CN113980925A - 利用10-去乙酰巴卡亭III 10β-O-乙酰转移酶突变体催化合成紫杉醇及其衍生物 - Google Patents
利用10-去乙酰巴卡亭III 10β-O-乙酰转移酶突变体催化合成紫杉醇及其衍生物 Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供了10‑去乙酰巴卡亭III 10β‑O‑乙酰转移酶(DBAT)一系列突变体蛋白，这些蛋白均能专一性地将乙酰辅酶A等的酰基转移到10‑去乙酰紫杉烷的C10位羟基上，生成紫杉醇或其类似物。本发明涉及DBAT突变体的氨基酸序列以及编码这些氨基酸序列的核苷酸序列，以及DBAT突变体蛋白在利用10‑去乙酰紫杉醇等10‑去乙酰紫杉烷底物合成紫杉醇或其类似物上的应用；特别是将其与能够专一性水解7‑木糖‑10‑去乙酰紫杉醇的糖基水解酶相偶联，以7‑木糖‑10‑去乙酰紫杉醇为底物，以乙酰辅酶A为酰基供体直接合成紫杉醇。
Description
本申请是申请日为2016年5月24日、发明名称为“10-去乙酰巴卡亭III 10β-O-乙酰转移酶突变体及其在催化合成紫杉醇及其类似物中的应用”的申请号为201610346558.4的专利申请的分案申请。
技术领域
本发明涉及10-去乙酰巴卡亭III 10β-O-乙酰转移酶(DBAT)的一系列突变体蛋白，将来自但不限于乙酰辅酶A的酰基转移到10-去乙酰紫杉烷上，生成紫杉醇或其类似物；突变体蛋白的酰基受体包括但不限于10-去乙酰紫杉醇和10-去乙酰巴卡亭III。本发明涉及这些突变体蛋白的氨基酸序列以及编码这些氨基酸序列的核苷酸序列及其应用。
技术背景
紫杉醇(paclitaxel,
)是一个具有确切抗肿瘤疗效、主要来自于红豆杉但天然含量极低的“重磅炸弹”式药物，而10-去乙酰巴卡亭III 10β-O-乙酰转移酶(DBAT)则是紫杉醇生物合成途径的一个重要酶，催化该途径的中间体10-去乙酰巴卡亭III(10-DAB)C10位上羟基的乙酰化反应，形成巴卡亭III，后者再经过若干步反应最终形成具有复杂结构的二萜类化合物——紫杉醇。
1996年，Zocher等首次报道以乙酰辅酶A为酰基供体、用来自欧洲红豆杉(Taxusbaccata)的根部(蛋白)粗提液催化10-DAB的C10位羟基乙酰化形成巴卡亭III，该粗提液显示区域选择性，即只对C10羟基具有乙酰化作用，而对于10-DAB的C1、C7和C13位的游离羟基不起作用[Zocher,R,et al.Biosynthesis of Taxol:enzymatic acetylation of 10-deacetylbaccatin-III to baccatin-III in crude extracts from roots of Taxusbaccata.Biochem Biophys Res Commun.,1996, 229(1):16-20]。之后，Pennington等报道分别从东北红豆杉(Taxus cuspidata)的叶和悬浮培养细胞中得到了部分纯化的DBAT，均能在乙酰辅酶A存在的条件下催化10-DAB形成巴卡亭III；但如果以10-去乙酰紫杉醇(DT)为底物，则不能得到肯定的结果，表现在产物紫杉醇生成的不确定性、或因其产量不足而无显著性统计学意义，并认为最有可能的解释是：紫杉醇的时隐时现源于粗酶液中污染一种尚未被表征的乙酰辅酶A:10-去乙酰紫杉醇-O-乙酰转移酶 [Pennington,JJ,etal.Acetyl CoA:10-deacetylbaccatin-III-10-O-acetyltransferase activity inleaves and cell suspension cultures of Taxus cuspidata.Phytochemistry,1998,49(8):2261-2266]。上述两篇文献仅涉及到DBAT粗酶液，其中夹杂着其他未被表征的蛋白质(或酶)，且其中的DBAT都未被表征；反应产物的鉴定也仅限于薄层色谱(TLC)、高效液相色谱(HPLC)和同位素扫描，没有进行严格的波谱学证明，因此证据尚不够充分。1999年Menhard等报道从中国红豆杉(Taxus chinensis)的悬浮细胞中纯化到DBAT，该酶为单体蛋白，表观分子量为71±1.5kDa，最适pH 和最适温度分别为9.0和35℃，pI为5.6[Menhard B,Zenk MH.Purification and characterization of acetyl coenzyme A:10-hydroxytaxane O-acetyltransferase from cell suspension cultures of Taxuschinensis.Phytochemistry,1999,50:763-774]。