CN114250219B

CN114250219B - 一种香树脂醇合酶突变体及其应用

Info

Publication number: CN114250219B
Application number: CN202210011919.5A
Authority: CN
Inventors: 于爱群; 孔婧; 苗琳; 王淑慧; 卢志慧; 赵栢祥; 肖冬光; 张翠英
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-01-06
Filing date: 2022-01-06
Publication date: 2023-02-03
Anticipated expiration: 2042-01-06
Also published as: CN114250219A

Abstract

本发明涉及一种定点突变改造的香树脂醇合酶突变体及其应用，属于酶工程技术领域。所述突变体是将SEQ ID NO.1所示氨基酸序列第323位的亮氨酸替换为丙氨酸；或第328位的亮氨酸替换为丙氨酸；或第331位的苯丙氨酸替换为丙氨酸；或第341位的半胱氨酸替换为丙氨酸。本发明构建的香树脂醇合酶突变体通过在解脂耶氏酵母中表达，与出发菌株即未突变菌株相比α‑香树脂醇和β‑香树脂醇的产量均有所提升，其中α‑香树脂醇的产量最多提升12.38倍，β‑香树脂醇的产量最多提产7.99倍。改造后的基因工程菌株，更适合工业应用，可降低生产成本，提高生产效率。

Description

一种香树脂醇合酶突变体及其应用

技术领域：

本发明涉及一种定点突变改造的香树脂醇合酶突变体及其应用，属于酶工程技术领域。

背景技术：

香树脂醇(C₃₀H₅₀O)是一类五环三萜化合物，是植物中丰富的次生代谢产物之一。在自然界中，香树脂醇是以游离或成酯形式存在于植物中的，并有α-香树脂醇和β-香树脂醇两种同分异构体。他们具有广泛的药理活性以及药用价值，包括抗炎、抗菌、抗癌、保护肝脏、提高免疫力等，市场应用价值极高。

本发明的香树脂醇合酶(CrMAS)基因从长春花中获取，通过分子模拟和分子对接预测其有五个关键结合口袋，选择结合力最高的口袋进行后续研究。香树脂醇合酶可以和底物2,3-氧化角鲨烯通过疏水作用结合并同时产生ɑ-香树脂醇和β-香树脂醇。

定点突变是指通过聚合酶链式反应(PCR)的方法向目的DNA片段(质粒或基因组)中引入所需变化，包括碱基的添加、删除、点突变等。定点突变能迅速、高效地提高DNA所表达的目的蛋白的性状及表征，是基因工程研究工作中一种非常有用的手段，也是研究蛋白质结构和功能之间复杂关系的有力工具。

发明内容：

本发明提供的技术方案之一，是一种香树脂醇合酶突变体，所述的香树脂醇合酶突变体，是在SEQ ID NO.1所示的野生型香树脂醇合酶基础上，发生如下突变中的至少一种获得的：

