Asp Asp Lys Ile His Met Asn Asn Gly Lys Asn Leu Asn Cys Gln
115 120 125
Leu Thr His Lys Lys Ile Ser Lys Val Ala Glu Ile Arg Val Ala Thr
130 135 140
Val Asn Ala Pro Pro Val His Asp Gln Asp Asp Ser Thr Glu Asn Gln
145 150 155 160
Cys His Asp Ala Val Asn Asn Ile Glu Asp Pro Ile Glu Tyr Ile Arg
165 170 175
Thr Leu Leu Arg Thr Thr Gly Asp Gly Arg Ile Ser Val Ser Pro Tyr
180 185 190
Asp Thr Ala Trp Val Ala Leu Ile Lys Asp Leu Gln Gly Arg Asp Ala
195 200 205
Pro Glu Phe Pro Ser Ser Leu Glu Trp Ile Ile Gln Asn Gln Leu Ala
210 215 220
Asp Gly Ser Trp Gly Asp Ala Lys Phe Phe Cys Val Tyr Asp Arg Leu
225 230 235 240
Val Asn Thr Ile Ala Cys Val Val Ala Leu Arg Ser Trp Asp Val His
245 250 255
Ala Glu Lys Val Glu Arg Gly Val Arg Tyr Ile Asn Glu Asn Val Glu
260 265 270
Lys Leu Arg Asp Gly Asn Glu Glu His Met Thr Cys Gly Phe Glu Val
275 280 285
Val Phe Pro Ala Leu Leu Gln Arg Ala Lys Ser Leu Gly Ile Gln Asp
290 295 300
Leu Pro Tyr Asp Ala Pro Val Ile Gln Glu Ile Tyr His Ser Arg Glu
305 310 315 320
Gln Lys Ser Lys Arg Ile Pro Leu Glu Met Met His Lys Val Pro Thr
325 330 335
Ser Leu Leu Phe Ser Leu Glu Gly Leu Glu Asn Leu Glu Trp Asp Lys
340 345 350
Leu Leu Lys Leu Gln Ser Ala Asp Gly Ser Phe Leu Thr Ser Pro Ser
355 360 365
Ser Thr Ala Phe Ala Phe Met Gln Thr Arg Asp Pro Lys Cys Tyr Gln
370 375 380
Phe Ile Lys Asn Thr Ile Gln Thr Phe Asn Gly Gly Ala Pro His Thr
385 390 395 400
Tyr Pro Val Asp Val Phe Gly Arg Leu Trp Ala Ile Asp Arg Leu Gln
405 410 415
Arg Leu Gly Ile Ser Arg Phe Phe Glu Ser Glu Ile Ala Asp Cys Ile
420 425 430
Ala His Ile His Arg Phe Trp Thr Glu Lys Gly Val Phe Ser Gly Arg
435 440 445
Glu Ser Glu Phe Cys Asp Ile Asp Asp Thr Ser Met Gly Val Arg Leu
450 455 460
Met Arg Met His Gly Tyr Asp Val Asp Pro Asn Val Leu Lys Asn Phe
465 470 475 480
Lys Lys Asp Asp Lys Phe Ser Cys Tyr Gly Gly Gln Met Ile Glu Ser
485 490 495
Pro Ser Pro Ile Tyr Asn Leu Tyr Arg Ala Ser Gln Leu Arg Phe Pro
500 505 510
Gly Glu Gln Ile Leu Glu Asp Ala Asn Lys Phe Ala Tyr Asp Phe Leu
515 520 525
Gln Glu Lys Leu Ala His Asn Gln Ile Leu Asp Lys Trp Val Ile Ser
530 535 540
Lys His Leu Pro Asp Glu Ile Lys Leu Gly Leu Glu Met Pro Trp Tyr
545 550 555 560
Ala Thr Leu Pro Arg Val Glu Ala Arg Tyr Tyr Ile Gln Tyr Tyr Ala
565 570 575
Gly Ser Gly Asp Val Trp Ile Gly Lys Thr Leu Tyr Arg Met Pro Glu
580 585 590
Ile Ser Asn Asp Thr Tyr His Glu Leu Ala Lys Thr Asp Phe Lys Arg
595 600 605
Cys Gln Ala Gln His Gln Phe Glu Trp Ile Tyr Met Gln Glu Trp Tyr
610 615 620
Glu Ser Cys Asn Met Glu Glu Phe Gly Ile Ser Arg Lys Glu Leu Leu
625 630 635 640
Val Ala Tyr Phe Leu Ala Thr Ala Ser Ile Phe Glu Leu Glu Arg Ala
645 650 655
Asn Glu Arg Ile Ala Trp Ala Lys Ser Gln Ile Ile Ser Thr Ile Ile
660 665 670
Ala Ser Phe Phe Asn Asn Gln Asn Thr Ser Pro Glu Asp Lys Leu Ala
675 680 685
Phe Leu Thr Asp Phe Lys Asn Gly Asn Ser Thr Asn Met Ala Leu Val
690 695 700
Thr Leu Thr Gln Phe Leu Glu Gly Phe Asp Arg Tyr Thr Ser His Gln
705 710 715 720
Leu Lys Asn Ala Trp Ser Val Trp Leu Arg Lys Leu Gln Gln Gly Glu
725 730 735
Gly Asn Gly Gly Ala Asp Ala Glu Leu Leu Val Asn Thr Leu Asn Ile
740 745 750
Cys Ala Gly His Ile Ala Phe Arg Glu Glu Ile Leu Ala His Asn Asp
755 760 765
Tyr Lys Thr Leu Ser Asn Leu Thr Ser Lys Ile Cys Arg Gln Leu Ser
770 775 780
Gln Ile Gln Asn Glu Lys Glu Leu Glu Thr Glu Gly Gln Lys Thr Ser
785 790 795 800
Ile Lys Asn Lys Glu Leu Glu Glu Asp Met Gln Arg Leu Val Lys Leu
805 810 815
Val Leu Glu Lys Ser Arg Val Gly Ile Asn Arg Asp Met Lys Lys Thr
820 825 830
Phe Leu Ala Val Val Lys Thr Tyr Tyr Tyr Lys Ala Tyr His Ser Ala
835 840 845
Gln Ala Ile Asp Asn His Met Phe Lys Val Leu Phe Glu Pro Val Ala
850 855 860
<210> 13
<211> 849
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 13
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Gly Asp
100 105 110
Asp Asp Asp Lys Ile His Met Cys Arg Val Ala Ser Leu Asp Ala Leu
115 120 125
Asn Gly Ile Gln Lys Val Gly Pro Ala Thr Ile Gly Thr Pro Glu Glu
130 135 140
Glu Asn Lys Lys Ile Glu Asp Ser Ile Glu Tyr Val Lys Glu Leu Leu
145 150 155 160
Lys Thr Met Gly Asp Gly Arg Ile Ser Val Ser Pro Tyr Asp Thr Ala
165 170 175
Ile Val Ala Leu Ile Lys Asp Leu Glu Gly Gly Asp Gly Pro Glu Phe
180 185 190
Pro Ser Cys Leu Glu Trp Ile Ala Gln Asn Gln Leu Ala Asp Gly Ser
195 200 205
Trp Gly Asp His Phe Phe Cys Ile Tyr Asp Arg Val Val Asn Thr Ala
210 215 220
Ala Cys Val Val Ala Leu Lys Ser Trp Asn Val His Ala Asp Lys Ile
225 230 235 240
Glu Lys Gly Ala Val Tyr Leu Lys Glu Asn Val His Lys Leu Lys Asp
245 250 255
Gly Lys Ile Glu His Met Pro Ala Gly Phe Glu Phe Val Val Pro Ala
260 265 270
Thr Leu Glu Arg Ala Lys Ala Leu Gly Ile Lys Gly Leu Pro Tyr Asp
275 280 285
Asp Pro Phe Ile Arg Glu Ile Tyr Ser Ala Lys Gln Thr Arg Leu Thr
290 295 300
Lys Ile Pro Lys Gly Met Ile Tyr Glu Ser Pro Thr Ser Leu Leu Tyr
305 310 315 320
Ser Leu Asp Gly Leu Glu Gly Leu Glu Trp Asp Lys Ile Leu Lys Leu
325 330 335
Gln Ser Ala Asp Gly Ser Phe Ile Thr Ser Val Ser Ser Thr Ala Phe
340 345 350
Val Phe Met His Thr Asn Asp Leu Lys Cys His Ala Phe Ile Lys Asn
355 360 365
Ala Leu Thr Asn Cys Asn Gly Gly Val Pro His Thr Tyr Pro Val Asp
370 375 380
Ile Phe Ala Arg Leu Trp Ala Val Asp Arg Leu Gln Arg Leu Gly Ile
385 390 395 400
Ser Arg Phe Phe Glu Pro Glu Ile Lys Tyr Leu Met Asp His Ile Asn
405 410 415
Asn Val Trp Arg Glu Lys Gly Val Phe Ser Ser Arg His Ser Gln Phe
420 425 430
Ala Asp Ile Asp Asp Thr Ser Met Gly Ile Arg Leu Leu Lys Met His
435 440 445
Gly Tyr Asn Val Asn Pro Asn Ala Leu Glu His Phe Lys Gln Lys Asp
450 455 460
Gly Lys Phe Thr Cys Tyr Ala Asp Gln His Ile Glu Ser Pro Ser Pro
465 470 475 480
Met Tyr Asn Leu Tyr Arg Ala Ala Gln Leu Arg Phe Pro Gly Glu Glu
485 490 495
Ile Leu Gln Gln Ala Leu Gln Phe Ala Tyr Asn Phe Leu His Glu Asn
500 505 510
Leu Ala Ser Asn His Phe Gln Glu Lys Trp Val Ile Ser Asp His Leu
515 520 525
Ile Asp Glu Val Arg Ile Gly Leu Lys Met Pro Trp Tyr Ala Thr Leu
530 535 540
Pro Arg Val Glu Ala Ser Tyr Tyr Leu Gln His Tyr Gly Gly Ser Ser
545 550 555 560
Asp Val Trp Ile Gly Lys Thr Leu Tyr Arg Met Pro Glu Ile Ser Asn
565 570 575
Asp Thr Tyr Lys Ile Leu Ala Gln Leu Asp Phe Asn Lys Cys Gln Ala
580 585 590
Gln His Gln Leu Glu Trp Met Ser Met Lys Glu Trp Tyr Gln Ser Asn
595 600 605
Asn Val Lys Glu Phe Gly Ile Ser Lys Lys Glu Leu Leu Leu Ala Tyr
610 615 620
Phe Leu Ala Ala Ala Thr Met Phe Glu Pro Glu Arg Thr Gln Glu Arg
625 630 635 640
Ile Met Trp Ala Lys Thr Gln Val Val Ser Arg Met Ile Thr Ser Phe
645 650 655
Leu Asn Lys Glu Asn Thr Met Ser Phe Asp Leu Lys Ile Ala Leu Leu
660 665 670
Thr Gln Pro Gln His Gln Ile Asn Gly Ser Glu Met Lys Asn Gly Leu
675 680 685
Ala Gln Thr Leu Pro Ala Ala Phe Arg Gln Leu Leu Lys Glu Phe Asp
690 695 700
Lys Tyr Thr Arg His Gln Leu Arg Asn Thr Trp Asn Lys Trp Leu Met
705 710 715 720
Lys Leu Lys Gln Gly Asp Asp Asn Gly Gly Ala Asp Ala Glu Leu Leu
725 730 735
Ala Asn Thr Leu Asn Ile Cys Ala Gly His Asn Glu Asp Ile Leu Ser
740 745 750
His Tyr Glu Tyr Thr Ala Leu Ser Ser Leu Thr Asn Lys Ile Cys Gln
755 760 765
Arg Leu Ser Gln Ile Gln Asp Lys Lys Met Leu Glu Ile Glu Glu Gly
770 775 780
Ser Ile Lys Asp Lys Glu Met Glu Leu Glu Ile Gln Thr Leu Val Lys
785 790 795 800
Leu Val Leu Gln Glu Thr Ser Gly Gly Ile Asp Arg Asn Ile Lys Gln
805 810 815
Thr Phe Leu Ser Val Phe Lys Thr Phe Tyr Tyr Arg Ala Tyr His Asp
820 825 830
Ala Lys Thr Ile Asp Ala His Ile Phe Gln Val Leu Phe Glu Pro Val
835 840 845
Val
<210> 14
<211> 920
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 14
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Gly Asp
100 105 110
Asp Asp Asp Lys Ile His Met Gln Val Ile Ile Thr Ser Ser His Arg
115 120 125
Phe Phe Cys His His Leu His Gln Leu Lys Ser Pro Thr Ser Leu Ser
130 135 140
Ala Gln Lys Ala Glu Phe Lys Lys His Gly Pro Arg Asn Trp Leu Phe
145 150 155 160
Gln Thr Glu Gly Ser Leu Leu Tyr Lys Pro Val Arg Leu Asn Cys Ala
165 170 175
Thr Ser Asp Ala Ser Tyr Leu Gly Asn Val Asn Glu Tyr Leu Glu Ser
180 185 190
Asp His Ser Lys Asn Ser Glu Glu Lys Asp Ile Gln Val Ser Arg Thr
195 200 205
Ile Gln Met Lys Gly Leu Thr Glu Glu Ile Lys His Met Leu Asn Ser
210 215 220
Met Glu Asp Gly Arg Leu Asn Val Leu Ala Tyr Asp Thr Ala Trp Val
225 230 235 240
Ser Phe Ile Pro Asn Thr Thr Asn Asn Gly Asn Asp Gln Arg Pro Met
245 250 255
Phe Pro Ser Cys Leu Gln Trp Ile Ile Asp Asn Gln Leu Ser Asp Gly
260 265 270
Ser Trp Gly Glu Glu Ile Val Phe Cys Ile Tyr Asp Arg Leu Leu Asn
275 280 285
Thr Leu Val Cys Val Ile Ala Leu Thr Leu Trp Asn Thr Cys Leu His
290 295 300
Lys Arg Asn Lys Gly Val Met Phe Ile Lys Glu Asn Leu Ser Lys Leu
305 310 315 320
Glu Thr Gly Glu Val Glu Asn Met Thr Ser Gly Phe Glu Leu Val Phe
325 330 335
Pro Thr Leu Leu Glu Lys Ala Gln Gln Leu Asp Ile Asp Ile Pro Tyr
340 345 350
Asp Ala Pro Val Leu Lys Asp Ile Tyr Ala Arg Arg Glu Val Lys Leu
355 360 365
Thr Arg Ile Pro Lys Asp Val Ile His Thr Ile Pro Thr Thr Val Leu
370 375 380
Phe Ser Leu Glu Gly Leu Arg Asp Asp Leu Asp Trp Gln Arg Leu Leu
385 390 395 400
Lys Leu Gln Met Pro Asp Gly Ser Phe Leu Ile Ser Pro Ala Ser Thr
405 410 415
Ala Phe Ala Phe Met Glu Thr Asn Asp Glu Lys Cys Leu Ala Tyr Leu
420 425 430
Gln Asn Val Val Glu Lys Ser Asn Gly Gly Ala Arg Gln Tyr Pro Phe
435 440 445
Asp Leu Val Thr Arg Leu Trp Ala Ile Asp Arg Leu Gln Arg Leu Gly
450 455 460
Ile Ser Tyr Tyr Phe Ala Glu Glu Phe Lys Glu Leu Leu Asn His Val
465 470 475 480
Phe Arg Tyr Trp Asp Glu Glu Asn Gly Ile Phe Ser Gly Arg Asn Ser
485 490 495
Asn Val Ser Asp Val Asp Asp Thr Cys Met Ala Ile Arg Leu Leu Arg
500 505 510
Leu His Gly Tyr Asp Val Ser Pro Asp Ala Leu Asn Asn Phe Lys Asp
515 520 525
Gly Asp Gln Phe Val Cys Phe Arg Gly Glu Val Asp Gly Ser Pro Thr
530 535 540
His Met Phe Asn Leu Tyr Arg Cys Ser Gln Val Leu Phe Pro Gly Glu
545 550 555 560
Lys Ile Leu Glu Glu Ala Lys Asn Phe Thr Tyr Asn Phe Leu Gln Gln
565 570 575
Cys Leu Ala Asn Asn Arg Cys Leu Asp Lys Trp Val Ile Ala Lys Asp
580 585 590
Ile Pro Gly Glu Ile Trp Tyr Ala Leu Glu Phe Pro Trp Tyr Ala Ser
595 600 605
Leu Pro Arg Val Glu Ala Arg Tyr Tyr Ile Glu Gln Tyr Gly Gly Ala
610 615 620
Asp Asp Ile Trp Ile Gly Lys Thr Leu Tyr Arg Met Pro Asp Val Asn
625 630 635 640
Asn Asn Val Tyr Leu Gln Ala Ala Lys Leu Asp Tyr Asn Arg Cys Gln
645 650 655
Ser Gln His Arg Phe Glu Trp Leu Ile Met Gln Glu Trp Phe Glu Lys
660 665 670
Cys Asn Phe Gln Gln Phe Gly Ile Ser Lys Lys Tyr Leu Leu Val Ser
675 680 685
Tyr Phe Leu Ala Ala Ala Ser Ile Phe Glu Val Glu Lys Ser Arg Glu
690 695 700
Arg Leu Ala Trp Ala Lys Ser Arg Ile Ile Cys Lys Met Ile Thr Ser
705 710 715 720
Tyr Tyr Asn Asp Glu Ala Thr Thr Trp Thr Thr Arg Asn Ser Leu Leu
725 730 735
Met Glu Phe Lys Val Ser His Asp Pro Thr Arg Lys Asn Gly Asn Glu
740 745 750
Thr Lys Glu Ile Leu Val Leu Lys Asn Leu Arg Gln Phe Leu Arg Gln
755 760 765
Leu Ser Glu Glu Thr Phe Glu Asp Leu Gly Lys Asp Ile His His Gln
770 775 780
Leu Gln Asn Ala Trp Glu Thr Trp Leu Val Phe Leu Arg Glu Glu Lys
785 790 795 800
Asn Ala Cys Gln Glu Glu Thr Glu Leu Leu Val Arg Thr Ile Asn Leu
805 810 815
Ser Gly Gly Tyr Met Thr His Asp Glu Ile Leu Phe Asp Ala Asp Tyr
820 825 830
Glu Asn Leu Ser Asn Leu Thr Asn Lys Val Cys Gly Lys Leu Asn Glu
835 840 845
Leu Gln Asn Asp Lys Val Thr Gly Gly Ser Lys Asn Thr Asn Ile Glu
850 855 860
Leu Asp Met Gln Ala Leu Val Lys Leu Val Phe Gly Asn Thr Ser Ser
865 870 875 880
Asn Ile Asn Gln Asp Ile Lys Gln Thr Phe Phe Ala Val Val Lys Thr
885 890 895
Phe Tyr Tyr Ser Ala His Val Ser Glu Glu Ile Met Asn Phe His Ile
900 905 910
Ser Lys Val Leu Phe Gln Gln Val
915 920
<210> 15
<211> 366
<212> PRT
<213> 巨柏（Cupressus gigantea）
<400> 15
Met Ser Ser Phe Asp Ala His Asp Leu Asp Leu Asp Lys Phe Pro Glu
1 5 10 15
Val Val Arg Asp Arg Leu Thr Gln Phe Leu Asp Ala Gln Glu Leu Thr
20 25 30
Ile Ala Asp Ile Gly Ala Pro Val Thr Asp Ala Val Ala His Leu Arg
35 40 45
Ser Phe Val Leu Asn Gly Gly Lys Arg Ile Arg Pro Leu Tyr Ala Trp
50 55 60
Ala Gly Phe Leu Ala Ala Gln Gly His Lys Asn Ser Ser Glu Lys Leu
65 70 75 80
Glu Ser Val Leu Asp Ala Ala Ala Ser Leu Glu Phe Ile Gln Ala Cys
85 90 95
Ala Leu Ile His Asp Asp Ile Ile Asp Ser Ser Asp Thr Arg Arg Gly
100 105 110
Ala Pro Thr Val His Arg Ala Val Glu Ala Asp His Arg Ala Asn Asn
115 120 125
Phe Glu Gly Asp Pro Glu His Phe Gly Val Ser Val Ser Ile Leu Ala
130 135 140
Gly Asp Met Ala Leu Val Trp Ala Glu Asp Met Leu Gln Asp Ser Gly
145 150 155 160
Leu Ser Ala Glu Ala Leu Ala Arg Thr Arg Asp Ala Trp Arg Gly Met
165 170 175
Arg Thr Glu Val Ile Gly Gly Gln Leu Leu Asp Ile Tyr Leu Glu Ser
180 185 190
His Ala Asn Glu Ser Val Glu Leu Ala Asp Ser Val Asn Arg Phe Lys
195 200 205
Thr Ala Ala Tyr Thr Ile Ala Arg Pro Leu His Leu Gly Ala Ser Ile
210 215 220
Ala Gly Gly Ser Pro Gln Leu Ile Asp Ala Leu Leu His Tyr Gly His
225 230 235 240
Asp Ile Gly Ile Ala Phe Gln Leu Arg Asp Asp Leu Leu Gly Val Phe
245 250 255
Gly Asp Pro Ala Ile Thr Gly Lys Pro Ala Gly Asp Asp Ile Arg Glu
260 265 270
Gly Lys Arg Thr Val Leu Leu Ala Leu Ala Leu Gln Arg Ala Asp Lys
275 280 285
Gln Ser Pro Glu Ala Ala Thr Ala Ile Arg Ala Gly Val Gly Lys Val
290 295 300
Thr Ser Pro Glu Asp Ile Ala Val Ile Thr Glu His Ile Arg Ala Thr
305 310 315 320
Gly Ala Glu Glu Glu Val Glu Gln Arg Ile Ser Gln Leu Thr Glu Ser
325 330 335
Gly Leu Ala His Leu Asp Asp Val Asp Ile Pro Asp Glu Val Arg Ala
340 345 350
Gln Leu Arg Ala Leu Ala Ile Arg Ser Thr Glu Arg Arg Met
355 360 365
<210> 16
<211> 2769
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 16
atgaagatcg aggaaggcaa gctcgtgatc tggatcaacg gcgacaaggg ctacaacggc 60
ctggccgagg tgggcaagaa gttcgagaag gacaccggca tcaaggtgac ggtggagcac 120
ccggacaagc tcgaggagaa gttcccgcag gtggcggcca cgggcgacgg cccggacatc 180
atcttctggg cccatgaccg cttcggcggc tacgcccagt cgggcctgct ggccgagatc 240
accccggaca aggcgttcca ggacaagctc tatcccttca cgtgggacgc cgtgcgctac 300
aacggcaagc tgatcgcgta tcccatcgcg gtggaggccc tgtcgctcat ctataacaag 360
gacctgctcc cgaacccgcc caagacctgg gaggagatcc ccgccctcga caaggagctg 420
aaggccaagg gcaagtcggc gctcatgttc aacctgcagg agccgtactt cacctggccc 480
ctgatcgcgg ccgacggcgg ctacgcgttc aagtatgaga acggcaagta tgacatcaag 540
gacgtgggcg tggacaacgc gggcgccaag gccggcctga ccttcctcgt ggacctgatc 600
aagaacaagc acatgaacgc cgacacggac tactcgatcg cggaggccgc gttcaacaag 660
ggcgagactg ccatgacgat caacggcccg tgggcgtggt cgaacatcga cacctcgaag 720
gtgaactatg gcgtgaccgt gctccccacg ttcaagggcc agccctcgaa gcccttcgtg 780