该细胞系在优化条件下可产生高达150mg/L云南紫杉烷C(taxuyunnanine C，该化合物不含四元氧环)，以该化合物为原料水解制备出一系列去乙酰化的化合物(10-deacetyltaxuyunnanine C,10,14-deacetyltaxuyunnanine C, 5,10,14-deacetyltaxuyunnanine C,2,10,14-deacetyltaxuyunnanine C, 2,5,10,14-deacetyltaxuyunnanine C)。催化实验证明该酶均能将这些化合物的C10位羟基乙酰化，但不能在其他位置上乙酰化，说明该酶具有区域选择性。还发现该酶也能将10-DAB的C10位羟基乙酰化，但对10表-10-DAB(10-epi-10-DAB)则不起作用，表现为立体选择性[Menhard B, Zenk MH.Purification andcharacterization of acetyl coenzyme A:10-hydroxytaxane O-acetyltransferasefrom cell suspension cultures of Taxus chinensis.Phytochemistry,1999, 50:763-774]。
2000年Croteau实验室首次报道从东北红豆杉(Taxus cuspidata)中克隆到DBATcDNA [Walker K,Croteau R.Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyltransferase cDNA from Taxus and functional expression in Escherichiacoli.Proc Natl Acad Sci USA.,2000, 97(2):583-587；Croteau et al.Transacylasesof the paclitaxel biosynthetic pathway.US7,153,676B1, Date of patent:Dec.26,2006]，并在大肠杆菌中实现异源表达。该重组酶的最适pH为7.4，可以将乙酰辅酶A上的乙酰基转移到10-DAB上，得到产物巴卡亭III。该酶同样具有区域选择性，对于10-DAB的1β-、7β-、13α-位羟基则不能进行乙酰化。之后，其他实验室也相继从欧洲红豆杉等植物中克隆到该酶的编码基因[Fang J,Ewald D.Expression cloned cDNA for 10-deacetylbaccatinIII-10-O-acetyltransferase in Escherichia coli:a comparative study of threefusion systems.Protein Expr Purif.,2004,35(1):17-24；Guo,BH,et al.Molecularcloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyltransferase cDNA from Taxus x media.Mol Biol Rep.,2007,34(2):89-95；程抒劼,等.南方红豆杉10-去乙酰巴卡亭III-10-乙酰转移酶基因的克隆与生物信息学分析.生物技术通报,2011,(1):107-112]。Walker研究团队发现，在以10-DAB为酰基受体时，DBAT对于酰基供体具有一定的宽泛性(promiscuity)，但酰基辅酶A的碳链长度与催化效率呈负相关，其中以乙酰辅酶A作为酰基供体时的催化效率最高；当dbat基因被导入大肠杆菌后，产生的重组酶可利用大肠杆菌内源性乙酰辅酶A实现底物10-DAB到产物巴卡亭III的转化[Loncaric C,et al.Profiling a Taxol pathway10β-acetyltransferase:Assessmentof the specificity and the production of baccatin III by in vivo acetylationin E.coli.Chem Biol.,2006,13:1-9；Loncaric C,et al.Expression of an acetyl-CoAsynthase and a CoA-transferase in Escherichia coli to produce modifiedtaxanes in vivo.Biotechnol J.,2006,2(2):266-274]；该研究团队还发现DBAT具有一定的区域选择宽泛性，也能对4-DAB 的C4位羟基乙酰化[Ondari ME,Walker KD.The taxolpathway 10-O-acetyltransferase shows regioselective promiscuity with theoxetane hydroxyl of 4-deacetyltaxanes.J Am Chem Soc.,2008, 130(50):17187-17194]。