ggcgtgctgt cggcgggcat caacgccgcg tcgccgaaca aggagctcgc gaaggagttc 840
ctggagaact acctgctcac cgacgagggc ctggaggccg tgaacaagga caagcccctg 900
ggcgccgtgg ccctgaagtc gtatgaggaa gagctggtga aggacccgcg catcgcggcc 960
accatggaga acgcgcagaa gggcgagatc atgccgaaca tcccccagat gtcggccttc 1020
tggtatgcgg tgcgcaccgc cgtgatcaac gcggcctcgg gccgccagac cgtggacgag 1080
gccctcaagg acgcccagac cggcggaggt gctgccatgg agaacaagtc gttcggctcg 1140
atgtgcatcg acgacaacat cacccgcatc aacatgctct tcctcgagat caagcgccgc 1200
tactcgtcgc tctcggccta cgacaccgtg cacggcgcct acccgatcaa cctcatcctc 1260
ggcctctcga tggtgaacca cctcgagtcg ctcggcatcg cccgccactt caagctcgag 1320
atcaaggagg tgctcgacga cgtgtaccgc ttctggatcg agaacgagat ggtgaagggc 1380
atgccgtcgg cctcggacat catgacctcg tcgatctcgt tcaagatcct ccgctggaac 1440
ggctacgacg tgtcgccgga cgtgttcttc tcgtacctcc gcgactcgga cttcctcccg 1500
ttcctcgaga actcgggcca ggccactatg gccctcctca acctctacaa ggcctcgcag 1560
atgatgttcc cgggcgagcg catcctcgag gaggccaagt cgttctcgca gaactacctc 1620
gagaacatca aggtgggcga ccagtcgggc gccctcaagg acatcgaggc cgaggtgaag 1680
cacgccctcg acgtgccgtg gatggccggc ctcgagcgca tcgagcacct ccgctacatg 1740
aaggccttcg ccttcgacga catctggatc ggcgagacgt cgtcgcgcat cccgttcacc 1800
ggcaaggacc tcttcctctc gctcgccaag caggacttca acatctgcca ggaggcccag 1860
caggaggacc tcaaggccct cgagaagtgg tcggtggact cgaagctcgg cgacctccac 1920
ttcgcccgcc agaagctcgt ggcctgctac ttctcggccg cctcgaccct cttctcgccg 1980
gagatgtcga ccacccgcat cgtgtggacc aagaacgccg tgctcaccac cctcatggac 2040
gacttctacg acgtggaggg ctcgatcgag gacatccagt cgttcgtgga ggccgtgaag 2100
cgctgggacc cgatcgtgct ctgcaactgc tcggccaacg tgaagaagct cttctcggcc 2160
atctacaaca ccgtgaacga catcgcccag gacatctgga tcttccaggg ctgggacatc 2220
tcgacccagc tccgcgagat ctggtacgag ctcgccatct cgatgatgaa ggaggccgag 2280
tggaccaaga ccggcttcat cccgtcgctc gaggagtaca tggagaacgg ccgcacctcg 2340
atcgccctcg agccgatcat cttcacctcg ctcttcttcc tcggcccgaa gctctcggag 2400
cagatgatcc acgagtacaa gcgcctcatg cagctcctca acacctgcgg caggctcctc 2460
aacgactcgc agtcgtacaa gcgcgagatc aagcagggca agctcaactc gctctcgctc 2520
ttcgtgaagg agtacccggc cacctcgatc gaggacgcca ccgagtggat caggctcacc 2580
gtgtcggagt cgcgccagaa catcctccgc aacctcctcc agccgtcgga gatcccgcgc 2640
gagtgcaagc agatcttctg gaacatggtg aagatcatcc agctcttcta cctccactcg 2700
gacgagttca cctcgccgac cgccatgctc gagcacatgc aggccgtgct cttcaacccg 2760
gtgctctaa 2769
<210> 17
<211> 2769
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 17
atgaagatcg aggaaggcaa gctcgtgatc tggatcaacg gcgacaaggg ctacaacggc 60
ctggccgagg tgggcaagaa gttcgagaag gacaccggca tcaaggtgac ggtggagcac 120
ccggacaagc tcgaggagaa gttcccgcag gtggcggcca cgggcgacgg cccggacatc 180
atcttctggg cccatgaccg cttcggcggc tacgcccagt cgggcctgct ggccgagatc 240
accccggaca aggcgttcca ggacaagctc tatcccttca cgtgggacgc cgtgcgctac 300
aacggcaagc tgatcgcgta tcccatcgcg gtggaggccc tgtcgctcat ctataacaag 360
gacctgctcc cgaacccgcc caagacctgg gaggagatcc ccgccctcga caaggagctg 420
aaggccaagg gcaagtcggc gctcatgttc aacctgcagg agccgtactt cacctggccc 480
ctgatcgcgg ccgacggcgg ctacgcgttc aagtatgaga acggcaagta tgacatcaag 540
gacgtgggcg tggacaacgc gggcgccaag gccggcctga ccttcctcgt ggacctgatc 600
aagaacaagc acatgaacgc cgacacggac tactcgatcg cggaggccgc gttcaacaag 660
ggcgagactg ccatgacgat caacggcccg tgggcgtggt cgaacatcga cacctcgaag 720
gtgaactatg gcgtgaccgt gctccccacg ttcaagggcc agccctcgaa gcccttcgtg 780
ggcgtgctgt cggcgggcat caacgccgcg tcgccgaaca aggagctcgc gaaggagttc 840
ctggagaact acctgctcac cgacgagggc ctggaggccg tgaacaagga caagcccctg 900
ggcgccgtgg ccctgaagtc gtatgaggaa gagctggtga aggacccgcg catcgcggcc 960
accatggaga acgcgcagaa gggcgagatc atgccgaaca tcccccagat gtcggccttc 1020
tggtatgcgg tgcgcaccgc cgtgatcaac gcggcctcgg gccgccagac cgtggacgag 1080
gccctcaagg acgcccagac cggcggaggt gctgccatgg agaacaagtc gttcggctcg 1140
atgtgcatcg acgacaacat cacccgcatc aacatgctct tcctcgagat caagcgccgc 1200
tactcgtcgc tctcggccta cgacaccgtg cacggcgcct acccgatcaa cctcatcctc 1260
ggcctctcga tggtgaacca cctcgagtcg ctcggcatcg cccgccactt caagctcgag 1320
atcaaggagg tgctcgacga cgtgtaccgc ttctggatcg agaacgagat ggtgaagggc 1380
atgccgtcgg cctcggacat catgacctcg tcgatctcgt tcaagatcct ccgctggaac 1440
ggctacgacg tgtcgccgga cgtgttcttc tcgtacctcc gcgactcgga cttcctcccg 1500
ttcctcgaga actcgggcca ggccactatg gccctcctca acctctacaa ggcctcgcag 1560
atgatgttcc cgggcgagcg catcctcgag gaggccaagt cgttctcgca gaactacctc 1620
gagaacatca aggtgggcga ccagtcgggc gccctcaagg acatcgaggc cgaggtgaag 1680
cacgccctcg acgtgccgtg gatggccggc ctcgagcgca tcgagcacct ccgctacatg 1740
aaggccttcg ccttcgacga catctggatc ggcgagacgt cgtcgcgcat cccgttcacc 1800
ggcaaggacc tcttcctctc gctcgccaag caggacttca acatctgcca ggaggcccag 1860
caggaggacc tcaaggccct cgagaagtgg tcggtggact cgaagctcgg cgacctccac 1920
ttcgcccgcc agaagctcgt ggcctgctac ttctcggccg cctcgaccct cttctcgccg 1980
gagatgtcga ccacccgcat cgtgtggacc aagaacgccg tgctcaccac cctcatggac 2040
gacttctacg acgtggaggg ctcgatcgag gacatccagt cgttcgtgga ggccgtgaag 2100
cgctgggacc cgatcgtgct ctgcaactgc tcggccaacg tgaagaagct cttctcggcc 2160
atctacaaca ccgtgaacga catcgcccag gacatctgga tcttccaggg ctgggacatc 2220
tcgacccagc tccgcgagat ctggtacgag ctcgccatct cgatgatgaa ggaggccgag 2280
tggaccaaga ccggcttcat cccgtcgctc gaggagtaca tggagaacgg ccgcacctcg 2340
atcgccctcg agccgatcat cttcacctcg ctcttcttcc tcggcccgaa gctctcggag 2400
cagatgatcc acgagtacaa gcgcctcatg cagctcctca acacctgcgg caggctcctc 2460
aacgactcgc agtcgtacaa gcgcgagatc aagcagggca agctcacctc gctctcgctc 2520
ttcgtgaagg agtacccggc ccacctcatc gaggacgcca ccgagtggat caggctcacc 2580
gtgtcggagt cgcgccagaa catcctccgc aacctcctcc agccgtcgga gatcccgcgc 2640
gagtgcaagc agatcttctg gaacatggtg aagatcatcc agctcttcta cctccactcg 2700
gacgagttca cctcgccgac cgccatgctc gagcacatgc aggccgtgct cttcaacccg 2760
gtgctctaa 2769
<210> 18
<211> 2766
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 18
atgaagatcg aggaaggcaa gctcgtgatc tggatcaacg gcgacaaggg ctacaacggc 60
ctggccgagg tgggcaagaa gttcgagaag gacaccggca tcaaggtgac ggtggagcac 120
ccggacaagc tcgaggagaa gttcccgcag gtggcggcca cgggcgacgg cccggacatc 180
atcttctggg cccatgaccg cttcggcggc tacgcccagt cgggcctgct ggccgagatc 240
accccggaca aggcgttcca ggacaagctc tatcccttca cgtgggacgc cgtgcgctac 300
aacggcaagc tgatcgcgta tcccatcgcg gtggaggccc tgtcgctcat ctataacaag 360
gacctgctcc cgaacccgcc caagacctgg gaggagatcc ccgccctcga caaggagctg 420
aaggccaagg gcaagtcggc gctcatgttc aacctgcagg agccgtactt cacctggccc 480
ctgatcgcgg ccgacggcgg ctacgcgttc aagtatgaga acggcaagta tgacatcaag 540
gacgtgggcg tggacaacgc gggcgccaag gccggcctga ccttcctcgt ggacctgatc 600
aagaacaagc acatgaacgc cgacacggac tactcgatcg cggaggccgc gttcaacaag 660
ggcgagactg ccatgacgat caacggcccg tgggcgtggt cgaacatcga cacctcgaag 720
gtgaactatg gcgtgaccgt gctccccacg ttcaagggcc agccctcgaa gcccttcgtg 780
ggcgtgctgt cggcgggcat caacgccgcg tcgccgaaca aggagctcgc gaaggagttc 840
ctggagaact acctgctcac cgacgagggc ctggaggccg tgaacaagga caagcccctg 900
ggcgccgtgg ccctgaagtc gtatgaggaa gagctggtga aggacccgcg catcgcggcc 960
accatggaga acgcgcagaa gggcgagatc atgccgaaca tcccccagat gtcggccttc 1020
tggtatgcgg tgcgcaccgc cgtgatcaac gcggcctcgg gccgccagac cgtggacgag 1080
gccctcaagg acgcccagac cggcggaggt gctgccatgg agaagaagtc gttcggctcg 1140
atgtgcatcg acgacgacat caccaagatc aagatgctct tctcggagat caagcgccgc 1200
tactcgtcgc tctcggccta cgacaccgtg cacggcgcct acccgatcaa cctcatcctc 1260
ggcctctcga tggtgaacca cctcgagtcg ctcggcatcg cccgccactt caagctcgag 1320
atcaaggagg tgctcgacga cgtgtaccgc ttctggatcg agaacgagat ggtggagggc 1380
atgccgtcgg cctcggacat catgacctcg tcgatctcgt tccgcatcct ccgctggaac 1440
ggctacgacg tgtcgccgga cgtgttcttc tcgtacctcc gcgactcgga cttcctcctc 1500
ttcctcgaga actcggacca ggccaccatc gccctcctca acctctacaa ggcctcgcag 1560
atgatgttcc cgggcgagcg catcctcgag gaggccaagc tcttctcgca gaactacctc 1620
gagaacatca agatgggcaa ccagtcgggc gccctcaagg acatcgaggc cgaggtgaag 1680
cacgccctcg acgtgccgtg gatggccaac ctcgagcgca tcgagcacct ccgctacatg 1740
aaggccttcg ccttcgacaa catctggatc ggcgagacgt cgtcgcgcat cccgttcacc 1800
ggcaaggacc tcttcctctc gttcgccaag caggacttca acatctgcca ggccgcccag 1860
caggaggacc tcaaggccct cgagaagtgg tcggtggact cgaagctcgg cgacctccac 1920
ttcgcccgcc agaagctcgt ggcctgctac ttctcggccg cctcgatcct cttctcgccg 1980
gagatgtcgg ccacccgcat cgtgtggacc aagaacgccg tgctcatcac cctcatggac 2040
gacttctacg acgtggaggg ctcgatcgag gacatccagt cgttcgtgga ggccgtgaag 2100
cgctgggacc cgatcgtgct ctgcaactgc tcggccaacg tgaagatcct cttctcggcc 2160
atctacaaca ccgtgaacga catcgcccag gacatctgga tcttccaggg ctgggacatc 2220
tcgacccagc tccgcgagat ctggtacgag ctcgccatct cgatgatgaa ggaggccgag 2280
tggaccaaga ccggcttcat cccgtcgctc gaggagtaca tggagaacgg ccgcacctcg 2340
atcgccctcg agccgatcat cttcacctcg ctcttcttca tcggcccgaa gctctcggag 2400
cagatgatcc accaccacga gtacaagcgc ctcctccagc tcatgaacac ctgcggccgc 2460
ctcctcaacg acatccagtc ggacaagcgc gagatgaagc agggcaagct cacctcggtg 2520
tcgctcttcg tgaaggagta cccgatcgag gacgccacca agtggatccg cctcaccgtg 2580
tcggagaaca cccagaacat cctcaagaag atcctccagc cgtcggagat cccgcgcgag 2640
tgcaagcaga tcttctggaa catggtgaag atcatccagc tcttctgcct ccactcggac 2700
gagttcacct cgccgaccac catgctcgag cacatgaagg ccgtgctctt ctacccggtg 2760
ctctga 2766
<210> 19
<211> 1578
<212> DNA
<213> 南欧丹参（Salvia sclarea）
<400> 19
atggccaaga tgaaggagaa cttcaagcgc gaggacgaca agttcccgac caccaccacc 60
ctccgctcgg aggacatccc gtcgaacctc tgcatcatcg acaccctcca gcgcctcggc 120
gtggaccagt tcttccagta cgagatcaac accatcctcg acaacacctt ccgcctctgg 180
caggagaagc acaaggtgat ctacggcaac gtgaccaccc acgccatggc cttccgcctc 240
ctccgcgtga agggctacga ggtgtcgtcg gaggagctcg ccccgtacgg caaccaggag 300
gccgtgtcgc agcagaccaa cgacctcccg atgatcatcg agctctaccg cgccgccaac 360
gagcgcatct acgaggagga gcgctcgctc gagaagatcc tcgcctggac caccatcttc 420
ctcaacaagc aggtgcagga caactcgatc ccggacaaga agctccacaa gctcgtggag 480
ttctacctcc gcaactacaa gggcatcacc atccgcctcg gcgcccgccg caacctcgag 540
ctctacgaca tgacctacta ccaggccctc aagtcgacca accgcttctc gaacctctgc 600
aacgaggact tcctcgtgtt cgccaagcag gacttcgaca tccacgaggc ccagaaccag 660
aagggcctcc agcagctcca gcgctggtac gccgactgcc gcctcgacac cctcaacttc 720
ggccgcgacg tggtgatcat cgccaactac ctcgcctcgc tcatcatcgg cgaccacgcc 780
ttcgactacg tgcgcctcgc cttcgccaag acctcggtgc tcgtgaccat catggacgac 840
ttcttcgact gccacggctc gtcgcaggag tgcgacaaga tcatcgagct cgtgaaggag 900
tggaaggaga acccggacgc cgagtacggc tcggaggagc tcgagatcct cttcatggcc 960
ctctacaaca ccgtgaacga gctcgccgag cgcgcccgcg tggagcaggg ccgctcggtg 1020
aaggagttcc tcgtgaagct ctgggtggag atcctctcgg ccttcaagat cgagctcgac 1080
acctggtcga acggcaccca gcagtcgttc gacgagtaca tctcgtcgtc gtggctctcg 1140
aacggctcgc gcctcaccgg cctcctcacc atgcagttcg tgggcgtgaa gctctcggac 1200
gagatgctca tgtcggagga gtgcaccgac ctcgcccgcc acgtgtgcat ggtgggccgc 1260
ctcctcaacg acgtgtgctc gtcggagcgc gagcgcgagg agaacatcgc cggcaagtcg 1320
tactcgatcc tcctcgccac cgagaaggac ggccgcaagg tgtcggagga cgaggccatc 1380
gccgagatca acgagatggt ggagtaccac tggcgcaagg tgctccagat cgtgtacaag 1440
aaggagtcga tcctcccgcg ccgctgcaag gacgtgttcc tcgagatggc caagggcacc 1500
ttctacgcct acggcatcaa cgacgagctc acctcgccgc agcagtcgaa ggaggacatg 1560
aagtcgttcg tgttctga 1578
<210> 20
<211> 2550
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 20
atgtcggaca agatcatcca cctgaccgac gacagcttcg acaccgacgt gctgaaggcc 60
gacggcgcca tcctcgtcga tttctgggcc gaatggtgcg gcccctgcaa gatgatcgcg 120
ccgatcctcg acgagatcgc cgacgaatat cagggcaagc tgaccgtcgc caagctgaac 180
atcgaccaga acccgggcac ggcgccgaaa tacggcatcc gcggcatccc gacgctgctg 240
ctcttcaaga acggcgaggt ggcggccacc aaggtcggcg cgctgtcgaa gggccagctg 300
aaggagttcc tcgatgcgaa cctcgccggt ggtgatgacg acgacaagat tcatatgtgc 360
cgcgtggcct cgctcgacgc cctcaacggc atccagaagg tgggcccggc caccatcggc 420
accccggagg aggagaacaa gaagatcgag gactcgatcg agtacgtgaa ggagctcctc 480
aagaccatgg gcgacggccg catctcggtg tcgccgtacg acaccgccat cgtggccctc 540
atcaaggacc tcgagggcgg cgacggcccg gagttcccgt cgtgcctcga gtggatcgcc 600
cagaaccagc tcgccgacgg ctcgtggggc gaccacttct tctgcatcta cgaccgcgtg 660
gtgaacaccg ccgcctgcgt ggtggccctc aagtcgtgga acgtgcacgc cgacaagatc 720
gagaagggcg ccgtgtacct caaggagaac gtgcacaagc tcaaggacgg caagatcgag 780
cacatgccgg ccggcttcga gttcgtggtg ccggccaccc tcgagcgcgc caaggccctc 840
ggcatcaagg gcctcccgta cgacgacccg ttcatccgcg agatctactc ggccaagcag 900
acccgcctca ccaagatccc gaagggcatg atctacgagt cgccgacctc gctcctctac 960
tcgctcgacg gcctcgaggg cctcgagtgg gacaagatcc tcaagctcca gtcggccgac 1020
ggctcgttca tcacctcggt gtcgtcgacc gccttcgtgt tcatgcacac caacgacctc 1080
aagtgccacg ccttcatcaa gaacgccctc accaactgca acggcggcgt gccgcacacc 1140
tacccggtgg acatcttcgc ccgcctctgg gccgtggacc gcctccagcg cctcggcatc 1200
tcgcgcttct tcgagccgga gatcaagtac ctcatggacc acatcaacaa cgtgtggcgc 1260
gagaagggcg tgttctcgtc gcgccactcg cagttcgccg acatcgacga cacctcgatg 1320
ggcatccgcc tcctcaagat gcacggctac aacgtgaacc cgaacgccct cgagcacttc 1380
aagcagaagg acggcaagtt cacctgctac gccgaccagc acatcgagtc gccgtcgccg 1440
atgtacaacc tctaccgcgc cgcccagctc cgcttcccgg gcgaggagat cctccagcag 1500
gccctccagt tcgcctacaa cttcctccac gagaacctcg cctcgaacca cttccaggag 1560
aagtgggtga tctcggacca cctcatcgac gaggtgcgca tcggcctcaa gatgccgtgg 1620
tacgccaccc tcccgcgcgt ggaggcctcg tactacctcc agcactacgg cggctcgtcg 1680
gacgtgtgga tcggcaagac cctctaccgc atgccggaga tctcgaacga cacctacaag 1740
atcctcgccc agctcgactt caacaagtgc caggcccagc accagctcga gtggatgtcg 1800
atgaaggagt ggtaccagtc gaacaacgtg aaggagttcg gcatctcgaa gaaggagctc 1860
ctcctcgcct acttcctcgc cgccgccacc atgttcgagc cggagcgcac ccaggagcgc 1920
atcatgtggg ccaagaccca ggtggtgtcg cgcatgatca cctcgttcct caacaaggag 1980
aacaccatgt cgttcgacct caagatcgcc ctcctcaccc agccgcagca ccagatcaac 2040
ggctcggaga tgaagaacgg cctcgcccag accctcccgg ccgccttccg ccagctcctc 2100
aaggagttcg acaagtacac ccgccaccag ctccgcaaca cctggaacaa gtggctcatg 2160
aagctcaagc agggcgacga caacggcggc gccgacgccg agctcctcgc caacaccctc 2220
aacatctgcg ccggccacaa cgaggacatc ctctcgcact acgagtacac cgccctctcg 2280
tcgctcacca acaagatctg ccagcgcctc tcgcagatcc aggacaagaa gatgctcgag 2340
atcgaggagg gctcgatcaa ggacaaggag atggagctcg agatccagac cctcgtgaag 2400
ctcgtgctcc aggagacgtc gggcggcatc gaccgcaaca tcaagcagac cttcctctcg 2460
gtgttcaaga ccttctacta ccgcgcctac cacgacgcca agaccatcga cgcccacatc 2520
ttccaggtgc tcttcgagcc ggtggtgtga 2550
<210> 21
<211> 2595
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 21
atgtcggaca agatcatcca cctgaccgac gacagcttcg acaccgacgt gctgaaggcc 60
gacggcgcca tcctcgtcga tttctgggcc gaatggtgcg gcccctgcaa gatgatcgcg 120
ccgatcctcg acgagatcgc cgacgaatat cagggcaagc tgaccgtcgc caagctgaac 180
atcgaccaga acccgggcac ggcgccgaaa tacggcatcc gcggcatccc gacgctgctg 240
ctcttcaaga acggcgaggt ggcggccacc aaggtcggcg cgctgtcgaa gggccagctg 300
aaggagttcc tcgatgcgaa cctcgccggt ggtgatgacg acgacaagat tcatatgaac 360
aacggcaaga acctcaactg ccagctcacc cacaagaaga tctcgaaggt ggccgagatc 420
cgcgtggcca ccgtgaacgc cccgccggtg cacgaccagg acgactcgac cgagaaccag 480
tgccacgacg ccgtgaacaa catcgaggac ccgatcgagt acatccgcac cctcctccgc 540
accaccggcg acggccgcat ctcggtgtcg ccgtacgaca ccgcctgggt ggccctcatc 600
aaggacctcc agggccgcga cgccccggag ttcccgtcgt cgctcgagtg gatcatccag 660
aaccagctcg ccgacggctc gtggggcgac gccaagttct tctgcgtgta cgaccgcctc 720
gtgaacacca tcgcctgcgt ggtggccctc cgctcgtggg acgtgcacgc cgagaaggtg 780
gagcgcggcg tgcgctacat caacgagaac gtggagaagc tccgcgacgg caacgaggag 840
cacatgacct gcggcttcga ggtggtgttc ccggccctcc tccagcgcgc caagtcgctc 900
ggcatccagg acctcccgta cgacgccccg gtgatccagg agatctacca ctcgcgcgag 960
cagaagtcga agcgcatccc gctcgagatg atgcacaagg tgccgacctc gctcctcttc 1020
tcgctcgagg gcctcgagaa cctcgagtgg gacaagctcc tcaagctcca gtcggccgac 1080
ggctcgttcc tcacctcgcc gtcgtcgacc gccttcgcct tcatgcagac ccgcgacccg 1140
aagtgctacc agttcatcaa gaacaccatc cagaccttca acggcggcgc cccgcacacc 1200
tacccggtgg acgtgttcgg ccgcctctgg gccatcgacc gcctccagcg cctcggcatc 1260
tcgcgcttct tcgagtcgga gatcgccgac tgcatcgccc acatccaccg cttctggacc 1320
gagaagggcg tgttctcggg ccgcgagtcg gagttctgcg acatcgacga cacctcgatg 1380
ggcgtgcgcc tcatgcgcat gcacggctac gacgtggacc cgaacgtgct caagaacttc 1440
aagaaggacg acaagttctc gtgctacggc ggccagatga tcgagtcgcc gtcgccgatc 1500
tacaacctct accgcgcctc gcagctccgc ttcccgggcg agcagatcct cgaggacgcc 1560
aacaagttcg cctacgactt cctccaggag aagctcgccc acaaccagat cctcgacaag 1620
tgggtgatct cgaagcacct cccggacgag atcaagctcg gcctcgagat gccgtggtac 1680
gccaccctcc cgcgcgtgga ggcccgctac tacatccagt actacgccgg ctcgggcgac 1740
gtgtggatcg gcaagaccct ctaccgcatg ccggagatct cgaacgacac ctaccacgag 1800
ctcgccaaga ccgacttcaa gcgctgccag gcccagcacc agttcgagtg gatctacatg 1860
caggagtggt acgagtcgtg caacatggag gagttcggca tctcgcgcaa ggagctcctc 1920
gtggcctact tcctcgccac cgcctcgatc ttcgagctcg agcgcgccaa cgagcgcatc 1980
gcctgggcca agtcgcagat catctcgacc atcatcgcct cgttcttcaa caaccagaac 2040
acctcgccgg aggacaagct cgccttcctc accgacttca agaacggcaa ctcgaccaac 2100
atggccctcg tgaccctcac ccagttcctc gagggcttcg accgctacac ctcgcaccag 2160
ctcaagaacg cctggtcggt gtggctccgc aagctccagc agggcgaggg caacggcggc 2220
gccgacgccg agctcctcgt gaacaccctc aacatctgcg ccggccacat cgccttccgc 2280
gaggagatcc tcgcccacaa cgactacaag accctctcga acctcacctc gaagatctgc 2340
cgccagctct cgcagatcca gaacgagaag gagctcgaga cggagggcca gaagacctcg 2400
atcaagaaca aggagctcga ggaggacatg cagcgcctcg tgaagctcgt gctcgagaag 2460
tcgcgcgtgg gcatcaaccg cgacatgaag aagaccttcc tcgccgtggt gaagacctac 2520
tactacaagg cctaccactc ggcccaggcc atcgacaacc acatgttcaa ggtgctcttc 2580
gagccggtgg cctga 2595
<210> 22
<211> 1101
<212> DNA
<213> 巨柏（Cupressus gigantea）
<400> 22
atgtcgtcgt tcgacgccca cgacctcgac ctcgacaagt tcccggaggt ggtgcgcgac 60
cgcctcaccc agttcctcga cgcccaggag ctcaccatcg ccgacatcgg cgccccggtg 120
accgacgccg tggcccacct ccgctcgttc gtgctcaacg gcggcaagcg catccgcccg 180
ctctacgcct gggccggctt cctcgccgcc cagggccaca agaactcgtc ggagaagctc 240
gagtcggtgc tcgacgccgc cgcctcgctc gagttcatcc aggcctgcgc cctcatccac 300
gacgacatca tcgactcgtc ggacacccgc cgcggcgccc cgaccgtgca ccgcgccgtg 360
gaggccgacc accgcgccaa caacttcgag ggcgacccgg agcacttcgg cgtgtcggtg 420
tcgatcctcg ccggcgacat ggccctcgtg tgggccgagg acatgctcca ggactcgggc 480
ctctcggccg aggccctcgc ccgcacccgc gacgcctggc gcggcatgcg caccgaggtg 540
atcggcggcc agctcctcga catctacctc gagtcgcacg ccaacgagtc ggtggagctc 600
gccgactcgg tgaaccgctt caagaccgcc gcctacacca tcgcccgccc gctccacctc 660
ggcgcctcga tcgccggcgg ctcgccgcag ctcatcgacg ccctcctcca ctacggccac 720
gacatcggca tcgccttcca gctccgcgac gacctcctcg gcgtgttcgg cgacccggcc 780
atcaccggca agccggccgg cgacgacatc cgcgagggca agcgcaccgt gctcctcgcc 840
ctcgccctcc agcgcgccga caagcagtcg ccggaggccg ccaccgccat ccgcgccggc 900
gtgggcaagg tgacctcgcc ggaggacatc gccgtgatca ccgagcacat ccgcgccacc 960
ggcgccgagg aggaggtgga gcagcgcatc tcgcagctca ccgagtcggg cctcgcccac 1020
ctcgacgacg tggacatccc ggacgaggtg cgcgcccagc tccgcgccct cgccatccgc 1080
tcgaccgagc gccgcatgtg a 1101
<210> 23
<211> 2763
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 23
atgtcggaca agatcatcca cctgaccgac gacagcttcg acaccgacgt gctgaaggcc 60
gacggcgcca tcctcgtcga tttctgggcc gaatggtgcg gcccctgcaa gatgatcgcg 120
ccgatcctcg acgagatcgc cgacgaatat cagggcaagc tgaccgtcgc caagctgaac 180
atcgaccaga acccgggcac ggcgccgaaa tacggcatcc gcggcatccc gacgctgctg 240
ctcttcaaga acggcgaggt ggcggccacc aaggtcggcg cgctgtcgaa gggccagctg 300
aaggagttcc tcgatgcgaa cctcgccggt ggtgatgacg acgacaagat tcatatgcag 360
gtgatcatca cctcgtcgca ccgcttcttc tgccaccacc tccaccagct caagtcgccg 420
acctcgctct cggcccagaa ggccgagttc aagaagcacg gcccgcgcaa ctggctcttc 480
cagaccgagg gctcgctcct ctacaagccg gtgcgcctca actgcgccac ctcggacgcc 540
tcgtacctcg gcaacgtgaa cgagtacctc gagtcggacc actcgaagaa ctcggaggag 600
aaggacatcc aggtgtcgcg caccatccag atgaagggcc tcaccgagga gatcaagcac 660
atgctcaact cgatggagga cggccgcctc aacgtgctcg cctacgacac cgcctgggtg 720
tcgttcatcc cgaacaccac caacaacggc aacgaccagc gcccgatgtt cccgtcgtgc 780
ctccagtgga tcatcgacaa ccagctctcg gacggctcgt ggggcgagga gatcgtgttc 840
tgcatctacg accgcctcct caacaccctc gtgtgcgtga tcgccctcac cctctggaac 900
acctgcctcc acaagcgcaa caagggcgtg atgttcatca aggagaacct ctcgaagctc 960
gagacgggcg aggtggagaa catgacctcg ggcttcgagc tcgtgttccc gaccctcctc 1020
gagaaggccc agcagctcga catcgacatc ccgtacgacg ccccggtgct caaggacatc 1080
tacgcccgcc gcgaggtgaa gctcacccgc atcccgaagg acgtgatcca caccatcccg 1140
accaccgtgc tcttctcgct cgagggcctc cgcgacgacc tcgactggca gcgcctcctc 1200
aagctccaga tgccggacgg ctcgttcctc atctcgccgg cctcgaccgc cttcgccttc 1260
atggagacga acgacgagaa gtgcctcgcc tacctccaga acgtggtgga gaagtcgaac 1320
ggcggcgccc gccagtaccc gttcgacctc gtgacccgcc tctgggccat cgaccgcctc 1380
cagcgcctcg gcatctcgta ctacttcgcc gaggagttca aggagctcct caaccacgtg 1440
ttccgctact gggacgagga gaacggcatc ttctcgggcc gcaactcgaa cgtgtcggac 1500
gtggacgaca cctgcatggc catccgcctc ctccgcctcc acggctacga cgtgtcgccg 1560
gacgccctca acaacttcaa ggacggcgac cagttcgtgt gcttccgcgg cgaggtggac 1620
ggctcgccga cccacatgtt caacctctac cgctgctcgc aggtgctctt cccgggcgag 1680
aagatcctcg aggaggccaa gaacttcacc tacaacttcc tccagcagtg cctcgccaac 1740
aaccgctgcc tcgacaagtg ggtgatcgcc aaggacatcc cgggcgagat ctggtacgcc 1800
ctcgagttcc cgtggtacgc ctcgctcccg cgcgtggagg cccgctacta catcgagcag 1860
tacggcggcg ccgacgacat ctggatcggc aagaccctct accgcatgcc ggacgtgaac 1920
aacaacgtgt acctccaggc cgccaagctc gactacaacc gctgccagtc gcagcaccgc 1980
ttcgagtggc tcatcatgca ggagtggttc gagaagtgca acttccagca gttcggcatc 2040
tcgaagaagt acctcctcgt gtcgtacttc ctcgccgccg cctcgatctt cgaggtggag 2100
aagtcgcgcg agcgcctcgc ctgggccaag tcgcgcatca tctgcaagat gatcacctcg 2160
tactacaacg acgaggccac cacctggacc acccgcaact cgctcctcat ggagttcaag 2220
gtgtcgcacg acccgacccg caagaacggc aacgagacga aggagatcct cgtgctcaag 2280
aacctccgcc agttcctccg ccagctctcg gaggagacgt tcgaggacct cggcaaggac 2340
atccaccacc agctccagaa cgcctgggag acgtggctcg tgttcctccg cgaggagaag 2400
aacgcctgcc aggaggagac ggagctcctc gtgcgcacca tcaacctctc gggcggctac 2460
atgacccacg acgagatcct cttcgacgcc gactacgaga acctctcgaa cctcaccaac 2520
aaggtgtgcg gcaagctcaa cgagctccag aacgacaagg tgaccggcgg ctcgaagaac 2580
accaacatcg agctcgacat gcaggccctc gtgaagctcg tgttcggcaa cacctcgtcg 2640
aacatcaacc aggacatcaa gcagaccttc ttcgccgtgg tgaagacctt ctactactcg 2700
gcccacgtgt cggaggagat catgaacttc cacatctcga aggtgctctt ccagcaggtg 2760
tga 2763
<210> 24
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 结构域
<400> 24
Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe
1 5 10
<210> 25
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 结构域
<400> 25
Ser Trp Val Ser Ile Gly Cys Arg Ile Cys Ile Leu
1 5 10
<210> 26
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 结构域
<400> 26
Ser Trp Leu Ser Asn Gly Ser Arg Leu Thr Gly Leu
1 5 10
<210> 27
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 结构域
<400> 27
Ser Trp Leu Ser Ile Gly Ser Arg Leu Thr Gly Leu
1 5 10
<210> 28
<211> 940
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 28
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly
355 360 365
Gly Gly Ala Ala Met Leu Ser Val Glu Asp Ser Ser Asn Val Leu Ile
370 375 380
Lys Ala Glu Lys Glu Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys
385 390 395 400
Ile Asp Asp Asn Ile Thr Arg Ile Asn Met Leu Phe Leu Glu Ile Lys
405 410 415
Arg Arg Tyr Ser Ser Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr
420 425 430
Pro Ile Asn Leu Ile Leu Gly Leu Ser Met Val Asn His Leu Glu Ser
435 440 445
Leu Gly Ile Ala Arg His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp
450 455 460
Asp Val Tyr Arg Phe Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro
465 470 475 480
Ser Ala Ser Asp Ile Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg
485 490 495
Trp Asn Gly Tyr Asp Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg
500 505 510
Asp Ser Asp Phe Leu Pro Phe Leu Glu Asn Ser Gly Gln Ala Thr Met
515 520 525
Ala Leu Leu Asn Leu Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu
530 535 540
Arg Ile Leu Glu Glu Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn
545 550 555 560
Ile Lys Val Gly Asp Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu
565 570 575
Val Lys His Ala Leu Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile
580 585 590
Glu His Leu Arg Tyr Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile
595 600 605
Gly Glu Thr Ser Ser Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu
610 615 620
Ser Leu Ala Lys Gln Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu
625 630 635 640
Asp Leu Lys Ala Leu Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp
645 650 655
Leu His Phe Ala Arg Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala
660 665 670
Ser Thr Leu Phe Ser Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr
675 680 685
Lys Asn Ala Val Leu Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu
690 695 700
Gly Ser Ile Glu Asp Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp
705 710 715 720
Asp Pro Ile Val Leu Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe
725 730 735
Ser Ala Ile Tyr Asn Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile
740 745 750
Phe Gln Gly Trp Asp Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu
755 760 765
Leu Ala Ile Ser Met Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe
770 775 780
Ile Pro Ser Leu Glu Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala
785 790 795 800
Leu Glu Pro Ile Ile Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu
805 810 815
Ser Glu Gln Met Ile His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn
820 825 830
Thr Cys Gly Arg Leu Leu Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile
835 840 845
Lys Gln Gly Lys Leu Asn Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro
850 855 860
Ala Thr Ser Ile Glu Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Ser
865 870 875 880
Glu Ser Arg Gln Asn Ile Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile
885 890 895
Pro Arg Glu Cys Lys Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln
900 905 910
Leu Phe Tyr Leu His Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu
915 920 925
Glu His Met Gln Ala Val Leu Phe Asn Pro Val Leu
930 935 940
<210> 29
<211> 964
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 29
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly
355 360 365
Gly Gly Ala Ala Met Ala Gly Ala Ser Asn Phe Asn Phe Asn Gly Arg
370 375 380
Lys Lys Phe Asn Pro Arg Thr His Gln Lys Val Lys Ser Leu Ser Val
385 390 395 400
Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu Asn Arg Glu
405 410 415
Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile Thr Arg Ile
420 425 430
Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser Leu Ser Ala
435 440 445
Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile Leu Gly Leu
450 455 460
Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg His Phe Lys
465 470 475 480
Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe Trp Ile Glu
485 490 495
Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile Met Thr Ser
500 505 510
Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp Val Ser Pro
515 520 525
Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu Pro Phe Leu
530 535 540
Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu Tyr Lys Ala
545 550 555 560
Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu Ala Lys Ser
565 570 575
Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp Gln Ser Gly
580 585 590
Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu Asp Val Pro
595 600 605
Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr Met Lys Ala
610 615 620
Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser Arg Ile Pro
625 630 635 640
Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln Asp Phe Asn
645 650 655
Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu Glu Lys Trp
660 665 670
Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg Gln Lys Leu
675 680 685
Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser Pro Glu Met
690 695 700
Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu Thr Thr Leu
705 710 715 720
Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp Ile Gln Ser
725 730 735
Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu Cys Asn Cys
740 745 750
Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn Thr Val Asn
755 760 765
Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp Ile Ser Thr
770 775 780
Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met Met Lys Glu
785 790 795 800
Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu Glu Tyr Met
805 810 815
Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe Thr Ser
820 825 830
Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Ile His Glu Tyr
835 840 845
Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu Leu Asn Asp
850 855 860
Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln Gly Lys Leu Asn Ser Leu
865 870 875 880
Ser Leu Phe Val Lys Glu Tyr Pro Ala Thr Ser Ile Glu Asp Ala Thr
885 890 895
Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn Ile Leu Arg
900 905 910
Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln Ile Phe
915 920 925
Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His Ser Asp Glu
930 935 940
Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala Val Leu Phe
945 950 955 960
Asn Pro Val Leu
<210> 30
<211> 964
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 30
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly
355 360 365
Gly Gly Ala Ala Met Ala Gly Ala Ser Asn Phe Asn Phe Asn Gly Arg
370 375 380
Lys Lys Phe Asn Pro Arg Thr His Gln Lys Val Lys Ser Leu Ser Arg
385 390 395 400
Arg Asp Ser Ser Asn Val Leu Ile Lys Trp Glu Lys Glu Asn Arg Glu
405 410 415
Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile Thr Arg Ile
420 425 430
Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser Leu Ser Ala
435 440 445
Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile Leu Gly Leu
450 455 460
Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg His Phe Lys
465 470 475 480
Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe Trp Ile Glu
485 490 495
Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile Met Thr Ser
500 505 510
Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp Val Ser Pro
515 520 525
Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu Pro Phe Leu
530 535 540
Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu Tyr Lys Ala
545 550 555 560
Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu Ala Lys Ser
565 570 575
Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp Gln Ser Gly
580 585 590
Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu Asp Val Pro
595 600 605
Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr Met Lys Ala
610 615 620
Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser Arg Ile Pro
625 630 635 640
Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln Asp Phe Asn
645 650 655
Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu Glu Lys Trp
660 665 670
Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg Gln Lys Leu
675 680 685
Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser Pro Glu Met
690 695 700
Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu Thr Thr Leu
705 710 715 720
Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp Ile Gln Ser
725 730 735
Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu Cys Asn Cys
740 745 750
Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn Thr Val Asn
755 760 765
Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp Ile Ser Thr
770 775 780
Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met Met Lys Glu
785 790 795 800
Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu Glu Tyr Met
805 810 815
Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe Thr Ser
820 825 830
Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Ile His Glu Tyr
835 840 845
Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu Leu Asn Asp
850 855 860
Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln Gly Lys Leu Asn Ser Leu
865 870 875 880
Ser Leu Phe Val Lys Glu Tyr Pro Ala Thr Ser Ile Glu Asp Ala Thr
885 890 895
Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn Ile Leu Arg
900 905 910
Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln Ile Phe
915 920 925
Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His Ser Asp Glu
930 935 940
Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala Val Leu Phe
945 950 955 960
Asn Pro Val Leu
<210> 31
<211> 2823
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 31
atgaagatcg aggaaggcaa gctcgtgatc tggatcaacg gcgacaaggg ctacaacggc 60
ctggccgagg tgggcaagaa gttcgagaag gacaccggca tcaaggtgac ggtggagcac 120
ccggacaagc tcgaggagaa gttcccgcag gtggcggcca cgggcgacgg cccggacatc 180
atcttctggg cccatgaccg cttcggcggc tacgcccagt cgggcctgct ggccgagatc 240
accccggaca aggcgttcca ggacaagctc tatcccttca cgtgggacgc cgtgcgctac 300
aacggcaagc tgatcgcgta tcccatcgcg gtggaggccc tgtcgctcat ctataacaag 360
gacctgctcc cgaacccgcc caagacctgg gaggagatcc ccgccctcga caaggagctg 420
aaggccaagg gcaagtcggc gctcatgttc aacctgcagg agccgtactt cacctggccc 480
ctgatcgcgg ccgacggcgg ctacgcgttc aagtatgaga acggcaagta tgacatcaag 540
gacgtgggcg tggacaacgc gggcgccaag gccggcctga ccttcctcgt ggacctgatc 600
aagaacaagc acatgaacgc cgacacggac tactcgatcg cggaggccgc gttcaacaag 660
ggcgagactg ccatgacgat caacggcccg tgggcgtggt cgaacatcga cacctcgaag 720
gtgaactatg gcgtgaccgt gctccccacg ttcaagggcc agccctcgaa gcccttcgtg 780
ggcgtgctgt cggcgggcat caacgccgcg tcgccgaaca aggagctcgc gaaggagttc 840
ctggagaact acctgctcac cgacgagggc ctggaggccg tgaacaagga caagcccctg 900
ggcgccgtgg ccctgaagtc gtatgaggaa gagctggtga aggacccgcg catcgcggcc 960
accatggaga acgcgcagaa gggcgagatc atgccgaaca tcccccagat gtcggccttc 1020
tggtatgcgg tgcgcaccgc cgtgatcaac gcggcctcgg gccgccagac cgtggacgag 1080
gccctcaagg acgcccagac cggcggaggt gctgccatgc tctcggtgga ggactcgtcg 1140
aacgtgctca tcaaggccga gaaggagaac cgcgagaaga agtcgttcgg ctcgatgtgc 1200
atcgacgaca acatcacccg catcaacatg ctcttcctcg agatcaagcg ccgctactcg 1260
tcgctctcgg cctacgacac cgtgcacggc gcctacccga tcaacctcat cctcggcctc 1320
tcgatggtga accacctcga gtcgctcggc atcgcccgcc acttcaagct cgagatcaag 1380
gaggtgctcg acgacgtgta ccgcttctgg atcgagaacg agatggtgaa gggcatgccg 1440
tcggcctcgg acatcatgac ctcgtcgatc tcgttcaaga tcctccgctg gaacggctac 1500
gacgtgtcgc cggacgtgtt cttctcgtac ctccgcgact cggacttcct cccgttcctc 1560
gagaactcgg gccaggccac tatggccctc ctcaacctct acaaggcctc gcagatgatg 1620
ttcccgggcg agcgcatcct cgaggaggcc aagtcgttct cgcagaacta cctcgagaac 1680
atcaaggtgg gcgaccagtc gggcgccctc aaggacatcg aggccgaggt gaagcacgcc 1740
ctcgacgtgc cgtggatggc cggcctcgag cgcatcgagc acctccgcta catgaaggcc 1800
ttcgccttcg acgacatctg gatcggcgag acgtcgtcgc gcatcccgtt caccggcaag 1860
gacctcttcc tctcgctcgc caagcaggac ttcaacatct gccaggaggc ccagcaggag 1920
gacctcaagg ccctcgagaa gtggtcggtg gactcgaagc tcggcgacct ccacttcgcc 1980
cgccagaagc tcgtggcctg ctacttctcg gccgcctcga ccctcttctc gccggagatg 2040
tcgaccaccc gcatcgtgtg gaccaagaac gccgtgctca ccaccctcat ggacgacttc 2100
tacgacgtgg agggctcgat cgaggacatc cagtcgttcg tggaggccgt gaagcgctgg 2160
gacccgatcg tgctctgcaa ctgctcggcc aacgtgaaga agctcttctc ggccatctac 2220
aacaccgtga acgacatcgc ccaggacatc tggatcttcc agggctggga catctcgacc 2280
cagctccgcg agatctggta cgagctcgcc atctcgatga tgaaggaggc cgagtggacc 2340
aagaccggct tcatcccgtc gctcgaggag tacatggaga acggccgcac ctcgatcgcc 2400
ctcgagccga tcatcttcac ctcgctcttc ttcctcggcc cgaagctctc ggagcagatg 2460
atccacgagt acaagcgcct catgcagctc ctcaacacct gcggcaggct cctcaacgac 2520
tcgcagtcgt acaagcgcga gatcaagcag ggcaagctca actcgctctc gctcttcgtg 2580
aaggagtacc cggccacctc gatcgaggac gccaccgagt ggatcaggct caccgtgtcg 2640
gagtcgcgcc agaacatcct ccgcaacctc ctccagccgt cggagatccc gcgcgagtgc 2700
aagcagatct tctggaacat ggtgaagatc atccagctct tctacctcca ctcggacgag 2760
ttcacctcgc cgaccgccat gctcgagcac atgcaggccg tgctcttcaa cccggtgctc 2820
taa 2823
<210> 32
<211> 2895
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 32
atgaagatcg aggaaggcaa gctcgtgatc tggatcaacg gcgacaaggg ctacaacggc 60
ctggccgagg tgggcaagaa gttcgagaag gacaccggca tcaaggtgac ggtggagcac 120
ccggacaagc tcgaggagaa gttcccgcag gtggcggcca cgggcgacgg cccggacatc 180
atcttctggg cccatgaccg cttcggcggc tacgcccagt cgggcctgct ggccgagatc 240
accccggaca aggcgttcca ggacaagctc tatcccttca cgtgggacgc cgtgcgctac 300
aacggcaagc tgatcgcgta tcccatcgcg gtggaggccc tgtcgctcat ctataacaag 360
gacctgctcc cgaacccgcc caagacctgg gaggagatcc ccgccctcga caaggagctg 420
aaggccaagg gcaagtcggc gctcatgttc aacctgcagg agccgtactt cacctggccc 480
ctgatcgcgg ccgacggcgg ctacgcgttc aagtatgaga acggcaagta tgacatcaag 540
gacgtgggcg tggacaacgc gggcgccaag gccggcctga ccttcctcgt ggacctgatc 600
aagaacaagc acatgaacgc cgacacggac tactcgatcg cggaggccgc gttcaacaag 660
ggcgagactg ccatgacgat caacggcccg tgggcgtggt cgaacatcga cacctcgaag 720
gtgaactatg gcgtgaccgt gctccccacg ttcaagggcc agccctcgaa gcccttcgtg 780
ggcgtgctgt cggcgggcat caacgccgcg tcgccgaaca aggagctcgc gaaggagttc 840
ctggagaact acctgctcac cgacgagggc ctggaggccg tgaacaagga caagcccctg 900
ggcgccgtgg ccctgaagtc gtatgaggaa gagctggtga aggacccgcg catcgcggcc 960
accatggaga acgcgcagaa gggcgagatc atgccgaaca tcccccagat gtcggccttc 1020
tggtatgcgg tgcgcaccgc cgtgatcaac gcggcctcgg gccgccagac cgtggacgag 1080
gccctcaagg acgcccagac cggcggaggt gctgccatgg ccggcgcctc gaacttcaac 1140
ttcaacggcc gcaagaagtt caacccgcgc acccaccaga aggtgaagtc gctctcggtg 1200
gaggactcgt cgaacgtgct catcaaggcc gagaaggaga accgcgagaa gaagtcgttc 1260
ggctcgatgt gcatcgacga caacatcacc cgcatcaaca tgctcttcct cgagatcaag 1320
cgccgctact cgtcgctctc ggcctacgac accgtgcacg gcgcctaccc gatcaacctc 1380
atcctcggcc tctcgatggt gaaccacctc gagtcgctcg gcatcgcccg ccacttcaag 1440
ctcgagatca aggaggtgct cgacgacgtg taccgcttct ggatcgagaa cgagatggtg 1500
aagggcatgc cgtcggcctc ggacatcatg acctcgtcga tctcgttcaa gatcctccgc 1560
tggaacggct acgacgtgtc gccggacgtg ttcttctcgt acctccgcga ctcggacttc 1620
ctcccgttcc tcgagaactc gggccaggcc actatggccc tcctcaacct ctacaaggcc 1680
tcgcagatga tgttcccggg cgagcgcatc ctcgaggagg ccaagtcgtt ctcgcagaac 1740
tacctcgaga acatcaaggt gggcgaccag tcgggcgccc tcaaggacat cgaggccgag 1800
gtgaagcacg ccctcgacgt gccgtggatg gccggcctcg agcgcatcga gcacctccgc 1860
tacatgaagg ccttcgcctt cgacgacatc tggatcggcg agacgtcgtc gcgcatcccg 1920
ttcaccggca aggacctctt cctctcgctc gccaagcagg acttcaacat ctgccaggag 1980
gcccagcagg aggacctcaa ggccctcgag aagtggtcgg tggactcgaa gctcggcgac 2040
ctccacttcg cccgccagaa gctcgtggcc tgctacttct cggccgcctc gaccctcttc 2100
tcgccggaga tgtcgaccac ccgcatcgtg tggaccaaga acgccgtgct caccaccctc 2160
atggacgact tctacgacgt ggagggctcg atcgaggaca tccagtcgtt cgtggaggcc 2220
gtgaagcgct gggacccgat cgtgctctgc aactgctcgg ccaacgtgaa gaagctcttc 2280
tcggccatct acaacaccgt gaacgacatc gcccaggaca tctggatctt ccagggctgg 2340
gacatctcga cccagctccg cgagatctgg tacgagctcg ccatctcgat gatgaaggag 2400
gccgagtgga ccaagaccgg cttcatcccg tcgctcgagg agtacatgga gaacggccgc 2460
acctcgatcg ccctcgagcc gatcatcttc acctcgctct tcttcctcgg cccgaagctc 2520
tcggagcaga tgatccacga gtacaagcgc ctcatgcagc tcctcaacac ctgcggcagg 2580
ctcctcaacg actcgcagtc gtacaagcgc gagatcaagc agggcaagct caactcgctc 2640
tcgctcttcg tgaaggagta cccggccacc tcgatcgagg acgccaccga gtggatcagg 2700
ctcaccgtgt cggagtcgcg ccagaacatc ctccgcaacc tcctccagcc gtcggagatc 2760
ccgcgcgagt gcaagcagat cttctggaac atggtgaaga tcatccagct cttctacctc 2820
cactcggacg agttcacctc gccgaccgcc atgctcgagc acatgcaggc cgtgctcttc 2880
aacccggtgc tctaa 2895
<210> 33
<211> 2895
<212> DNA
<213> 人工序列
<220>
<223> 融合
<400> 33
atgaagatcg aggaaggcaa gctcgtgatc tggatcaacg gcgacaaggg ctacaacggc 60
ctggccgagg tgggcaagaa gttcgagaag gacaccggca tcaaggtgac ggtggagcac 120
ccggacaagc tcgaggagaa gttcccgcag gtggcggcca cgggcgacgg cccggacatc 180
atcttctggg cccatgaccg cttcggcggc tacgcccagt cgggcctgct ggccgagatc 240
accccggaca aggcgttcca ggacaagctc tatcccttca cgtgggacgc cgtgcgctac 300
aacggcaagc tgatcgcgta tcccatcgcg gtggaggccc tgtcgctcat ctataacaag 360
gacctgctcc cgaacccgcc caagacctgg gaggagatcc ccgccctcga caaggagctg 420
aaggccaagg gcaagtcggc gctcatgttc aacctgcagg agccgtactt cacctggccc 480
ctgatcgcgg ccgacggcgg ctacgcgttc aagtatgaga acggcaagta tgacatcaag 540
gacgtgggcg tggacaacgc gggcgccaag gccggcctga ccttcctcgt ggacctgatc 600
aagaacaagc acatgaacgc cgacacggac tactcgatcg cggaggccgc gttcaacaag 660
ggcgagactg ccatgacgat caacggcccg tgggcgtggt cgaacatcga cacctcgaag 720
gtgaactatg gcgtgaccgt gctccccacg ttcaagggcc agccctcgaa gcccttcgtg 780
ggcgtgctgt cggcgggcat caacgccgcg tcgccgaaca aggagctcgc gaaggagttc 840
ctggagaact acctgctcac cgacgagggc ctggaggccg tgaacaagga caagcccctg 900
ggcgccgtgg ccctgaagtc gtatgaggaa gagctggtga aggacccgcg catcgcggcc 960
accatggaga acgcgcagaa gggcgagatc atgccgaaca tcccccagat gtcggccttc 1020
tggtatgcgg tgcgcaccgc cgtgatcaac gcggcctcgg gccgccagac cgtggacgag 1080
gccctcaagg acgcccagac cggcggaggt gctgccatgg ccggcgcctc gaacttcaac 1140
ttcaacggcc gcaagaagtt caacccgcgc acccaccaga aggtgaagtc gctctcgcgc 1200
cgcgactcgt cgaacgtgct catcaagtgg gagaaggaga accgcgagaa gaagtcgttc 1260
ggctcgatgt gcatcgacga caacatcacc cgcatcaaca tgctcttcct cgagatcaag 1320
cgccgctact cgtcgctctc ggcctacgac accgtgcacg gcgcctaccc gatcaacctc 1380
atcctcggcc tctcgatggt gaaccacctc gagtcgctcg gcatcgcccg ccacttcaag 1440
ctcgagatca aggaggtgct cgacgacgtg taccgcttct ggatcgagaa cgagatggtg 1500
aagggcatgc cgtcggcctc ggacatcatg acctcgtcga tctcgttcaa gatcctccgc 1560
tggaacggct acgacgtgtc gccggacgtg ttcttctcgt acctccgcga ctcggacttc 1620
ctcccgttcc tcgagaactc gggccaggcc actatggccc tcctcaacct ctacaaggcc 1680
tcgcagatga tgttcccggg cgagcgcatc ctcgaggagg ccaagtcgtt ctcgcagaac 1740
tacctcgaga acatcaaggt gggcgaccag tcgggcgccc tcaaggacat cgaggccgag 1800
gtgaagcacg ccctcgacgt gccgtggatg gccggcctcg agcgcatcga gcacctccgc 1860
tacatgaagg ccttcgcctt cgacgacatc tggatcggcg agacgtcgtc gcgcatcccg 1920
ttcaccggca aggacctctt cctctcgctc gccaagcagg acttcaacat ctgccaggag 1980
gcccagcagg aggacctcaa ggccctcgag aagtggtcgg tggactcgaa gctcggcgac 2040
ctccacttcg cccgccagaa gctcgtggcc tgctacttct cggccgcctc gaccctcttc 2100
tcgccggaga tgtcgaccac ccgcatcgtg tggaccaaga acgccgtgct caccaccctc 2160
atggacgact tctacgacgt ggagggctcg atcgaggaca tccagtcgtt cgtggaggcc 2220
gtgaagcgct gggacccgat cgtgctctgc aactgctcgg ccaacgtgaa gaagctcttc 2280
tcggccatct acaacaccgt gaacgacatc gcccaggaca tctggatctt ccagggctgg 2340
gacatctcga cccagctccg cgagatctgg tacgagctcg ccatctcgat gatgaaggag 2400
gccgagtgga ccaagaccgg cttcatcccg tcgctcgagg agtacatgga gaacggccgc 2460
acctcgatcg ccctcgagcc gatcatcttc acctcgctct tcttcctcgg cccgaagctc 2520
tcggagcaga tgatccacga gtacaagcgc ctcatgcagc tcctcaacac ctgcggcagg 2580
ctcctcaacg actcgcagtc gtacaagcgc gagatcaagc agggcaagct caactcgctc 2640
tcgctcttcg tgaaggagta cccggccacc tcgatcgagg acgccaccga gtggatcagg 2700
ctcaccgtgt cggagtcgcg ccagaacatc ctccgcaacc tcctccagcc gtcggagatc 2760
ccgcgcgagt gcaagcagat cttctggaac atggtgaaga tcatccagct cttctacctc 2820
cactcggacg agttcacctc gccgaccgcc atgctcgagc acatgcaggc cgtgctcttc 2880
aacccggtgc tctaa 2895
<210> 34
<211> 631
<212> PRT
<213> 巨柏（Cupressus gigantea）
<400> 34