由于10-去乙酰紫杉醇(DT)仅需一步C10位羟基乙酰化即成为紫杉醇，因此，研究和开发这类非天然底物C10位羟基的酶促乙酰化反应具有重要的理论和实际意义。但DBAT究竟能不能催化非天然底物DT的C10位羟基乙酰化、或者即使能够完成此催化反应但催化效率如何，这是一个亟待解决的问题。为此，本发明以DT为酰基受体，以乙酰辅酶A为酰基供体，应用重组的DBAT结合LC-MS等分析技术进行了催化研究，发现DBAT确实能够催化非天然底物DT的C10位羟基乙酰化反应而生成紫杉醇，但催化效率极低。之后，利用蛋白质工程对DBAT进行改造，已获得13个对于DT等非天然酰基受体底物的催化活性比野生型 DBAT有显著提高的突变体蛋白(DBATm系列)(产物为紫杉醇)，其中的一些突变体蛋白对于天然酰基受体底物10-DAB的催化活性也有显著提高(产物为巴卡亭III)。将这些突变体蛋白与来自香菇的一种糖基水解酶LXYL-P1-2[Cheng HL,et al.Cloning and characterization ofthe glycoside hydrolases that remove xylosyl group from 7-β-xylosyl-10-deacetyltaxol and its analogues. Mol Cell Proteomics,2013,12(8):2236-2248]相偶联，以乙酰辅酶A为酰基供体，通过“一锅法”反应，还可以将天然含量较为丰富的7-木糖-10-去乙酰紫杉醇(XDT)直接转变为紫杉醇。
发明内容
针对DBAT究竟能不能催化非天然底物DT的C10位羟基乙酰化和如何提高此乙酰化效率，本发明解决的技术问题是提供了一类DBAT系列突变体蛋白、编码该突变体蛋白的核苷酸序列、含有该核苷酸序列的重组质粒、含有该核苷酸序列或重组质粒的重组细胞，以及以上所述的突变体蛋白、其核苷酸序列、其重组质粒或重组细胞在催化合成紫杉醇或其类似物方面的应用。
为解决本发明的技术问题，提供了如下技术方案：
本发明技术方案的第一方面是：应用纯化的(HPLC色谱纯)重组DBAT为催化剂，分别以10-去乙酰紫杉醇(DT)和乙酰辅酶A为乙酰基受体和供体进行催化反应，对产物进行LC-MS 鉴定，证明产物为紫杉醇，证明了重组DBAT可以催化非天然底物DT的C10位羟基乙酰化。
为了提高DBAT的乙酰化效率，本发明技术方案的第二方面是提供一种10-去乙酰巴卡亭 III 10β-O-乙酰转移酶DBAT的突变体蛋白，其特征在于，所述的突变体蛋白具有与SEQ ID NO1所示的氨基酸序列至少90％以上的一致性，但不包括SEQ ID NO1。优选的突变体蛋白具有与SEQ ID NO1所示的氨基酸序列至少95％以上的一致性。最优选的突变体蛋白的氨基酸序列选自SEQ ID NO2～SEQ ID NO23所示的氨基酸序列。
以上所述的突变体蛋白上可进行常规修饰；或者在这些突变体蛋白上连接有用于检测或纯化的标签；所述的常规修饰包括乙酰化、酰胺化、环化、糖基化、磷酸化、烷基化、生物素化、荧光基团修饰、聚乙二醇PEG修饰、固定化修饰；所述的标签包括6×His、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc、Profinity eXact。
以上所述的突变体蛋白与野生型蛋白DBAT相比，其氨基酸突变包括：G38R、G38W、G38Y、G38I、G38T、G38E、G38M、G38Q、G38C、G38S、G38D、G38H、G38A、F301C、 F301V、F301A、F301M、F301L、F301T、F301S、C216R，以及以上氨基酸突变的组合；所述的组合包括但不限于G38R/F301V双突变。
本发明技术方案的第三方面是：提供了编码第二方面所述突变体蛋白的核苷酸序列，优选SEQ ID NO 25～SEQ ID 46所示的核苷酸序列。
本发明技术方案的第四方面是：提供含有第三方面所述核苷酸序列的重组质粒。
本发明技术方案的第五方面是：提供含有第三方面所述核苷酸序列或第四方面所述重组质粒的重组细胞。
本发明技术方案的第六方面是：提供本发明第二方面所述突变体蛋白、第三方面所述核苷酸序列、第四方面所述重组质粒、第五方面所述重组细胞在催化合成紫杉醇或其类似物方面的应用；进一步的，在催化10-去乙酰紫杉醇及其类似物的C10位羟基酰基化生成紫杉醇或其类似物中的应用；所述的突变体蛋白可以与能够专一性水解7-木糖-10-去乙酰紫杉烷的糖基水解酶相偶联，以7-木糖-10-去乙酰紫杉烷为底物，以酰基辅酶A为酰基供体生成紫杉醇或其类似物；所述的酰基受体包括但不限于10-去乙酰紫杉醇、10-去乙酰巴卡亭III；所述的酰基供体包括但不限于乙酰辅酶A、丙酰辅酶A和丁酰辅酶A。