Met Ala Ala Leu Ser Phe Ser Thr Ile Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Lys Leu Pro Ser Ser Asn Leu Gln Gly Arg Ser Arg Asp Thr
20 25 30
Lys Tyr His Val Leu Ile Arg Ser Ala Gly Ala Ser Asn Phe Asn Phe
35 40 45
Asn Gly Arg Lys Lys Phe Asn Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Arg Ile Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser
100 105 110
Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu
195 200 205
Pro Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Ile
500 505 510
His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Tyr Lys Arg Glu Ile Lys Gln Gly Lys Leu
530 535 540
Thr Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala His Leu Ile Glu
545 550 555 560
Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys
580 585 590
Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala
610 615 620
Val Leu Phe Asn Pro Val Leu
625 630
<210> 35
<211> 1896
<212> DNA
<213> 巨柏（Cupressus gigantea）
<400> 35
atggcagcac ttagtttttc taccatctcc accactgcca actatgcaat tcctaagctg 60
ccatcttcaa atcttcaagg gcggtccaga gatacaaaat accatgtctt gataagatct 120
gcaggggcct ctaatttcaa cttcaatgga agaaagaagt tcaatccaag aacacatcag 180
aaagtcaaat ctttgagtgt tgaagattcc tccaatgtac ttattaaggc tgagaaagag 240
aacagagaga aaaaatcatt tggttcgatg tgcatagacg ataatattac aaggataaat 300
atgttgttct tggaaattaa gagaagatat tcatctcttt ctgcttatga tacagtgcac 360
ggtgcttatc ctatcaattt gattcttggc ctctcaatgg taaatcattt ggagagcttg 420
ggtatagcaa gacatttcaa acttgagata aaagaggttc tagatgatgt ctatagattt 480
tggatagaga atgaaatggt taaaggaatg ccatcagcat cagacataat gaccagttct 540
atcagtttca agatcttgcg gtggaatgga tatgacgtgt ctccagatgt ctttttcagt 600
tacctgaggg atagtgactt cctaccattt ctagaaaata gtggccaagc tactatggca 660
ttgctgaatt tatacaaggc atctcagatg atgttcccag gggaaagaat tcttgaagaa 720
gcaaaatcgt tctcccaaaa ttatcttgaa aatatcaagg tgggcgatca aagtggagct 780
ctcaaagata ttgaagcaga ggttaagcat gcactggatg ttccttggat ggctggtttg 840
gagagaatag aacatctgcg atacatgaaa gcatttgctt ttgatgatat ttggattgga 900
gagacctctt ctaggatccc atttacagga aaggatttat ttttatcttt agccaaacaa 960
gacttcaata tctgccaaga agctcaacaa gaggatttga aagcgttgga aaaatggagt 1020
gtagactcta aacttggtga cctacacttt gctagacaaa agcttgtagc ctgctatttc 1080
tctgccgcct caacactttt tagccctgaa atgtctacta ctcggattgt gtggactaaa 1140
aatgctgttc tcactactct aatggatgac ttttatgatg tggagggttc cattgaagat 1200
attcaatcct ttgttgaagc agttaagaga tgggacccaa tagttttatg taattgttca 1260
gctaatgtga aaaaattatt ctctgctatt tataacactg tgaatgatat tgctcaagat 1320
atatggattt tccagggctg ggacattagc actcagttaa gagaaatttg gtacgaactg 1380
gcaatctcaa tgatgaagga ggctgaatgg accaaaactg gatttatacc atcgctggaa 1440
gaatacatgg aaaatgggag aacgtcaatt gccttggaac caattatttt tacatcacta 1500
ttttttcttg gccctaagct ttccgagcaa atgatccacg agtataagag acttatgcaa 1560
ctcctgaata cttgtgggcg ccttttgaat gacagtcaga gctacaagag ggagataaag 1620
caaggaaagc tcacctcttt atcattgttc gtgaaagaat atcccgcaca tcttattgag 1680
gatgctactg agtggattag actcactgta agtgaaagca gacaaaatat actcagaaat 1740
ttattgcaac caagtgagat ccctcgtgaa tgcaagcaga tattttggaa tatggtcaaa 1800
attatccaac tattctacct ccacagtgat gaattcacat ctcctactgc aatgcttgaa 1860
catatgcaag cagttctttt taatcctgtt ttataa 1896
<210> 36
<211> 1650
<212> DNA
<213> 人工序列
<220>
<223> 截短
<400> 36
atggagaaaa aatcatttgg ttcgatgtgc atagatgatg atattacaaa gataaaaatg 60
ttgttctcgg aaattaaaag aagatattca tctctttctg cttatgatac agtgcacggt 120
gcttatccta tcaatttgat tcttggcctc tcaatggtaa accacttgga gagcttgggt 180
atagcaagac atttcaaact tgagataaaa gaggttctag atgatgtcta tagattttgg 240
atagagaatg aaatggttga aggaatgcca tcagcatcag acataatgac cagttctatc 300
agtttcagga tcttgcggtg gaatggatat gacgtgtctc cagatgtctt tttcagttac 360
ctgagggata gtgacttcct actattttta gaaaatagtg accaagctac tatagcattg 420
ctgaatttat acaaggcatc tcagatgatg ttcccaggcg aaagaattct tgaagaagca 480
aaattgttct cccaaaatta tcttgaaaat atcaagatgg gcaatcaaag tggagctctc 540
aaagacattg aagcagaggt taagcatgca ctggatgtcc cttggatggc taatttggag 600
agaatagaac atctgcgata catgaaagca tttgcttttg acaatatttg gattggagag 660
acctcttcta ggatcccatt tacaggaaag gatttatttt tatcttttgc caaacaagac 720
ttcaatatct gccaagcagc tcaacaagag gatttgaagg cgttggaaaa atggagtgta 780
gactctaaac ttggtgacct acactttgct agacaaaagc ttgtagcctg ctatttctct 840
gccgcctcaa tactttttag ccctgaaatg tctgctactc ggattgtgtg gactaaaaat 900
gctgttctca ttacactaat ggatgacttt tatgatgtgg agggttccat ygaagatatt 960
caatcctttg ttgaagcggt taagagatgg gacccaatag ttttatgtaa ttgttcagct 1020
aatgttaaaa ttttattctc tgctatttat aacactgtga atgatattgc tcaagatata 1080
tggattttcc agggctggga cattagcact cagttaagag aaatttggta cgaactggca 1140
atctcaatga tgaaggaggc tgaatggacc aaaactggat ttataccatc cttggaagaa 1200
tacatggaaa atgggagaac atcaattgcc ttggaaccaa ttatttttac atcactattt 1260
tttattggcc ctaagctttc cgagcaaatg atccaccatc acgagtataa gaggcttttg 1320
caactcatga atacttgtgg gcgccttttg aatgacattc agagcgacaa gagggagatg 1380
aagcaaggaa agctcacctc tgtatcattg ttcgtgaaag aatacccaat tgaggatgct 1440
actaagtgga ttagactcac tgtaagtgaa aacacacaaa atatactcaa aaaaatattg 1500
caaccaagtg agatccctcg tgaatgcaag cagatatttt ggaatatggt caaaattatc 1560
caactattct gcctccacag tgatgaattc acatctccta ccacaatgct tgaacatatg 1620
aaggcagttc ttttttatcc tgttttataa 1650
<210> 37
<211> 1653
<212> DNA
<213> 人工序列
<220>
<223> 截短
<400> 37
atggagaaaa aatcatttgg ttcgatgtgc atagacgata atattacaag gataaatatg 60
ttgttcttgg aaattaagag aagatattca tctctttctg cttatgatac agtgcacggt 120
gcttatccta tcaatttgat tcttggcctc tcaatggtaa atcatttgga gagcttgggt 180
atagcaagac atttcaaact tgagataaaa gaggttctag atgatgtcta tagattttgg 240
atagagaatg aaatggttaa aggaatgcca tcagcatcag acataatgac cagttctatc 300
agtttcaaga tcttgcggtg gaatggatat gacgtgtctc cagatgtctt tttcagttac 360
ctgagggata gtgacttcct accatttcta gaaaatagtg gccaagctac tatggcattg 420
ctgaatttat acaaggcatc tcagatgatg ttcccagggg aaagaattct tgaagaagca 480
aaatcgttct cccaaaatta tcttgaaaat atcaaggtgg gcgatcaaag tggagctctc 540
aaagatattg aagcagaggt taagcatgca ctggatgttc cttggatggc tggtttggag 600
agaatagaac atctgcgata catgaaagca tttgcttttg atgatatttg gattggagag 660
acctcttcta ggatcccatt tacaggaaag gatttatttt tatctttagc caaacaagac 720
ttcaatatct gccaagaagc tcaacaagag gatttgaaag cgttggaaaa atggagtgta 780
gactctaaac ttggtgacct acactttgct agacaaaagc ttgtagcctg ctatttctct 840
gccgcctcaa cactttttag ccctgaaatg tctactactc ggattgtgtg gactaaaaat 900
gctgttctca ctactctaat ggatgacttt tatgatgtgg agggttccat tgaagatatt 960
caatcctttg ttgaagcagt taagagatgg gacccaatag ttttatgtaa ttgttcagct 1020
aatgtgaaaa aattattctc tgctatttat aacactgtga atgatattgc tcaagatata 1080
tggattttcc agggctggga cattagcact cagttaagag aaatttggta cgaactggca 1140
atctcaatga tgaaggaggc tgaatggacc aaaactggat ttataccatc gctggaagaa 1200
tacatggaaa atgggagaac gtcaattgcc ttggaaccaa ttatttttac atcactattt 1260
tttcttggcc ctaagctttc cgagcaaatg atccacgagt ataagagact tatgcaactc 1320
ctgaatactt gtgggcgcct tttgaatgac agtcagagct acaagaggga gataaagcaa 1380
ggaaagctca actctttatc attgttcgtg aaagaatatc ccgcaacatc tattgaggat 1440
gctactgagt ggattagact cactgtaagt gaaagcagac aaaatatact cagaaattta 1500
ttgcaaccaa gtgagatccc tcgtgaatgc aagcagatat tttggaatat ggtcaaaatt 1560
atccaactat tctacctcca cagtgatgaa ttcacatctc ctactgcaat gcttgaacat 1620
atgcaagcag ttctttttaa tcctgtttta taa 1653
<210> 38
<211> 1653
<212> DNA
<213> 人工序列
<220>
<223> 截短
<400> 38
atggagaaaa aatcatttgg ttcgatgtgc atagacgata atattacaag gataaatatg 60
ttgttcttgg aaattaagag aagatattca tctctttctg cttatgatac agtgcacggt 120
gcttatccta tcaatttgat tcttggcctc tcaatggtaa atcatttgga gagcttgggt 180
atagcaagac atttcaaact tgagataaaa gaggttctag atgatgtcta tagattttgg 240
atagagaatg aaatggttaa aggaatgcca tcagcatcag acataatgac cagttctatc 300
agtttcaaga tcttgcggtg gaatggatat gacgtgtctc cagatgtctt tttcagttac 360
ctgagggata gtgacttcct accatttcta gaaaatagtg gccaagctac tatggcattg 420
ctgaatttat acaaggcatc tcagatgatg ttcccagggg aaagaattct tgaagaagca 480
aaatcgttct cccaaaatta tcttgaaaat atcaaggtgg gcgatcaaag tggagctctc 540
aaagatattg aagcagaggt taagcatgca ctggatgttc cttggatggc tggtttggag 600
agaatagaac atctgcgata catgaaagca tttgcttttg atgatatttg gattggagag 660
acctcttcta ggatcccatt tacaggaaag gatttatttt tatctttagc caaacaagac 720
ttcaatatct gccaagaagc tcaacaagag gatttgaaag cgttggaaaa atggagtgta 780
gactctaaac ttggtgacct acactttgct agacaaaagc ttgtagcctg ctatttctct 840
gccgcctcaa cactttttag ccctgaaatg tctactactc ggattgtgtg gactaaaaat 900
gctgttctca ctactctaat ggatgacttt tatgatgtgg agggttccat tgaagatatt 960
caatcctttg ttgaagcagt taagagatgg gacccaatag ttttatgtaa ttgttcagct 1020
aatgtgaaaa aattattctc tgctatttat aacactgtga atgatattgc tcaagatata 1080
tggattttcc agggctggga cattagcact cagttaagag aaatttggta cgaactggca 1140
atctcaatga tgaaggaggc tgaatggacc aaaactggat ttataccatc gctggaagaa 1200
tacatggaaa atgggagaac gtcaattgcc ttggaaccaa ttatttttac atcactattt 1260
tttcttggcc ctaagctttc cgagcaaatg atccacgagt ataagagact tatgcaactc 1320
ctgaatactt gtgggcgcct tttgaatgac agtcagagct acaagaggga gataaagcaa 1380
ggaaagctca cctctttatc attgttcgtg aaagaatatc ccgcacatct tattgaggat 1440
gctactgagt ggattagact cactgtaagt gaaagcagac aaaatatact cagaaattta 1500
ttgcaaccaa gtgagatccc tcgtgaatgc aagcagatat tttggaatat ggtcaaaatt 1560
atccaactat tctacctcca cagtgatgaa ttcacatctc ctactgcaat gcttgaacat 1620
atgcaagcag ttctttttaa tcctgtttta taa 1653
<210> 39
<211> 1550
<212> DNA
<213> 人工序列
<220>
<223> 截短
<400> 39
atggagaaga agtcgttcgg ctcgatgtgc atcgacgacg acatcaccaa gatcaagatg 60
ctcttctcgg agatcaagcg ccgctactcg tcgctctcgg cctacgacac cgtgcacggc 120
gcctacccga tcaacctcat cctcggcctc tcgatggtga accacctcga gtcgctcggc 180
atcgcccgcc acttcaagct cgagatcaag gaggtgctcg acgacgtgta ccgcttctgg 240
atcgagaacg agatggtgga gggcatgccg tcggcctcgg acatcatgac ctcgtcgatc 300
tcgttccgca tcctccgctg gaacggctac gacgtgtcgc cggacgtgtt cttctcgtac 360
ctccgcgact cggacttcct cctcttcctc gagaactcgg accaggccac catcgccctc 420
ctcaacctct acaaggcctc gcagatgatg ttcccgggcg agcgcatcct cgaggaggcc 480
aagctcttct cgcagaacta cctcgagaac atcaagatgg gcaaccagtc gggcgccctc 540
aaggacatcg aggccgaggt gaagcacgcc ctcgacgtgc cgtggatggc caacctcgag 600
cgcatcgagc acctccgcta catgaaggcc ttcgccttcg acaacatctg gatcggcgag 660
acgtcgtcgc gcatcccgtt caccggcaag gacctcttcc tctcgttcgc caagcaggac 720
ttcaacatct gccaggccgc ccagcaggag gacctcaagg ccctcgagaa gtggtcggtg 780
gactcgaagc tcggcgacct ccacttcgcc cgccagaagc tcgtggcctg ctacttctcg 840
gccgcctcga tcctcttctc gccggagatg tcggccaccc gcatcgtgtg gaccaagaac 900
gccgtgctca tcaccctcat ggacgacttc tacgacgtgg agggctcgat cgaggacatc 960
cagtcgttcg tggaggccgt gaagcgctgg gacccgatcg tgctctgcaa ctgctcggcc 1020
aacgtgaaga tcctcttctc ggccatctac aacaccgtga acgacatcgc ccaggacatc 1080
tggatcttcc agggctggga catctcgacc cagctccgcg agatctggta cgagctcgcc 1140
atctcgatga tgaaggaggc cgagtggacc aagaccggct tcatcccgtc gctcgaggag 1200
tacatggaga acggccgcac ctcgatcgcc ctcgagccga tcatcttcac ctcgctcttc 1260
ttcatcggcc cgaagctctc ggagcagatg atccaccacc acgagtacaa gcgcctcctc 1320
cagctcatga acacctgcgg ccgcctcctc aacgacatcc agtcggacaa gcgcgagatg 1380
aagcagggca agctcacctc ggtgtcgctc ttcgtgaagg agtacccgat cgaggacgcc 1440
accaagtgga tccgcctcac cgtgtcggag aacacccaga acatcctcaa gaagatcctc 1500
cagccgtcgg agatcccgcg cgagtgcaag cagatcttct ggaacatggt 1550
<210> 40
<211> 516
<212> PRT
<213> 人工序列
<220>
<223> 截短
<400> 40
Met Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asp Ile Thr
1 5 10 15
Lys Ile Lys Met Leu Phe Ser Glu Ile Lys Arg Arg Tyr Ser Ser Leu
20 25 30
Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile Leu
35 40 45
Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg His
50 55 60
Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe Trp
65 70 75 80
Ile Glu Asn Glu Met Val Glu Gly Met Pro Ser Ala Ser Asp Ile Met
85 90 95
Thr Ser Ser Ile Ser Phe Arg Ile Leu Arg Trp Asn Gly Tyr Asp Val
100 105 110
Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Leu Leu
115 120 125
Phe Leu Glu Asn Ser Asp Gln Ala Thr Ile Ala Leu Leu Asn Leu Tyr
130 135 140
Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu Ala
145 150 155 160
Lys Leu Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Met Gly Asn Gln
165 170 175
Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu Asp
180 185 190
Val Pro Trp Met Ala Asn Leu Glu Arg Ile Glu His Leu Arg Tyr Met
195 200 205
Lys Ala Phe Ala Phe Asp Asn Ile Trp Ile Gly Glu Thr Ser Ser Arg
210 215 220
Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Phe Ala Lys Gln Asp
225 230 235 240
Phe Asn Ile Cys Gln Ala Ala Gln Gln Glu Asp Leu Lys Ala Leu Glu
245 250 255
Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg Gln
260 265 270
Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Ile Leu Phe Ser Pro
275 280 285
Glu Met Ser Ala Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu Ile
290 295 300
Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp Ile
305 310 315 320
Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu Cys
325 330 335
Asn Cys Ser Ala Asn Val Lys Ile Leu Phe Ser Ala Ile Tyr Asn Thr
340 345 350
Val Asn Asp Ile Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp Ile
355 360 365
Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met Met
370 375 380
Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Ser Leu Glu Glu
385 390 395 400
Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile Phe
405 410 415
Thr Ser Leu Phe Phe Ile Gly Pro Lys Leu Ser Glu Gln Met Ile His
420 425 430
His His Glu Tyr Lys Arg Leu Leu Gln Leu Met Asn Thr Cys Gly Arg
435 440 445
Leu Leu Asn Asp Ile Gln Ser Asp Lys Arg Glu Met Lys Gln Gly Lys
450 455 460
Leu Thr Ser Val Ser Leu Phe Val Lys Glu Tyr Pro Ile Glu Asp Ala
465 470 475 480
Thr Lys Trp Ile Arg Leu Thr Val Ser Glu Asn Thr Gln Asn Ile Leu
485 490 495
Lys Lys Ile Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys Gln Ile
500 505 510
Phe Trp Asn Met
515
<210> 41
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体1
<400> 41
Met Ala Ala Leu Ser Phe Ser Thr Ile Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Arg Ile Pro Ser Ser Asn Leu Gln Gly Arg Ser Arg Asp Thr
20 25 30
Lys Tyr His Val Leu Ile Arg Ser Ala Gly Gly Ser Asn Phe Asn Phe
35 40 45
Asn Gly Arg Lys Lys Phe Asn Pro Arg Thr His Gln Lys Ile Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Gln Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Arg Ile Gln Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser
100 105 110
Leu Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile
115 120 125
Ile Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Gly Arg
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Lys Asp Ser Asp Phe Leu
195 200 205
Pro Phe Leu Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Asp Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Ala Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Leu Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Trp Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Arg Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Glu Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Ser Leu Phe Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Leu Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Arg Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Gln Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Ile Ala Gln Asp Val Trp Ile Phe Gln Gly Phe Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Trp Phe Glu Leu Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Ile Pro Thr Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Asn Met Ile
500 505 510
His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Tyr Lys Arg Glu Val Lys Gln Gly Lys Leu
530 535 540
Asn Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala Ser Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Thr Glu Ser Arg Gln Asn
565 570 575
Ile Leu Arg Asn Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys
580 585 590
Gln Ile Phe Trp Asn Lys Val Lys Leu Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala
610 615 620
Val Val Tyr Asn Pro Leu Leu
625 630
<210> 42
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体2
<400> 42
Met Gly Gly Leu Ser Phe Ser Thr Ile Ser Thr Thr Ala Asn Trp Ala
1 5 10 15
Leu Pro Lys Leu Pro Ser Ser Asn Leu Gln Gly Met Ser Arg Asp Thr
20 25 30
Lys Tyr His Val Leu Ile Arg Ser Ala Ala Ala Ser Asn Phe Asn Phe
35 40 45
Asn Gly Arg Arg Lys Phe Asn Pro Arg Thr His Gln Lys Ile Lys Ser
50 55 60
Leu Ser Val Glu Asp Thr Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Asn Arg Asp Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Arg Leu Asn Met Leu Phe Leu Glu Ile Lys Lys Lys Tyr Ser Ser
100 105 110
Leu Ser Ala Phe Asp Thr Val His Gly Ala Tyr Pro Ile Asn Val Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Met
130 135 140
His Phe Met Leu Glu Ile Lys Glu Ile Leu Asp Asp Val Tyr Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Leu Lys Gly Lys Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Met Asp Ser Asp Phe Leu
195 200 205
Pro Tyr Val Glu Asn Ser Ala Gln Ala Ser Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Asp Asn Ile Lys Ile Gly Asp
245 250 255
Gln Ser Ala Ala Leu Lys Asp Ile Glu Ala Glu Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Ile Tyr Ile Gly Glu Thr Ser Ser
290 295 300
Met Ile Pro Phe Thr Gly Arg Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Arg Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Val Gly Cys Tyr Phe Thr Ala Ala Ser Thr Leu Phe Ser
355 360 365
Pro Asp Met Ser Thr Thr Arg Ile Leu Phe Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Gln Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Ile Asn Asp Ile Ala Gln Asp Val Tyr Ile Phe Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Tyr Tyr Glu Leu Ala Val Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Ala Tyr Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Trp Leu Gly Pro Lys Leu Ser Glu Gln Met Ile
500 505 510
His Glu Phe Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Trp Lys Arg Glu Ile Lys Asn Gly Lys Leu
530 535 540
Gln Ser Leu Thr Leu Phe Val Lys Asp Tyr Pro Ala Thr Ser Ile Glu
545 550 555 560
Asp Ala Thr Asp Tyr Ile Arg Val Thr Val Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Lys Asn Leu Leu Gln Pro Ser Glu Leu Pro Arg Glu Cys Arg
580 585 590
Gln Ile Phe Trp Asn Met Val Lys Ile Ile Asn Leu Phe Tyr Leu His
595 600 605
Ser Asp Asp Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Gly
610 615 620
Leu Leu Tyr Asn Pro Val Ile
625 630
<210> 43
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体3
<400> 43
Met Ala Ala Leu Ser Phe Thr Thr Ile Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Lys Leu Pro Ser Thr Asn Val Asn Gly Arg Ser Arg Asp Thr
20 25 30
Arg Tyr His Val Leu Ile Arg Ser Ala Ala Gly Ser Asn Phe Gln Phe
35 40 45
Asn Gly Arg Lys Lys Phe Asn Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Val Asp Asp Asn Leu
85 90 95
Thr Arg Ile Gln Met Leu Phe Leu Glu Ile Met Arg Arg Trp Ser Ser
100 105 110
Leu Ser Ala Tyr Asp Thr Val His Gly Ala Trp Pro Leu Asn Leu Ile
115 120 125
Val Gly Leu Ser Arg Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Leu Tyr Arg Tyr
145 150 155 160
Tyr Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Glu Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Thr Asp Phe Leu
195 200 205
Pro Phe Leu Asp Asn Thr Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Arg Phe Pro Gly Glu Arg Ile Leu Asp Glu
225 230 235 240
Ala Met Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Arg His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Gly Phe Gly Tyr Asp Asp Ile Tyr Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Trp Thr Gly Lys Asp Ile Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Leu Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Asp Met Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Met Leu Val Gly Cys Tyr Phe Ser Ala Ala Ser Thr Leu Phe Ser
355 360 365
Pro Glu Met Ser Ser Thr Arg Ile Val Phe Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Lys Asp Asp Phe Tyr Asp Val Glu Gly Ser Leu Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Ile Lys Arg Trp Asp Pro Ile Val Val
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Arg Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Ser Val Asn Asp Ile Gly Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Val Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Met Thr Gly Phe Leu Pro Thr Val Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Leu Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Tyr Leu Gly Pro Lys Ile Ser Glu Gln Arg Ile
500 505 510
His Glu Tyr Lys Arg Leu Arg Gln Val Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Tyr Lys Met Glu Ile Lys Gln Gly Lys Leu
530 535 540
Asn Ser Leu Ser Leu Phe Val Lys Glu Tyr Pro Ala Thr Ser Ile Glu
545 550 555 560
Asp Ala Ser Glu Trp Ile Arg Leu Thr Val Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Arg Asn Ile Leu Gln Pro Ser Glu Leu Pro Arg Glu Cys Lys
580 585 590
Gln Ile Phe Trp Asn Met Val Lys Ile Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala
610 615 620
Val Leu Trp Asn Pro Val Leu
625 630
<210> 44
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体4
<400> 44
Met Ala Ala Leu Ser Phe Ser Thr Ile Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Lys Ile Pro Thr Ser Asn Leu Gln Gly Arg Ser Arg Asp Thr
20 25 30
Met Tyr His Val Leu Ile Arg Ser Ala Gly Ala Thr Asn Tyr Asn Phe
35 40 45
Gln Gly Arg Arg Lys Phe Gln Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Val Ile Lys Ala Glu Asn Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Val
85 90 95
Thr Arg Leu Asn Met Leu Phe Leu Glu Ile Lys Arg Arg Tyr Ser Ser
100 105 110
Leu Thr Gly Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Met Leu Glu Ile Arg Glu Val Leu Asp Asp Val Phe Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Val Lys Gly Lys Pro Ser Ala Ser Asp Ile
165 170 175
Met Ser Thr Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Glu Ser Asp Phe Leu
195 200 205
Pro Phe Leu Glu Asn Ser Gly Gln Ala Ser Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Met Ile Leu Glu Glu
225 230 235 240
Ala Arg Ser Phe Ser Gln Asn Trp Leu Glu Gln Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Ala Leu Lys Asp Ile Glu Ala Glu Val Met His Ala Leu
260 265 270
Asp Val Pro Phe Met Ala Gly Leu Glu Arg Ile Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Val Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Lys Glu Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Thr Arg Ile Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Met Leu Leu Gly Cys Tyr Phe Ser Ala Ala Ser Thr Leu Tyr Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Lys Asp Asp Phe Tyr Asp Val Asp Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Ile Lys Arg Trp Asp Pro Leu Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Thr Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Val Ala Gln Asp Ile Trp Ile Phe Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Ile Trp Tyr Glu Val Ala Ile Thr Arg
450 455 460
Met Lys Glu Ala Glu Trp Thr Met Thr Gly Tyr Val Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Phe Leu Gly Pro Lys Val Ser Glu Gln Met Ile
500 505 510
His Glu Tyr Lys Arg Leu Lys Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Thr Gln Thr Tyr Lys Arg Glu Ile Met Gln Gly Lys Leu
530 535 540
Gln Thr Leu Ser Leu Phe Val Arg Glu Tyr Pro Ala Thr Ser Ile Glu
545 550 555 560
Asp Ala Ser Glu Trp Ile Arg Leu Thr Leu Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Arg Gln Leu Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys
580 585 590
Gln Ile Tyr Trp Asn Met Ile Lys Val Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Gln Ala
610 615 620
Val Leu Phe Asn Pro Val Leu
625 630
<210> 45
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体5
<400> 45
Met Ala Ala Leu Ser Phe Ser Thr Ile Ser Thr Thr Gly Asn Tyr Ala
1 5 10 15
Ile Pro Lys Leu Pro Ser Ser Asn Leu Gln Gly Arg Ser Arg Glu Thr
20 25 30
Lys Tyr His Val Leu Val Arg Ser Gly Gly Ala Ser Asn Phe Asn Phe
35 40 45
Asn Ala Arg Lys Met Phe Asn Pro Arg Thr His Gln Lys Val Met Ser
50 55 60
Leu Ser Val Glu Glu Ser Ser Asn Val Leu Ile Lys Ala Glu Lys Glu
65 70 75 80
Gln Arg Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Glu Asp Asn Ile
85 90 95
Thr Met Ile Asn Met Leu Phe Val Glu Ile Arg Arg Arg Tyr Ser Ser
100 105 110
Val Thr Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Leu
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Gly Lys
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Val Trp Arg Phe
145 150 155 160
Trp Ile Asp Asn Asp Met Leu Met Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Lys Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Leu Phe Phe Thr Tyr Ile Arg Asp Ser Asp Phe Leu
195 200 205
Pro Phe Val Glu Gln Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Lys Met Phe Pro Gly Glu Arg Leu Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Met Leu Gly Asp
245 250 255
Gln Ser Gly Gly Leu Lys Asp Val Asp Gly Glu Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Gly Ile Glu Arg Val Glu His Leu Arg Phe
275 280 285
Met Met Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Asn
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Asn Glu Asp Leu Lys Gly Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Arg Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Asn Lys Ile Ile Ala Cys Tyr Phe Ser Gly Ala Thr Thr Leu Phe Ser
355 360 365
Pro Glu Met Ser Thr Ser Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Arg Asp Asp Phe Tyr Asp Val Asp Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Ile Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Val Ala Gln Glu Ile Trp Ile Tyr Gln Ala Phe Glu
435 440 445
Val Ser Thr Gln Leu Arg Glu Ile Trp Phe Glu Val Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Ser Gly Phe Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Gly Leu Glu Pro Ile Ile
485 490 495
Phe Ser Ser Ile Phe Phe Leu Gly Pro Arg Ile Thr Asp Gln Met Ile
500 505 510
His Glu Tyr Lys Arg Leu Met Gln Leu Val Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Thr Phe Lys Arg Glu Val Met Gln Gly Lys Val
530 535 540
Asn Ser Leu Ser Leu Phe Val Arg Glu Tyr Pro Ala Ser Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Trp Val Arg Leu Thr Val Ser Glu Ser Arg Gln Asn
565 570 575
Leu Leu Arg Asn Ile Leu Asn Pro Ser Glu Val Pro Arg Glu Cys Lys
580 585 590
Gln Leu Phe Trp Asn Met Val Lys Ile Leu Gln Leu Phe Tyr Leu His
595 600 605
Thr Asp Asp Phe Thr Ser Pro Thr Ala Met Leu Glu His Met Asn Ala
610 615 620
Val Leu Trp Gln Pro Val Ile
625 630
<210> 46
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体6
<400> 46
Met Ala Ala Val Ser Trp Ser Thr Ile Ser Thr Thr Ala Gln Tyr Ala
1 5 10 15
Leu Pro Lys Ile Pro Ser Ser Asn Leu Asn Ala Arg Ser Met Asp Thr
20 25 30
Arg Tyr His Val Leu Ile Arg Ser Ala Gly Ala Ser Asn Trp Asn Phe
35 40 45
Asn Ala Arg Lys Lys Trp Gln Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Asp Ser Thr Asn Leu Leu Ile Lys Ala Glu Arg Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Arg Ile Gln Met Leu Trp Leu Glu Ile Met Arg Arg Tyr Ser Ser
100 105 110
Ile Ser Ala Tyr Asp Ser Val His Ala Ala Tyr Pro Leu Asn Ile Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Asp Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Lys Leu Glu Ile Lys Glu Val Leu Asp Asp Leu Tyr Met Phe
145 150 155 160
Phe Ile Glu Gln Glu Met Val Arg Gly Met Pro Ser Ala Thr Asp Ile
165 170 175
Met Ser Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Gln Gly Tyr Glu
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Thr Asp Phe Leu
195 200 205
Pro Trp Ile Asp Asn Ser Gly Asn Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Lys Ile Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Gly Leu Met Asp Ile Glu Ala Glu Val Lys His Ala Leu
260 265 270
Asp Leu Pro Trp Met Gly Gly Leu Glu Arg Val Glu His Leu Arg Tyr
275 280 285
Met Met Gly Phe Ala Phe Glu Asp Leu Trp Ile Gly Glu Thr Ser Ser
290 295 300
Lys Ile Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Asn
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Glu Lys Trp Ser Leu Asp Ser Lys Leu Gly Glu Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Val Ala Cys Tyr Trp Ser Ala Ala Thr Thr Leu Tyr Ser
355 360 365
Pro Glu Met Ser Ser Thr Arg Leu Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Ile Met Asp Asp Phe Tyr Asp Val Asp Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Gln Cys Ser Ala Asn Val Lys Lys Leu Phe Ser Ala Ile Phe Asn
420 425 430
Thr Val Asn Asp Ile Gly Gln Asp Ile Trp Ile Phe Gln Ala Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Leu Trp Phe Asp Leu Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Phe Val Pro Ser Val Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Leu Ala Ile Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Tyr Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Val
500 505 510
His Glu Tyr Lys Met Leu Lys Gln Leu Val Asn Thr Cys Gly Arg Leu
515 520 525
Ile Asn Asp Thr Gln Ser Tyr Lys Met Glu Val Lys Gln Gly Arg Val
530 535 540
Asn Ser Val Ser Leu Phe Val Lys Glu Tyr Pro Ala Ser Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Trp Ile Arg Leu Thr Val Ser Glu Thr Arg Gln Asn
565 570 575
Leu Leu Lys Asn Leu Val Gln Pro Ser Glu Ile Pro Lys Glu Cys Lys
580 585 590
Gln Leu Phe Trp Asn Met Val Lys Ile Ile Asn Leu Phe Tyr Val His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Lys Leu Glu His Met Gln Ala
610 615 620
Ile Leu Phe Asn Pro Val Leu
625 630
<210> 47
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体7
<400> 47
Met Gly Gly Leu Ser Trp Ser Thr Ile Thr Thr Thr Ala Asn Trp Ala
1 5 10 15
Ile Pro Lys Leu Pro Ser Ser Asn Val Gln Gly Arg Ser Arg Asp Thr
20 25 30
Met Tyr His Val Leu Ile Lys Ser Gly Gly Ala Thr Asn Phe Asn Phe
35 40 45
Asn Gly Arg Lys Arg Phe Gln Pro Arg Thr His Gln Lys Ile Arg Ser
50 55 60
Leu Ser Val Asp Asp Ser Ser Asn Val Leu Ile Arg Ala Glu Lys Asp
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Arg Ile Asn Lys Leu Tyr Leu Asp Ile Lys Met Arg Tyr Ser Ser
100 105 110
Ile Ser Ala Tyr Asp Thr Val His Gly Ala Tyr Pro Ile Asn Leu Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Lys Leu Glu Ile Arg Glu Val Leu Asp Asp Leu Tyr Arg Phe
145 150 155 160
Trp Ile Glu Asn Glu Met Val Lys Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Thr Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Asp Ser Asp Phe Val
195 200 205
Pro Phe Leu Glu Asn Ser Ala Gln Ala Ser Met Ala Leu Leu Gln Leu
210 215 220
Tyr Lys Ala Ser Gln Arg Met Phe Pro Gly Glu Arg Val Leu Glu Glu
225 230 235 240
Ala Met Ser Phe Ser Gln Asn Tyr Leu Glu Asn Leu Met Val Gly Glu
245 250 255
Gln Ser Gly Ala Val Met Glu Ile Glu Ala Glu Val Lys His Ala Leu
260 265 270
Asp Leu Pro Trp Met Ala Gly Leu Glu Arg Ile Asp His Leu Lys Trp
275 280 285
Met Arg Ala Phe Ala Phe Asp Asp Ile Trp Leu Gly Glu Ser Ser Ser
290 295 300
Arg Val Pro Phe Thr Gly Lys Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Met Gly Val
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Ile Ala Asp Leu His Phe Ala Arg
340 345 350
Gln Lys Ile Val Ala Cys Tyr Tyr Ser Ala Ala Ser Thr Leu Phe Ser
355 360 365
Pro Glu Met Ser Ser Thr Arg Ile Val Trp Thr Lys Asn Ala Leu Leu
370 375 380
Ser Thr Leu Met Asp Asp Phe Tyr Asp Leu Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Thr Phe Ile Glu Ala Ile Lys Arg Trp Asp Pro Ile Leu Leu
405 410 415
Cys Asn Cys Ser Ala Asn Ile Lys Lys Leu Phe Ser Ala Ile Tyr Gln
420 425 430
Thr Val Asn Asp Ile Ala Asn Asp Ile Trp Ile Trp Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Arg Glu Leu Trp Tyr Glu Val Gly Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Tyr Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Trp Leu Gly Pro Arg Leu Ser Glu Gln Met Ile
500 505 510
His Glu Tyr Lys Lys Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Thr Tyr Lys Arg Glu Val Lys Gln Gly Lys Leu
530 535 540
Asn Ser Val Thr Leu Phe Val Lys Glu Tyr Pro Ala Thr Ser Val Glu
545 550 555 560
Glu Ala Ser Glu Trp Ile Met Leu Thr Leu Thr Glu Ser Arg Gln Gln
565 570 575
Ile Leu Arg Asn Ile Leu Gln Pro Ser Glu Ile Pro Arg Glu Cys Lys
580 585 590
Gln Ile Phe Trp Asn Arg Val Lys Ile Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Trp Ser Ser Pro Thr Ala Met Leu Glu His Met Asn Ala
610 615 620
Ile Ile Trp Gln Pro Val Leu
625 630
<210> 48
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体8
<400> 48
Met Gly Ala Val Thr Phe Ser Ser Ile Ser Thr Thr Ala Asn Tyr Gly
1 5 10 15
Ile Pro Met Ile Pro Ser Ser Asn Leu Gln Gly Arg Ser Arg Asp Thr
20 25 30
Arg Phe His Val Leu Ile Met Ser Ala Ala Ala Ser Gln Phe Asn Trp
35 40 45
Gln Gly Lys Met Met Phe Asn Pro Lys Thr His Gln Arg Val Met Ser
50 55 60
Leu Ser Val Glu Asp Ser Ser Asn Val Leu Val Met Ala Glu Lys Glu
65 70 75 80
Asn Arg Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Asp Glu Asn Leu
85 90 95
Thr Arg Ile Asn Met Leu Phe Leu Asp Ile Met Arg Met Tyr Ser Ser
100 105 110
Val Ser Gly Phe Asp Thr Val His Gly Ala Tyr Pro Val Asn Leu Ile
115 120 125
Val Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Met
130 135 140
His Phe Lys Val Glu Ile Arg Glu Leu Leu Asp Asp Val Trp Met Phe
145 150 155 160
Trp Ile Glu Gln Glu Met Ile Arg Gly Met Pro Thr Gly Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Thr Tyr Val Arg Asp Ser Glu Phe Leu
195 200 205
Pro Phe Ile Glu Asn Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Lys Tyr Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Asp Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Gly Ala Ile Arg Asp Leu Glu Gly Asp Val Lys His Ala Leu
260 265 270
Asp Val Pro Trp Met Gly Gly Leu Asp Arg Leu Glu His Leu Lys Tyr
275 280 285
Met Arg Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Arg Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Val Cys Gln Glu Ala Gln Gln Glu Asp Leu Arg Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Asn Met Leu Val Ala Cys Tyr Trp Ser Gly Ala Ser Ser Ile Trp Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Val Val Trp Thr Lys Asn Ala Ile Ile
370 375 380
Thr Thr Leu Met Asp Asp Phe Tyr Asp Ile Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Lys Arg Trp Asp Pro Leu Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Val Lys Met Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Asp Ile Ala Gln Glu Ile Trp Ile Tyr Asn Gly Trp Asp
435 440 445
Leu Thr Thr Gln Leu Met Glu Ile Tyr Phe Asp Leu Gly Ile Ser Met
450 455 460
Arg Lys Glu Ala Glu Trp Thr Met Thr Ala Phe Ile Pro Ser Leu Asp
465 470 475 480
Glu Tyr Lys Asp Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Ile Phe Phe Leu Gly Pro Arg Val Ser Glu Gln Arg Leu
500 505 510
His Glu Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Ala Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Trp Met Arg Glu Leu Arg Gln Gly Met Leu
530 535 540
Asn Ser Leu Ser Leu Phe Val Lys Glu Trp Pro Gly Thr Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Tyr Ile Arg Leu Thr Leu Ser Asp Thr Arg Gln Asn
565 570 575
Ile Leu Met Gln Leu Leu Gln Pro Thr Glu Val Pro Arg Asp Cys Arg
580 585 590
Gln Ile Phe Trp Asn Met Val Lys Val Val Gln Val Tyr Tyr Leu His
595 600 605
Ser Asp Asp Phe Thr Thr Pro Thr Ala Met Leu Glu His Met Gln Ala
610 615 620
Val Leu Trp Asn Pro Leu Ile
625 630
<210> 49
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体9
<400> 49
Met Ala Ala Val Ser Phe Ser Thr Ile Thr Ser Thr Ala Asn Tyr Ala
1 5 10 15
Ile Pro Met Leu Pro Thr Ser Gln Val Gln Gly Lys Ser Arg Asp Thr
20 25 30
Arg Phe His Val Leu Ile Lys Thr Ala Gly Ala Ser Gln Phe Asn Tyr
35 40 45
Gln Ala Arg Lys Lys Phe Gln Pro Arg Thr His Gln Lys Val Lys Ser
50 55 60
Leu Ser Leu Glu Asp Ser Ser Asn Val Leu Val Met Ala Asp Arg Glu
65 70 75 80
Gln Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Glu Asn Ile
85 90 95
Thr Arg Val Asn Met Leu Phe Leu Glu Ile Lys Arg Met Trp Ser Ser
100 105 110
Leu Ser Ala Phe Glu Thr Val His Gly Ala Trp Pro Ile Asn Ile Ile
115 120 125
Leu Gly Leu Ser Lys Val Asn His Leu Glu Ser Leu Gly Ile Ala Lys
130 135 140
His Phe Lys Ile Glu Ile Met Glu Leu Leu Asp Glu Val Trp Arg Tyr
145 150 155 160
Trp Ile Asp Asn Asp Lys Leu Met Gly Met Pro Ser Ala Ser Asp Ile
165 170 175
Met Ser Ser Ser Ile Thr Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Val Met Asp Ser Glu Phe Ile
195 200 205
Pro Phe Leu Glu Gln Ser Gly Gln Ala Thr Met Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Met Ser Phe Ser Gln Gln Tyr Leu Glu Asn Leu Arg Val Ala Asp
245 250 255
Gln Ser Ala Ala Ile Lys Asp Leu Glu Gly Glu Val Arg His Ala Leu
260 265 270
Asp Val Pro Tyr Arg Gly Gly Leu Glu Arg Leu Glu His Leu Arg Trp
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Ile Gly Glu Thr Ser Ser
290 295 300
Arg Ile Pro Phe Thr Gly Lys Asp Ile Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Asn Glu Asp Leu Arg Ala Ile
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Arg Leu Val Gly Cys Tyr Tyr Ser Ala Ala Ser Thr Ile Phe Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Ile Val Phe Thr Lys Asn Gly Val Leu
370 375 380
Ser Thr Leu Met Asp Asp Tyr Tyr Asp Val Glu Gly Ser Ile Asp Asp
385 390 395 400
Ile Gln Ser Phe Val Glu Ala Val Met Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Gly Asn Ile Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Glu Ile Ala Asn Asp Val Trp Ile Tyr Asn Gly Trp Asp
435 440 445
Ile Ser Thr Asn Leu Lys Glu Ile Phe Tyr Glu Val Ala Ile Ser Lys
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Thr Gly Trp Ile Pro Ser Leu Asp
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Leu Phe Phe Leu Ala Pro Lys Leu Thr Glu Gln Met Ile
500 505 510
His Glu Trp Lys Arg Leu Met Asn Leu Ile Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Trp Lys Arg Glu Ile Lys Gln Gly Lys Leu
530 535 540
Gln Thr Leu Ser Leu Phe Val Met Asp Phe Pro Ala Thr Ser Leu Glu
545 550 555 560
Asp Ala Thr Glu Trp Leu Met Leu Thr Val Ser Glu Ser Arg Gln Asn
565 570 575
Leu Leu Met Asn Leu Leu Gln Pro Ser Glu Leu Pro Lys Glu Cys Lys
580 585 590
Asn Ile Phe Phe Asn Met Ile Lys Ile Leu Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Trp Thr Ser Pro Thr Ala Arg Val Glu His Met Asn Gly
610 615 620
Val Leu Phe Asn Pro Leu Val
625 630
<210> 50
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体10
<400> 50
Met Gly Gly Leu Thr Phe Thr Thr Ile Ser Thr Thr Gly Asn Tyr Gly
1 5 10 15
Ile Pro Lys Leu Pro Ser Ser Asn Ile Gln Gly Lys Ser Met Asp Thr
20 25 30
Met Tyr His Leu Leu Ile Arg Ser Ala Gly Ala Thr Gln Trp Asn Tyr
35 40 45
Gln Ala Arg Arg Lys Phe Asn Pro Arg Thr His Gln Met Val Arg Ser
50 55 60
Leu Ser Ile Glu Asp Ser Ser Asn Val Leu Ile Lys Ala Glu Met Glu
65 70 75 80
Asn Arg Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Lys Leu Asn Arg Leu Trp Val Glu Ile Arg Arg Arg Tyr Ser Ser
100 105 110
Leu Ser Gly Trp Asp Thr Val His Ala Ala Tyr Pro Leu Asn Leu Ile
115 120 125
Leu Gly Leu Ser Met Val Asn His Leu Glu Ser Leu Gly Ile Ala Lys
130 135 140
His Phe Lys Val Glu Ile Lys Glu Val Leu Asp Asp Val Trp Lys Tyr
145 150 155 160
Trp Ile Glu Gln Glu Arg Leu Arg Ala Arg Pro Thr Ala Thr Asp Ile
165 170 175
Met Thr Thr Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Leu Arg Glu Ser Asp Phe Val
195 200 205
Pro Phe Leu Glu Gln Ser Gly Asn Ala Thr Met Ala Val Leu Gln Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Val Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Glu Asn Ile Met Val Gly Glu
245 250 255
Gln Ser Gly Gly Ile Met Asp Val Glu Gly Asp Val Lys His Gly Leu
260 265 270
Asp Leu Pro Trp Met Ala Gly Leu Glu Arg Ile Glu His Leu Met Tyr
275 280 285
Met Lys Ala Phe Ala Phe Asp Asp Ile Trp Val Gly Glu Thr Ser Thr
290 295 300
Arg Leu Pro Phe Thr Gly Arg Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Met Ala Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Lys Leu Gly Asp Leu His Phe Ala Arg
340 345 350
Gln Lys Leu Ile Ala Cys Tyr Trp Ser Ala Ala Ser Thr Val Tyr Ser
355 360 365
Pro Glu Met Ser Thr Ser Arg Leu Val Trp Thr Lys Gln Ala Val Ile
370 375 380
Thr Thr Ile Met Asp Asp Trp Tyr Asp Ile Glu Gly Ser Ile Asp Asp
385 390 395 400
Ile Gln Ser Phe Val Asp Ala Val Lys Arg Trp Asp Pro Ile Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Leu Lys Met Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Thr Val Asn Glu Leu Ala Gln Asp Ile Trp Ile Tyr Gln Gly Trp Asp
435 440 445
Ile Ser Thr Gln Leu Met Asp Ile Trp Tyr Glu Ile Gly Ile Ser Met
450 455 460
Lys Met Glu Ala Glu Trp Thr Lys Thr Ala Trp Val Pro Ser Leu Glu
465 470 475 480
Glu Tyr Lys Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Thr Ile Phe Phe Val Gly Pro Met Leu Ser Glu Gln Arg Ile
500 505 510
His Asp Tyr Lys Arg Leu Met Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Trp Lys Arg Glu Val Arg Gln Gly Lys Leu
530 535 540
Asn Thr Leu Ser Leu Phe Val Lys Glu Tyr Pro Gly Thr Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Tyr Ile Arg Val Thr Ile Ser Glu Ser Arg Gln Asn
565 570 575
Val Leu Met Asn Leu Ile Asn Pro Ser Glu Val Pro Arg Asp Cys Lys
580 585 590
Gln Ile Phe Phe Asn Arg Ile Lys Ile Ile Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Lys Leu Asp His Met Gln Ala
610 615 620
Ile Val Tyr Asn Pro Ile Leu
625 630
<210> 51
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体11
<400> 51
Met Gly Gly Leu Thr Phe Thr Thr Val Ser Thr Thr Ala Asn Tyr Ala
1 5 10 15
Val Pro Lys Leu Pro Ser Ser Asn Ile Gln Gly Lys Ser Arg Glu Thr
20 25 30
Met Phe His Ile Leu Leu Arg Thr Ala Ala Ala Thr Gln Phe Asn Trp
35 40 45
Gln Ala Arg Met Lys Phe Gln Pro Lys Thr His Asn Lys Val Met Thr
50 55 60
Leu Ser Val Glu Asp Ser Thr Asn Ile Val Leu Lys Ala Glu Lys Glu
65 70 75 80
Asn Arg Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Leu
85 90 95
Thr Arg Val Asn Met Leu Tyr Val Asp Ile Lys Met Arg Trp Ser Ser
100 105 110
Val Ser Ala Tyr Glu Thr Val His Gly Ala Tyr Pro Leu Asn Val Leu
115 120 125
Leu Gly Leu Thr Arg Val Asn His Leu Glu Ser Leu Gly Ile Ala Lys
130 135 140
His Phe Arg Val Glu Ile Arg Glu Ile Leu Asp Asp Val Tyr Arg Trp
145 150 155 160
Tyr Leu Asp Asn Asp Arg Val Lys Ala Lys Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Thr Ser Ile Ser Phe Lys Ile Leu Arg Tyr Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Tyr Ser Tyr Leu Arg Asp Ser Glu Phe Leu
195 200 205
Pro Phe Ile Asp Asn Ser Gly Asn Ala Ser Met Ala Val Leu Gln Leu
210 215 220
Tyr Lys Ala Ser Gln Met Met Phe Pro Gly Glu Arg Val Leu Glu Glu
225 230 235 240
Ala Lys Ser Phe Ser Gln Asn Tyr Leu Asp Asn Ile Lys Val Ala Asp
245 250 255
Gln Thr Ala Ala Leu Arg Asp Val Asp Ala Glu Val Met His Gly Leu
260 265 270
Asp Val Pro Tyr Met Ala Gly Val Asp Arg Ile Glu His Leu Arg Trp
275 280 285
Met Lys Ala Phe Gly Tyr Glu Asp Ile Tyr Val Gly Glu Thr Ser Ser
290 295 300
Met Ile Pro Phe Thr Ala Met Asp Leu Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Gly Gln Gln Glu Asp Leu Lys Ala Leu
325 330 335
Glu Met Trp Ser Leu Asp Ser Lys Leu Gly Glu Leu His Phe Ala Arg
340 345 350
Asn Met Leu Leu Ala Cys Tyr Phe Ser Ala Gly Ser Thr Leu Trp Ser
355 360 365
Pro Glu Met Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Lys Asp Asp Tyr Tyr Asp Ile Glu Gly Ser Val Glu Asp
385 390 395 400