优选的酰基供体底物为乙酰辅酶A，优选的酰基受体底物为10-去乙酰紫杉醇(DT)；
本发明技术方案的第六方面是提供一种酶促反应偶联体系，其特征在于，所述的酶促反应偶联体系是由权利要求1-7任一项的突变体蛋白与糖基水解酶系列蛋白相偶联形成的，所述的糖基水解酶系列蛋白包括克隆自香菇的LXYL-P1蛋白及其一系列活性突变体；所述的偶联形式包括：两种酶在同一反应体系中各自独立存在、或通过连接子形成的融合蛋白形式；优选的糖基水解酶包括LXYL-P1-1(见GenBank Accession:AET31457.1)、LXYL-P1-2(见 GenBank Accession:AET31459.1)、或其系列突变蛋白(即申请号201510268487.6的专利申请中提到的系列突变蛋白)。最优选的糖基水解酶为糖基水解酶LXYL-P1-2系列蛋白，将该突变体蛋白与糖基水解酶LXYL-P1-2系列蛋白相偶联，通过“一锅法”反应，以7-木糖-10-去乙酰紫杉醇(XDT)或其类似物为前体，生物合成紫杉醇或其类似物。本发明还可用于规模化制备紫杉醇中间体巴卡亭III或其类似物。
有益技术效果
本发明利用蛋白质工程对10-去乙酰巴卡亭III 10β-O-乙酰转移酶(DBAT)进行改造，获得13个对于非天然酰基受体底物DT等的催化活性比野生型DBAT有显著提高的突变体蛋白(DBATm系列)，其中的一些突变体蛋白对于天然酰基受体底物10-DAB的催化活性也有显著提高。将这些突变体蛋白与一种糖基水解酶相偶联，以乙酰辅酶A为酰基供体，通过“一锅法”反应，还可以将天然含量较为丰富的7-木糖-10-去乙酰紫杉醇(XDT)直接转变为紫杉醇。本发明可以简化紫杉醇或其类似物的合成步骤，解决紫杉醇或其类似物资源少，合成难的问题。
附图说明
图1重组DABT表达载体构建示意图
图2.DBAT催化天然底物10-DAB及非天然底物DT的HPLC和LC-MS分析
(注：在以DT为底物时DBAT的用量是以10-DAB为底物时的25倍)
图3.DBAT一段氨基酸序列的比对结果
图4.预测的DBAT三维结构(圆内所示为活性中心
内的氨基酸残基)
图5.DBAT突变体全质粒扩增示意图
图6.DBAT-C216R热稳定性测定结果
图7.DBAT-G38R/F301V催化DT的物质浓度-时间曲线
图8.DBAT-G38R/F301V催化DT体系中补加DBAT-G38R/F301V的物质浓度-时间曲线
图9.双酶催化体系中XDT、DT及紫杉醇含量变化情况
具体实施方式
本发明通过下列实施例予以进一步阐明，这些实施例是仅用于说明性的，而不是以任何方式限制本发明权利要求的范围。
实施例1：DBAT原核表达、纯化及催化天然底物10-DAB及非天然底物DT的HPLC-MS分析
人工合成东北红豆杉dbat基因序列(GenBank Accession:Q9M6E2.1)，利用引物F:GAATTCATGCATCATCATCATCATCATGCAGGCTCAAC及引物R: GCGGCCGCTCAAGGCTTAGT进行dbat的基因扩增，同时在DBAT的N-端引入His标签， PCR扩增的片段经Nde I及Xba I进行双酶切后与经同样双酶切的载体连接，转化大肠杆菌 JM109感受态，经菌落PCR筛选阳性转化子JM109-pCWori-dbat，提取阳性转化子的质粒DNA 并进行测序验证。基因dbat cDNA扩增及重组质粒构建过程见图1。
重组菌株的诱导培养：
1)挑取单菌落于含有氨苄青霉素(Amp)的10mL LB(Amp终浓度为100μg/mL)液体培养基中，37℃、200rpm摇瓶培养约12h；
2)将过夜培养的重组菌按1％的比例转接于含有Amp的100mL TB(Amp终浓度为100μg/mL)液体培养基中，37℃、摇瓶培养(200rpm)约2-3h；
3)待OD600≈0.8时，加入IPTG至终浓度为1mmol/L，诱导培养条件：18℃、200r/min、 18h；
4)诱导结束后培养物于8000rpm离心3min，菌体沉淀用ddH2O洗涤2次；得到的菌体沉淀进行超生破碎或-20℃保存备用。
镍亲和层析法纯化目的蛋白：
1)样品准备：将诱导表达后的菌体重悬于破碎缓冲液(同平衡缓冲液，1L菌液收集的菌体沉淀用50mL缓冲液悬浮)中，经高压破碎后(800bar，3次)，在4℃条件下12000rpm 离心30min，上清液用0.45μm滤膜过滤。
2)镍亲和层析柱平衡：2mL镍亲和层析柱经去离子水水洗后，用20mL平衡缓冲液平衡(20mM咪唑、100mM NaCl、20mM Tris-HCl，pH7.5)，流速2mL/min。
3)上样：蛋白样品反复上样5次，流速2mL/min。
4)洗脱：用20mL平衡缓冲液洗脱非特异结合蛋白；用20mL含20mM咪唑的缓冲液洗脱非特异结合蛋白；用20mL含200mM咪唑的缓冲液洗脱目的蛋白。
5)样品浓缩：将得到的目的蛋白洗脱液用截留分子量(Molecular WeightCutoff，MWCO)为30kDa的超滤管在4000g、30min的离心条件下进行浓缩；浓缩后的样品进行蛋白浓度测定。
DBAT催化天然底物10-DAB及非天然底物DT的HPLC-MS分析：
100μL反应体系中包含终浓度为0.02mg/mL(10-DAB测定体系)或0.5mg/mL(DT测定体系)的DBAT、500μM(404.5μg/mL)乙酰辅酶A、500μM底物(相当于10-DAB 272.30 μg/mL或DT 405.94μg/mL)，用pH 5.5的醋酸钠-醋酸缓冲液补齐至100μL，于37.5℃条件下反应12h后加入500μL甲醇终止反应，HPLC-MS检测转化产物(结果见图2)。
实施例2：DBAT蛋白一级序列同源比对及三维结构预测分析
将不同种红豆杉来源的DBAT进行一级序列比对分析(图3)，发现本研究所采用的东北红豆杉(Taxus cuspidata)来源的DBAT中第216位点在不同种属红豆杉中存在差异，该位点在Taxus baccata、Taxus canadensis、Taxus fauna及Taxus globosa等中均为精氨酸(Arg或R)，而在东北红豆杉中为半胱氨酸(Cys或C)。通过对预测的三维结构(图4)分析发现该位点的 Cys在空间上位于蛋白表面，且未与其他Cys形成二硫键，处于游离状态。文献报道蛋白中单独的Cys容易产生自氧化等导致蛋白失稳[Argos P,Rossmann MG,Grau UM,etal.Thermal stability and protein structure.Biochemistry,1979,18(25):5698-5703]；另据统计发现嗜热蛋白中带电荷氨基酸Glu、Arg、Asp、Lys的含量明显高于中温蛋白，而更多的带电残基可以为嗜热蛋白提供更多的盐桥[Kumar S,Tsai CJ,NussinovR.Factors enhancing protein thermostability. Protein Eng,2000,13(3):179-191]。因此，本发明尝试对此位点进行216位点Cys→Arg突变(见实施例3)。
目前，DBAT三维结构未知，为进一步研究DBAT的结构与功能关系，选用与DBAT一致性最高的HCT(GenBank Accession:ABO47805.1，与DBAT的一致性为30％)三维结构为模板，利用蛋白质三维结构在线预测软件Swissmodel(http://swissmodel.expasy.org/)对DBAT结构进行预测，结果见图4。通过对DBAT进行三维结构分析，推测距离DBAT活性中心
内的氨基酸位点(图4圆内表示
范围内的氨基酸)如38、301等可能参与酶与底物的结合或催化。
实施例3：DBAT第38及第301位点饱和突变及组合突变菌株的构建
根据一级序列比对及预测的三维结构分析结果，利用全质粒PCR扩增的方法，以pCWori-dbat为模板分别进行第38及第301位点饱和突变[Parikh A,GuengerichFP.Random mutagenesis by whole-plasmid PCR amplification.Biotechniques,1998,24(3):428-431.]和C216R 定点突变。以pCWori-dbatm-G38R为模板引入F301V突变，构建G38R/F301V组合突变体。以38位饱和突变体重组质粒pCWori-dbat-38X的构建为例，如图5所示。突变体构建所用引物序列如下：
表1.突变体构建所用引物序列
PCR扩增体系如下：
PCR扩增条件：
PCR产物以1.0％的琼脂糖凝胶电泳检测，纯化回收PCR产物。
PCR产物于37℃用Dpn I酶切处理5h。酶切体系如下：
转化及筛选：将酶切产物全部转化大肠杆菌JM109感受态细胞。采用菌落PCR方法进行阳性转化子的筛选并进行DNA序列测定。
实施例4：DBAT突变体蛋白催化非天然底物DT比活力测定
反应体系中DBATm的终浓度为0.5mg/mL，DT及乙酰辅酶A终浓度均为500μM，溶于pH5.5的醋酸钠-醋酸缓冲液中(共100μL)，37.5℃条件下反应3h后加入500μL甲醇终止反应，HPLC检测产物紫杉醇生成量。酶活力单位(U)定义为：在37.5℃、pH 5.5、以 DT为底物的条件下，每分钟产生1μmoL紫杉醇所需要的酶量。根据紫杉醇浓度-峰面积标准曲线，计算出酶反应体系中紫杉醇产生量，根据测得的蛋白质量浓度(mg/mL)，求出单位为 U/mg的比活力。表2为经过筛选获得的催化DT活性或催化特性有显著改善的突变体(以DBAT 为对照)，其中DBAT-G38R/F301V双突变的比活力是对照(DBAT)的3.7倍。
表2 DBAT及其突变体(DBATm)催化DT的比活力与相对酶活性
n＝3，*P<0.05vs DBAT，**P<0.01vs DBAT.