Ile Asn Ser Phe Val Glu Ala Leu Arg Arg Trp Asp Pro Leu Ile Val
405 410 415
Cys Asn Cys Ser Gly Asn Ile Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Ser Leu Asn Glu Leu Ala Gln Glu Ile Trp Leu Phe Gln Gly Trp Glu
435 440 445
Ile Ser Thr Asn Ile Met Glu Ile Trp Tyr Glu Leu Ala Ile Ser Met
450 455 460
Met Lys Glu Ala Glu Trp Thr Met Thr Ala Phe Ile Pro Thr Leu Asp
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Ser Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Ser Ile Trp Tyr Leu Gly Pro Lys Leu Ser Glu Gln Lys Ile
500 505 510
His Glu Tyr Met Arg Leu Met Gln Val Val Asn Thr Cys Ala Arg Leu
515 520 525
Leu Asn Asp Ser Gln Thr Tyr Met Arg Glu Leu Lys Asn Gly Lys Leu
530 535 540
Asn Ser Leu Ser Leu Phe Val Arg Glu Tyr Pro Gly Thr Ser Ile Glu
545 550 555 560
Asp Ala Thr Glu Trp Ile Arg Ile Thr Leu Thr Glu Thr Arg Gln Gln
565 570 575
Ile Leu Arg Asn Val Ile Gln Pro Ser Glu Ile Pro Arg Asp Cys Lys
580 585 590
Gln Ile Phe Trp Asn Arg Leu Lys Ile Val Gln Leu Phe Tyr Leu His
595 600 605
Ser Asp Glu Phe Thr Ser Pro Thr Ala Met Val Glu His Met Gln Ala
610 615 620
Ile Leu Phe Asn Pro Val Ile
625 630
<210> 52
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体12
<400> 52
Met Ala Gly Ile Ser Tyr Thr Thr Leu Ser Thr Thr Ala Asn Tyr Gly
1 5 10 15
Val Pro Arg Leu Pro Ser Ser Asn Ile Gln Gly Arg Thr Lys Asp Ser
20 25 30
Lys Trp His Val Leu Ile Arg Thr Gly Ala Gly Ser Gln Tyr Asn Tyr
35 40 45
Asn Gly Arg Lys Lys Tyr Gln Pro Arg Ser His Asn Lys Val Lys Ser
50 55 60
Leu Ser Val Glu Glu Thr Ser Asn Ile Leu Ile Lys Ala Asp Lys Asp
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Glu Asn Ile
85 90 95
Ser Lys Ile Asn Met Leu Tyr Leu Glu Ile Lys Arg Lys Tyr Ser Ser
100 105 110
Ile Thr Ala Phe Glu Thr Val His Gly Ala Trp Pro Leu Asn Leu Ile
115 120 125
Leu Gly Leu Ser Lys Val Asn His Leu Glu Ser Leu Gly Ile Ala Arg
130 135 140
His Phe Arg Leu Glu Ile Lys Glu Val Leu Asp Asp Val Tyr Arg Phe
145 150 155 160
Tyr Ile Glu Asn Glu Lys Val Lys Gly Lys Pro Ser Ala Ser Asp Ile
165 170 175
Met Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Trp Ser Tyr Val Arg Asp Thr Asp Phe Leu
195 200 205
Pro Phe Val Glu Asn Thr Gly Gln Ala Thr Met Ala Val Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Gln Arg Met Tyr Pro Gly Glu Lys Val Leu Glu Glu
225 230 235 240
Ala Arg Ser Phe Ser Gln Asn Tyr Leu Glu Asn Val Lys Val Gly Asp
245 250 255
Asn Ser Gly Ala Ile Lys Asp Ile Asp Ala Glu Val Arg His Ala Leu
260 265 270
Asp Val Pro Trp Arg Ala Ala Leu Glu Arg Val Glu His Leu Arg Tyr
275 280 285
Met Lys Ala Phe Gly Phe Glu Glu Leu Trp Leu Gly Glu Thr Ser Ser
290 295 300
Lys Ile Pro Phe Thr Ala Lys Asp Leu Phe Leu Thr Leu Ala Lys Asn
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Asp Asp Leu Met Ala Val
325 330 335
Glu Arg Trp Ser Leu Asp Ser Lys Ile Ala Asp Leu His Phe Ala Arg
340 345 350
Gln Arg Val Ile Ala Cys Tyr Phe Ser Ala Gly Ser Thr Leu Phe Ser
355 360 365
Pro Glu Lys Ser Thr Thr Arg Leu Val Tyr Thr Lys Asn Ala Val Leu
370 375 380
Thr Thr Leu Met Asp Asp Trp Tyr Asp Val Glu Gly Ser Leu Glu Asp
385 390 395 400
Ile Gln Ser Phe Val Asp Ala Val Lys Arg Trp Asp Pro Ile Val Val
405 410 415
Cys Asn Cys Ser Ala Asn Leu Lys Met Val Phe Ser Ala Ile Phe Asn
420 425 430
Thr Val Asn Asp Leu Ala Gln Glu Val Phe Val Phe Asn Ala Trp Asp
435 440 445
Leu Ser Thr Asn Leu Arg Asp Leu Trp Tyr Asp Val Ala Ile Ser Met
450 455 460
Met Arg Glu Ala Glu Trp Thr Lys Thr Gly Trp Leu Pro Thr Ile Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Lys Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Thr Thr Val Phe Phe Leu Gly Pro Met Ile Thr Glu Gln Met Val
500 505 510
His Asp Trp Arg Arg Leu Lys Gln Leu Leu Asn Thr Cys Gly Arg Leu
515 520 525
Ile Asn Asp Ser Asn Ser Phe Lys Met Glu Leu Lys Asn Gly Lys Val
530 535 540
Asn Ser Val Ser Leu Phe Val Lys Glu Trp Pro Ala Thr Ser Ile Glu
545 550 555 560
Asp Ala Ser Glu Tyr Ile Lys Val Thr Leu Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Lys Gln Leu Val Gln Pro Ser Glu Ile Pro Lys Glu Cys Lys
580 585 590
Gln Ile Phe Trp Asn Lys Val Lys Leu Leu Gln Val Phe Tyr Leu His
595 600 605
Ser Asp Asp Phe Thr Thr Pro Thr Gly Met Leu Glu His Met Gln Gly
610 615 620
Val Val Phe Asn Pro Val Val
625 630
<210> 53
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体13
<400> 53
Met Ala Ala Val Ser Phe Thr Thr Leu Thr Thr Ser Gly Asn Trp Ala
1 5 10 15
Leu Pro Lys Val Pro Ser Ser Gln Leu Gln Gly Arg Ser Met Glu Thr
20 25 30
Arg Trp His Leu Leu Ile Met Ser Ala Gly Ala Thr Gln Tyr Asn Phe
35 40 45
Asn Gly Met Met Lys Phe Gln Pro Arg Thr His Gln Lys Ile Arg Ser
50 55 60
Leu Ser Val Asp Asp Ser Thr Asn Val Val Val Met Ala Glu Arg Asp
65 70 75 80
Asn Arg Glu Asn Lys Ser Phe Gly Ser Met Cys Ile Glu Asp Gln Ile
85 90 95
Thr Arg Ile Asn Lys Leu Tyr Leu Asp Ile Arg Met Lys Phe Thr Ser
100 105 110
Leu Ser Ala Tyr Asp Thr Val His Gly Gly Phe Pro Val Asn Leu Ile
115 120 125
Ile Gly Leu Ser Arg Val Asn His Leu Glu Ser Leu Gly Ile Gly Arg
130 135 140
His Phe Lys Ile Glu Ile Lys Glu Leu Leu Glu Asp Val Phe Met Phe
145 150 155 160
Phe Leu Glu Asn Glu Met Val Arg Ala Lys Pro Thr Ala Ser Asp Ile
165 170 175
Lys Thr Ser Ser Ile Ser Phe Lys Ile Leu Arg Tyr Asn Gly Tyr Asp
180 185 190
Val Ser Pro Asp Val Phe Phe Ser Tyr Val Lys Asp Thr Asp Phe Val
195 200 205
Pro Trp Leu Glu Asn Thr Gly Gln Ala Thr Lys Ala Ile Leu Gln Leu
210 215 220
Tyr Lys Ala Ser Gln Met Lys Trp Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Thr Phe Ser Gln Asn Tyr Leu Glu Asn Leu Lys Ile Gly Asp
245 250 255
Gln Ser Gly Ala Leu Met Glu Ile Glu Ala Asp Val Met His Ala Leu
260 265 270
Asp Val Pro Trp Met Ala Gly Val Glu Arg Val Glu His Leu Arg Phe
275 280 285
Met Met Ala Phe Ala Phe Asp Glu Val Tyr Val Gly Glu Thr Ser Ser
290 295 300
Met Val Pro Phe Thr Gly Arg Asp Ile Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Ala Gln Gln Glu Asp Leu Lys Gly Leu
325 330 335
Glu Lys Trp Ser Val Asp Ser Met Val Gly Asp Leu His Phe Gly Arg
340 345 350
Gln Arg Leu Val Ala Cys Tyr Phe Ser Ala Ala Ser Thr Val Trp Ser
355 360 365
Pro Glu Arg Ser Thr Thr Arg Ile Val Trp Thr Lys Asn Ala Val Ile
370 375 380
Thr Thr Val Met Asp Asp Phe Tyr Asp Val Glu Gly Ser Ile Glu Asp
385 390 395 400
Ile Asn Ser Phe Ile Asp Ala Val Lys Arg Trp Asp Pro Leu Val Leu
405 410 415
Cys Asn Cys Ser Ala Asn Leu Lys Lys Leu Phe Ser Ala Ile Tyr Asn
420 425 430
Ser Val Gln Asp Ile Gly Gln Glu Ile Phe Val Trp Gln Gly Phe Asp
435 440 445
Ile Ser Thr Asn Leu Arg Glu Ile Tyr Trp Glu Leu Gly Ile Thr Met
450 455 460
Lys Lys Glu Ala Asp Trp Thr Met Ser Gly Tyr Leu Pro Thr Ile Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Phe Ser Ser Leu Phe Trp Val Gly Pro Lys Leu Thr Glu Asn Met Ile
500 505 510
His Glu Tyr Met Lys Ile Arg Gln Ile Ile Gln Thr Cys Gly Arg Leu
515 520 525
Ile Asn Asp Ser Gln Ser Tyr Lys Lys Glu Val Lys Gln Gly Lys Leu
530 535 540
Gln Ser Leu Ser Leu Phe Val Arg Glu Tyr Pro Gly Ser Ser Leu Glu
545 550 555 560
Asp Ala Thr Glu Phe Ile Met Val Ser Leu Ser Glu Thr Arg Gln Asn
565 570 575
Val Leu Arg Asn Leu Leu Gln Pro Ser Glu Val Pro Arg Glu Cys Lys
580 585 590
Gln Ile Tyr Phe Asn Lys Val Lys Leu Leu Asn Val Tyr Tyr Leu His
595 600 605
Ser Asp Glu Tyr Ser Ser Pro Thr Ala Arg Ile Glu His Met Asn Ala
610 615 620
Val Ile Trp Asn Pro Val Val
625 630
<210> 54
<211> 631
<212> PRT
<213> 人工序列
<220>
<223> 变体14
<400> 54
Met Gly Gly Ile Ser Tyr Ser Thr Val Thr Thr Thr Ala Gln Tyr Gly
1 5 10 15
Ile Pro Met Leu Pro Ser Thr Gln Val Asn Ala Met Ser Lys Asp Thr
20 25 30
Lys Tyr His Val Leu Ile Lys Thr Ala Gly Ala Ser Asn Trp Asn Phe
35 40 45
Asn Gly Met Arg Lys Tyr Asn Pro Arg Thr His Gln Lys Val Lys Thr
50 55 60
Val Ser Val Glu Asp Thr Ser Gln Val Leu Ile Lys Gly Asp Lys Glu
65 70 75 80
Asn Arg Glu Lys Lys Ser Phe Gly Ser Met Cys Ile Asp Asp Asn Ile
85 90 95
Thr Lys Ile Asn Met Leu Phe Val Asp Ile Lys Lys Arg Trp Thr Ser
100 105 110
Val Ser Ala Tyr Asp Thr Val His Gly Ala Trp Pro Ile Asn Leu Ile
115 120 125
Ile Gly Leu Ser Lys Val Asn His Leu Glu Ser Leu Gly Ile Ala Met
130 135 140
His Phe Arg Val Glu Ile Met Asp Val Leu Asp Asp Val Phe Lys Tyr
145 150 155 160
Tyr Ile Asp Asn Glu Met Val Arg Ala Met Pro Ser Ala Thr Asp Ile
165 170 175
Met Ser Ser Ser Ile Ser Phe Lys Ile Leu Arg Trp Gln Gly Tyr Asp
180 185 190
Val Ser Pro Asp Ile Phe Phe Ser Tyr Leu Lys Glu Ser Glu Phe Leu
195 200 205
Pro Trp Ile Asp Gln Ser Gly Gln Ala Thr Arg Ala Leu Leu Asn Leu
210 215 220
Tyr Lys Ala Ser Asn Met Met Phe Pro Gly Glu Arg Ile Leu Glu Glu
225 230 235 240
Ala Lys Thr Phe Ser Asn Asn Trp Leu Glu Asn Ile Lys Val Gly Asp
245 250 255
Gln Ser Ala Ala Leu Lys Asp Val Asp Ala Glu Val Met His Gly Leu
260 265 270
Asp Leu Pro Tyr Met Ala Gly Val Asp Arg Ile Glu His Leu Arg Phe
275 280 285
Met Arg Ala Phe Gly Phe Asp Asp Ile Trp Val Gly Glu Thr Thr Ser
290 295 300
Arg Ile Pro Trp Thr Gly Lys Asp Ile Phe Leu Ser Leu Ala Lys Gln
305 310 315 320
Asp Phe Asn Ile Cys Gln Glu Gly Gln Gln Glu Asp Leu Met Gly Ile
325 330 335
Asp Lys Trp Ser Val Asp Ser Lys Leu Gly Glu Leu His Phe Ala Arg
340 345 350
Gln Arg Leu Val Ala Cys Tyr Trp Ser Ala Ala Ser Ser Leu Trp Ser
355 360 365
Pro Asp Lys Thr Thr Thr Arg Ile Val Tyr Thr Lys Gln Ala Ile Leu
370 375 380
Thr Ser Val Lys Asp Asp Trp Tyr Asp Ile Asp Gly Ser Ile Glu Asp
385 390 395 400
Ile Gln Ser Phe Ile Glu Ala Val Lys Arg Trp Asp Pro Ile Leu Leu
405 410 415
Cys Asn Cys Ser Ala Asn Ile Lys Met Leu Phe Ser Ala Ile Trp Gln
420 425 430
Ser Ile Asn Asp Ile Gly Gln Glu Leu Trp Leu Tyr Asn Gly Tyr Asp
435 440 445
Ile Ser Thr Gln Val Arg Asp Ile Phe Tyr Glu Leu Gly Ile Ser Lys
450 455 460
Met Lys Glu Ala Glu Trp Thr Lys Ser Gly Phe Ile Pro Ser Leu Glu
465 470 475 480
Glu Tyr Met Glu Asn Gly Arg Thr Ser Ile Ala Leu Glu Pro Ile Ile
485 490 495
Trp Thr Ser Leu Phe Phe Leu Gly Pro Lys Leu Ser Glu Gln Met Leu
500 505 510
His Asp Tyr Arg Lys Leu Met Gln Val Leu Asn Ser Cys Ala Arg Leu
515 520 525
Leu Asn Asp Ser Gln Ser Trp Met Arg Glu Val Lys Gln Gly Met Ile
530 535 540
Asn Ser Leu Ser Leu Phe Val Lys Asp Trp Pro Gly Thr Ser Leu Glu
545 550 555 560
Asp Ala Thr Asp Phe Ile Lys Leu Thr Leu Ser Glu Ser Arg Gln Asn
565 570 575
Ile Leu Lys Asn Leu Val Asn Pro Ser Glu Ile Pro Arg Asp Cys Lys
580 585 590
Gln Val Trp Phe Asn Arg Ile Lys Val Val Gln Val Phe Tyr Ile His
595 600 605
Thr Asp Glu Phe Thr Ser Pro Ser Ala Lys Leu Asp His Met Gln Gly
610 615 620
Leu Val Trp Asn Pro Val Ile
625 630
<210> 55
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> Cup基序
<400> 55
Glu Asn Asn Ser Phe Gly Ser Met Cys Ile
1 5 10
<210> 56
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> Cup基序
<400> 56
Glu Lys Lys Ser Phe Gly Ser Met Cys Ile
1 5 10
<210> 57
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> Cup基序
<400> 57
Glu Lys Asn Ser Phe Gly Ser Met Cys Ile
1 5 10
<210> 58
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> Cup基序
<400> 58
Glu Asn Lys Ser Phe Gly Ser Met Cys Ile
1 5 10

Claims (16)

1.一种用于生产至少一种C-20萜醇的方法，该方法包括以下步骤：
a)将香叶基香叶基焦磷酸转化为柯巴基二磷酸(CPP)或半日花-13-烯-8-醇二磷酸(LPP)；以及
b)将CPP或LPP转化为至少一种C-20萜醇，其中所述转化是通过表现出二萜醇合酶活性的多肽实施的，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，
其中所述多肽包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
2.根据权利要求1所述的方法，其中所述表现出二萜醇合酶活性的多肽能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇，并且优选地，包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列；
b)与如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:2、16、17或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:2、16、17或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇的二萜醇合酶活性的多肽。
3.根据权利要求1所述的方法，其中所述表现出二萜醇合酶活性的多肽能够将LPP转化为冷杉醇，并且优选地，包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3、5或8所示的氨基酸序列；
b)与如SEQ ID NO:3、5或8所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1或18所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或18所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
4.根据权利要求1至3中任一项所述的方法，其中所述步骤a)中的转化是通过另外的多肽实施的，该另外的多肽表现出将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP的II型二萜合酶的酶活性。
5.根据权利要求1至4中任一项所述的方法，其中所述步骤b)或所述步骤a)和b)在宿主细胞或非人转基因生物体中实施。
6.一种组合物，该组合物包含宿主细胞或非人转基因生物体，以及可通过根据权利要求1至5中任一项所述的方法获得的所述至少一种C-20萜醇，优选地泪杉醇、香紫苏醇和/或冷杉醇，其中该宿主细胞或非人转基因生物体包含适于重组的至少一种表现出二萜醇合酶活性的多肽，该多肽具有以下氨基酸序列
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
7.一种表现出二萜醇合酶活性的多肽，其中所述二萜醇合酶活性能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇，所述多肽具有选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列；
b)与如SEQ ID NO:3至10或SEQ ID NO:34中任一个所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1、2、16、17、18或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1、2、16、17、18或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将柯巴基二磷酸(CPP)转化为泪杉醇、将半日花-13-烯-8-醇二磷酸(LPP)转化为香紫苏醇和/或将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
8.根据权利要求7所述的多肽，其中所述二萜醇合酶活性能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇，并且优选地，包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列；
b)与如SEQ ID NO:4、6、7、9、10或34所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:2、16、17或35所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:2、16、17或35所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将CPP转化为泪杉醇以及将LPP转化为香紫苏醇的二萜醇合酶活性的多肽。
9.根据权利要求7所述的多肽，其中所述二萜醇合酶活性能够将LPP转化为冷杉醇，并且优选地，包含选自由以下组成的组的氨基酸序列：
a)如SEQ ID NO:3、5或8所示的氨基酸序列；
b)与如SEQ ID NO:3、5或8所示的氨基酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性的氨基酸序列；
c)由如SEQ ID NO:1或18所示的核酸序列编码的氨基酸序列；
d)由核酸序列编码的氨基酸序列，该核酸序列与如SEQ ID NO:1或18所示的核酸序列具有至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一性；以及
e)(a)至(d)中任一项的片段的氨基酸序列，所述片段编码表现出能够将LPP转化为冷杉醇的二萜醇合酶活性的多肽。
10.一种融合多肽，该融合多肽包含该根据权利要求7至9中任一项所述的多肽和至少一种另外的多肽，该另外的多肽(i)表现出II型二萜合酶的酶活性，优选地，将香叶基香叶基焦磷酸(GGP)转化为LPP和/或CPP，(ii)具有麦芽糖结合特性，或者(iii)是硫氧还蛋白或硫氧还蛋白融合蛋白。
11.一种多核苷酸，该多核苷酸编码根据权利要求7至9中任一项所述的多肽或根据权利要求10所述的融合多肽或其反向互补或互补序列。
12.一种载体或基因构建体，该载体或基因构建体包含根据权利要求11所述的多核苷酸。
13.一种宿主细胞，该宿主细胞包含根据权利要求12所述的载体或基因构建体。
14.一种转基因非人生物体，该转基因非人生物体包含根据权利要求11所述的多核苷酸、根据权利要求12所述的载体或基因构建体、或根据权利要求13所述的宿主细胞。
15.根据权利要求7至9中任一项所述的多肽或根据权利要求10所述的融合多肽、根据权利要求11所述的多核苷酸、根据权利要求12所述的载体或基因构建体、根据权利要求13所述的宿主细胞或根据权利要求14所述的非人转基因生物体用于生产至少一种C-20萜醇，优选地冷杉醇、泪杉醇和/或香紫苏醇的用途。
16.一种用于制备具有二萜醇合酶活性的变体多肽的方法，该方法包括以下步骤：
a)选择根据权利要求11所述的核酸；
b)修饰该所选核酸以获得至少一种突变体核酸；
c)用突变体核酸序列转化宿主细胞或单细胞生物体以表达由该突变体核酸序列编码的多肽；
d)筛选该具有至少一种修饰的特性以及二萜醇合酶活性的多肽；以及，
e)任选地，如果该多肽不具有所需变体二萜醇合酶活性，则重复工艺步骤(a)至(d)，直到获得具有所需变体二萜醇合酶活性的多肽；
f)任选地，如果在步骤(d)中鉴定出具有所需变体二萜醇合酶活性的多肽，则分离在步骤(c)中获得的相应突变体核酸。
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