实施例5：DBAT突变体蛋白催化天然底物10-DAB比活力测定
反应体系中DBAT或突变体蛋白终浓度为0.02mg/mL，10-DAB及乙酰辅酶A终浓度均为500μM，溶于pH 5.5的醋酸钠-醋酸缓冲液中(共100μL)，40℃条件下反应20min后加入500μL甲醇终止反应，用HPLC检测产物生成量。催化10-DAB的酶活力单位(U)定义为：在40℃，pH5.5，以10-DAB为底物的条件下，每分钟产生1μmoL巴卡亭III所需要的酶量。根据巴卡亭III浓度-峰面积标准曲线，计算出酶反应体系中巴卡亭III生成量；根据测得的酶蛋白质量浓度(mg/mL)，求出单位为U/mg的比活力。表3为经过筛选获得的催化 10-DAB活性有显著提高的突变体。
表3.DBAT及其突变体(DBATm)催化10-DAB活性测定结果
n＝3，*P<0.05vs DBAT，**P<0.01vs DBAT.注：反应温度45℃
实施例6：DBAT-C216R突变体蛋白热稳定性及最适催化温度分析
将重组DBAT及突变体DBAT-C216R蛋白用pH 5.5的缓冲液稀释至0.1mg/mL，置于37℃静置12h，每隔1h检测蛋白残留活性，活性检测方法同实施例5，结果见图6。结果显示野生型DBAT酶的半衰期为1.7h，突变体DBAT-C216R的热稳定性显著增强，半衰期延长至4.5 h。
突变体催化10-DAB及DT的最适温度分析：酶催化体系分别同实施例5和实施例6。反应温度分别为25、30、35、40、45和50℃。以野生型DBAT为对照。结果显示DBAT-C216R 催化10-DAB及DT的最适温度分别为45℃和40℃，比突变前分别增加约5℃。
实施例7：DBAT-G38R/F301V催化体系中底物DT与产物紫杉醇的时间-浓度变化曲线
⑴催化体系中不补加DBAT-G38R/F301V
催化体系组成：DBAT-G38R/F301V 1.5mg/mL，DT及乙酰辅酶A浓度均为2mM，DMSO(5％ V/V)，pH 5.5醋酸-醋酸钠缓冲液补齐至1mL。
反应条件：37.5℃，分别于3h、6h、9h、12h及15h分别检测DT转化情况。
结果见图7，结果显示：反应6h后趋于平衡，紫杉醇产量最高为452.09±2.52μg/mL。
⑵催化体系中补加DBAT-G38R/F301V
催化体系组成：DBAT-G38R/F301V 1.5mg/mL，DT及乙酰辅酶A浓度均为2mM，DMSO(5％ V/V)，pH 5.5醋酸-醋酸钠缓冲液补齐至1mL，分别于3h、6h、9h补加DBAT-G38R/F301V150μL(酶溶液10mg/mL)。
反应条件：37.5℃，分别于3h、6h、9h、12h及15h分别检测DT转化情况。
结果见图8，结果显示：反应12h后趋于平衡，反应15h时，紫杉醇产量达到640.76±5.05μg/mL。
实施例8：LXYL-P1-2及DBAT突变体偶联反应催化XDT为紫杉醇(显示前体XDT、中间体DT和产物紫杉醇的时间-浓度变化曲线)
所用酶溶液及底物母液：LXYL-P1-2 5mg/mL，DBAT-G38R/F301V 10mg/mL，乙酰辅酶 A 100mM，XDT 100mM；反应体积为10mL。
催化体系组成：LXYL-P1-2 1mL，DBAT-G38R/F301V 1.5mL，XDT 200μL，乙酰辅酶A200 μL，DMSO 500μL，pH 5.5醋酸钠-醋酸缓冲液6.6mL。
分别于3h、6h、9h补加DBAT-G38R/F301V 1.5mL。
反应条件：37.5℃，分别于3h、6h、9h、12h及15h分别检测各物质浓度。
结果见图9。结果显示：反应12h后趋于平衡，反应15h时，紫杉醇产量达到637.24±5.10 μg/mL。
<110> 中国医学科学院药物研究所
<120> 10-去乙酰巴卡亭Ⅲ 10β-O-乙酰转移酶突变体及其在催化合成紫杉醇及其类似物中的应用
<210> SEQ ID NO 1 DBAT
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工合成序列
<400> SEQUENCE: 1
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 2 DBAT-G38R
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 2
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Arg Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 3 DBAT-G38W
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 3
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Trp Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 4 DBAT-G38Y
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 4
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Tyr Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 5 DBAT-G38I
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 5
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Ile Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 6 DBAT-G38T
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 6
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Thr Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 7 DBAT-G38E
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 7
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Glu Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 8 DBAT-G38M
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 8
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Met Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 9 DBAT-G38Q
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 9
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gln Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 10 DBAT-G38C
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 10
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Cys Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 11 DBAT-G38S
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 11
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Ser Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 12 DBAT-G38D
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 12
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Asp Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 13 DBAT-G38H
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 13
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro His Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 14 DBAT-G38A
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 14
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Ala Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 15 DBAT-F301C
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 15
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Cys Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 16 DBAT-F301V
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 16
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Arg Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Val Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 17 DBAT-F301A
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 17
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Ala Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 18 DBAT-F301M
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 18
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Met Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 19 DBAT-F301L
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 19
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Leu Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 20 DBAT-F301T
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 20
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Thr Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 21 DBAT-F301S
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 21
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Ser Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 22 DBAT-C216R
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 22
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Arg Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 23 DBAT-G38R/F301V
<211> LENGTH: 440
<212> TYPE: PRT
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 23
Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met Val Ala Pro Ser Gln Pro
5 10 15 20
Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr Leu Asp Asn Leu Pro Arg Val Arg Glu Asn Ile Phe
25 30 35 40
Asn Thr Leu Leu Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile Arg Gln
45 50 55 60 65
Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly Arg Leu Arg Lys Lys Glu Asn Gly
70 75 80 85
Asp Leu Glu Val Glu Cys Thr Gly Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu
90 95 100 105 110
Ser Val Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu Leu Phe Cys Leu Pro Pro
115 120 125 130
Asp Thr Asp Ile Glu Asp Ile His Pro Leu Val Val Gln Val Thr Arg Phe Thr Cys Gly Gly Phe
135 140 145 150
Val Val Gly Val Ser Phe Cys His Gly Ile Cys Asp Gly Leu Gly Ala Gly Gln Phe Leu Ile Ala
155 160 165 170 175
Met Gly Glu Met Ala Arg Gly Glu Ile Lys Pro Ser Ser Glu Pro Ile Trp Lys Arg Glu Leu Leu
180 185 190 195
Lys Pro Glu Asp Pro Leu Tyr Arg Phe Gln Tyr Tyr His Phe Gln Leu Ile Cys Pro Pro Ser Thr
200 205 210 215 220
Phe Gly Lys Ile Val Gln Gly Ser Leu Val Ile Thr Ser Glu Thr Ile Asn Cys Ile Lys Gln Cys
225 230 235 240
Leu Arg Glu Glu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val Val Ser Ala Leu Ala Trp Ile Ala
245 250 255 260
Arg Thr Arg Ala Leu Gln Ile Pro His Ser Glu Asn Val Lys Leu Ile Phe Ala Met Asp Met Arg
265 270 275 280 285
Lys Leu Phe Asn Pro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Val Val Gly Thr Val Cys Ala Met
290 295 300 305
Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu Arg Val Val Arg Ile Ile Lys Lys Ala Lys
310 315 320 325 330
Val Ser Leu Asn Glu His Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile
335 340 345 350
Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu Gly Phe Asp Glu Val Asp Phe Gly
355 360 365 370
Trp Gly His Ala Asp Asn Val Ser Leu Val Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser
375 380 385 390 395
Tyr Phe Leu Phe Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly Ile Lys Ile Leu Ser Phe Met Pro
400 405 410 415
Pro Ser Ile Val Lys Ser Phe Lys Phe Glu Met Glu Thr Met Thr Asn Lys Tyr Val Thr Lys Pro
420 425 430 435 440
<210> SEQ ID NO 24 DBAT
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工合成序列
<400> SEQUENCE: 24
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 25 DBAT-G38R
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 25
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc acgggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 26 DBAT-G38W
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 26
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc atgggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 27 DBAT-G38Y
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 27
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc atatgtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 28 DBAT-G38I
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 28
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aattgtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 29 DBAT-G38T
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 29
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aacggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 30 DBAT-G38E
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 30
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc agaggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 31 DBAT-G38M
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 31
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aatggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 32 DBAT-G38Q
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 32
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc acaggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 33 DBAT-G38C
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 33
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc atgtgtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 34 DBAT-G38S
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 34
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc atcggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 35 DBAT-G38D
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 35
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc agatgtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 36 DBAT-G38H
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 36
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc acatgtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 37 DBAT-G38A
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 37
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc agcggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 38 DBAT-F301C
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 38
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tgtgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 39 DBAT-F301V
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 39
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat gttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 40 DBAT-F301A
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 40
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat gctgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 41 DBAT-F301M
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 41
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat atggttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 42 DBAT-F301L
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 42
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat cttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 43 DBAT-F301T
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 43
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat actgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 44 DBAT-F301S
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 44
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat agtgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 45 DBAT-C216R
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 45
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc aggggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgattcgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat tttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
<210> SEQ ID NO 46 DBAT-G38R/F301V
<211> LENGTH: 1321
<212> TYPE: DNA
<213> ORGANISM: Taxus cuspidata人工突变序列
<400> SEQUENCE: 46
atggcaggct caacagaatt tgtggtaaga agcttagaga gagtgatggt ggctccaagc cagccatcgc 70
ccaaagcttt cctgcagctc tccacccttg acaatctacc acgggtgaga gaaaacattt ttaacacctt 140
gttagtctac aatgcctcag acagagtttc cgtagatcct gcaaaagtaa ttcggcaggc tctctccaag 210
gtgttggtgt actattcccc ttttgcaggg cgtctcagga aaaaagaaaa tggagatctt gaagtggagt 280
gcacagggga gggtgctctg tttgtggaag ccatggctga cactgacctc tcagtcttag gagatttgga 350
tgactacagt ccttcacttg agcaactact tttttgtctt ccgcctgata cagatattga ggacatccat 420
cctctggtgg ttcaggtaac tcgttttaca tgtggaggtt ttgttgtagg ggtgagtttc tgccatggta 490
tatgtgatgg actaggagca ggccagtttc ttatagccat gggagagatg gcaaggggag agattaagcc 560
ctcctcggag ccaatatgga agagagaatt gctgaagccg gaagaccctt tataccggtt ccagtattat 630
cactttcaat tgatttgccc gccttcaaca ttcgggaaaa tagttcaagg atctcttgtt ataacctctg 700
agacaataaa ttgtatcaaa caatgcctta gggaagaaag taaagaattt tgctctgcgt tcgaagttgt 770
atctgcattg gcttggatag caaggacaag ggctcttcaa attccacata gtgagaatgt gaagcttatt 840
tttgcaatgg acatgagaaa attatttaat ccaccacttt cgaagggata ctacggtaat gttgttggta 910
ccgtatgtgc aatggataat gtcaaggacc tattaagtgg atctcttttg cgtgttgtaa ggattataaa 980
gaaagcaaag gtctctttaa atgagcattt cacgtcaaca atcgtgacac cccgttctgg atcagatgag 1050
agtatcaatt atgaaaacat agttggattt ggtgatcgaa ggcgattggg atttgatgaa gtagactttg 1120
ggtgggggca tgcagataat gtaagtctcg tgcaacatgg attgaaggat gtttcagtcg tgcaaagtta 1190
ttttcttttc atacgacctc ccaagaataa ccccgatgga atcaagatcc tatcgttcat gcccccgtca 1260
atagtgaaat ccttcaaatt tgaaatggaa accatgacaa acaaatatgt aactaagcct tga
Claims (12)
1.一种10-去乙酰巴卡亭III 10β-O-乙酰转移酶DBAT的突变体蛋白，其特征在于，与氨基酸序列如SEQ ID NO 1所示的野生型蛋白DBAT相比，所述的突变体蛋白在301位氨基酸处具有选自下组中的一种突变：F301V、F301C、F301A、F301M、F301L、F301T、F301S。
2.根据权利要求1的突变体蛋白，其特征在于，所述的突变体蛋白具有F301V氨基酸突变。
3.根据权利要求1或2的突变体蛋白，其特征在于，所述的突变体蛋白与野生型蛋白DBAT相比，其氨基酸突变还包括：G38W、G38Y、G38I、G38T、G38E、G38M、G38Q、G38C、G38S、G38D、G38H、G38A、C216R，以及以上氨基酸突变的组合。
4.根据权利要求1-3任一项的突变体蛋白，其特征在于，所述的突变体蛋白的氨基酸序列选自SEQ ID NO 3～SEQ ID NO 14所示的氨基酸序列。
5.一种编码权利要求1-4任一项所述突变体蛋白的核苷酸序列。
6.根据权利要求6的核苷酸序列，其特征在于，所述的核苷酸序列选自SEQ ID NO 26～SEQ ID NO 37所示的核苷酸序列。
7.一种含有权利要求5-6任一项所述的核苷酸序列的重组质粒。
8.一种含有权利要求5-6任一项所述的核苷酸序列或权利要求7所述的重组质粒的重组细胞。
9.权利要求1-4任一项所述的突变体蛋白或权利要求5-6任一项所述的核苷酸序列或权利要求7所述的重组质粒或权利要求8所述的重组细胞在催化10-去乙酰紫杉醇及其类似物的C10位羟基酰基化生成紫杉醇或其类似物中的应用。
10.根据权利要求9的应用，其特征在于，所述的突变体蛋白可以与能够专一性水解7-木糖-10-去乙酰紫杉烷的糖基水解酶相偶联，以7-木糖-10-去乙酰紫杉烷为底物，以酰基辅酶A为酰基供体生成紫杉醇或其类似物。
11.根据权利要求10的应用，其特征在于，所述的酰基受体包括10-去乙酰紫杉醇、10-去乙酰巴卡亭III；所述的酰基供体包括乙酰辅酶A、丙酰辅酶A和丁酰辅酶A。
12.一种酶促反应偶联体系，其特征在于，所述的酶促反应偶联体系是由权利要求1-4任一项的突变体蛋白与糖基水解酶系列蛋白相偶联形成的，所述的糖基水解酶系列蛋白包括克隆自香菇的LXYL-P1蛋白及其一系列活性突变体；所述的偶联形式包括：两种酶在同一反应体系中各自独立存在、或通过连接子形成的融合蛋白形式；优选的糖基水解酶包括LXYL-P1-1或LXYL-P1-2或突变体蛋白。
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