(1)将SEQ ID NO.1所示氨基酸序列第323位的亮氨酸替换为丙氨酸；或

(2)将SEQ ID NO.1所示氨基酸序列第328位的亮氨酸替换为丙氨酸；或

(3)将SEQ ID NO.1所示氨基酸序列第331位的苯丙氨酸替换为丙氨酸；或

(4)将SEQ ID NO.1所示氨基酸序列第341位的半胱氨酸替换为丙氨酸。

进一步地，将氨基酸序列如SEQ ID NO.1所示的香树脂醇合酶的第323位亮氨酸替换为丙氨酸时，突变体的氨基酸序列如SEQ ID NO.2所示。

进一步地，将氨基酸序列如SEQ ID NO.1所示的香树脂醇合酶的第328位亮氨酸替换为丙氨酸时，突变体的氨基酸序列如SEQ ID NO.3所示。

进一步地，将氨基酸序列如SEQ ID NO.1所示的香树脂醇合酶的第331位苯丙氨酸替换为丙氨酸时，突变体的氨基酸序列如SEQ ID NO.4所示。

进一步地，将氨基酸序列如SEQ ID NO.1所示的香树脂醇合酶的第341位半胱氨酸替换为丙氨酸时，突变体的氨基酸序列如SEQ ID NO.5所示。

本发明提供的技术方案之二，是编码上述香树脂醇合酶突变体的基因；

进一步地，所述香树脂醇合酶突变体具有SEQ ID NO.7、SEQ ID NO.8、SEQ IDNO.9或SEQ ID NO.10任一所示的编码基因。

本发明提供的技术方案之三，是携带上述基因的重组质粒；

进一步地，所述重组质粒采用的表达质粒包括但不限于pYLEX1、pUG6等。

本发明提供的技术方案之四，是表达上述突变体的重组菌；

进一步地，所述重组菌采用的宿主细胞包括但不限于解脂耶氏酵母、酿酒酵母、乳酸克鲁维酵母、大肠杆菌等；

更进一步地，所述重组菌采用的宿主细胞为解脂耶氏酵母Po1gΔKU70；

优选地，所述重组菌以解脂耶氏酵母Po1gΔKU70为宿主，采用pYLEX1质粒对SEQID NO.7-10中的任一基因进行表达。

本发明提供的技术方案之五，是上述重组菌在生产香树脂醇中的应用。

本发明的有益效果：

本发明通过分子对接模拟确定香树脂醇合酶和底物2,3-氧化角鲨烯结合口袋和关键氨基酸残基，并通过定点突变改变蛋白质分子与底物结合位点的氨基酸残基，提高香树脂醇合酶催化效率，进一步提高香树脂醇的产量。同时本发明第一次将CrMAS基因在解脂耶氏酵母中进行异源表达。本发明构建的香树脂醇合酶突变体通过在解脂耶氏酵母中表达，与出发菌株即未突变菌株相比α-香树脂醇和β-香树脂醇的产量均有所提升，其中α-香树脂醇的产量最多提升12.38倍，β-香树脂醇的产量最多提产7.99倍。改造后的基因工程菌株，更适合工业应用，可降低生产成本，提高生产效率。

附图说明：

图1为香树脂醇合酶和2,3-氧化角鲨烯结合示意图；

图2为香树脂醇合酶和2,3-氧化角鲨烯在site1位点结合示意图；

图3为香树脂醇合酶突变质粒核酸凝胶电泳验证图

M，marker；1～5分别为：pYLCrMAS，pYLC323，pYLC328，pYLC331，pYLC341；

图4含有突变前、后香树脂醇合酶的阳性转化子的核酸凝胶电泳验证图

M，marker；1～5分别为：Po1g KCrMAS，Po1g KC323A，Po1g KC328A，Po1g KC331A，Po1g KC341A；

图5为突变后香树脂醇产量变化示意图

未突变菌株代表Po1g KCrMAS菌株、323代表Po1g KC323A菌株、328代表Po1gKC328A菌株、331代表Po1g KC331A菌株、341代表Po1g KC341A菌株。

具体实施方式：

下面结合实施例对本发明的技术内容做进一步说明。需要说明的是，在不冲突的情况下，本发明中的实施例仅为本发明中的一部分实施例，而不是全部的实施例。基于本发明的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的其他实施例，都属于本发明保护范围之内。

在本发明中采用如下定义：

1.氨基酸和DNA核酸序列的命名法

使用氨基酸残基的公认IUPAC命名法，用三字母代码形式。DNA核酸序列采用公认IUPAC命名法。

2.香树脂醇合酶突变体的标识

采用“原始氨基酸位置替换的氨基酸”来表示CrMAS突变体中突变的氨基酸。如Leu323Ala，表示位置323的氨基酸由野生型的Leu替换成Ala，位置的编号对应于SEQ IDNo.1中野生型CrMAS的氨基酸序列编号。

在本发明中，CrMAS代表野生型香树脂醇合酶，Leu323Ala、Leu328Ala、Phe331Ala、Cys341Ala分别代表四种突变体，具体信息如下表。

香树脂醇合酶	氨基酸突变位点	氨基酸SEQ ID NO.	核苷酸SEQ ID NO.
				CrMAS	—	1	6
Leu323Ala	Leu323Ala	2	7
				Leu328Ala	Leu328Ala	3	8
Phe331Ala	Phe331Ala	4	9
				Cys341Ala	Cys341Ala	5	10

以下将结合附图和实施例对本发明作进一步地解释说明。

实施例1长春花来源香树脂醇合酶结构模拟和2,3-氧化角鲨烯结合口袋分子模拟

以香树脂醇合酶基因(GenBank:AFJ19235)为模板，使用

软件ProteinPreparation Wizard Panel模块导入CrMAS蛋白三维结构，采用

软件Sitemap模块对CrMAS蛋白上的结合口袋进行预测，如图1，从对接得分来看，一共有五处结合良好的位点，其中2,3-氧化角鲨烯与Site 1的结合力最强，细节图如图2，故选择该口袋进行定点突变。

实施例2香树脂醇合酶菌株和定点突变菌株的获得

1)根据已知的香树脂醇合酶基因经密码子优化后(SEQ ID NO.6)，将其导入pYLEX1质粒中，构建获得含有香树脂醇合酶基因的重组质粒pYLEX1-CrMAS。香树脂醇合酶基因通过基因合成获取，重组质粒的构建采用本领域的常用方法即可。

2)采用全质粒定点突变的方法获得香树脂醇合酶突变体基因。以pYLEX1-CrMAS质粒为模板，利用Primer Premier 5.0进行引物设计，设计引物序列如下表：

3)PCR扩增目的基因：

反应体系为：(采用Q5定点突变试剂盒)

组分	体积
		ddH<sub>2</sub>O	8.5μL
pYLEX1-CrMAS	0.5μL
		上游引物	0.35μL
下游引物	0.35μL
		KOD	0.25μL
dNTP	1.25μL
		10×BufferiPCR	1.25μL

反应程序为：

PCR产物加入0.25μL的DpnⅠ消化去除甲基化，然后转化E.coli DH5α菌株，培养12小时候挑取单菌落，过夜培养后提质粒并通过DNA测序验证是否成功将香树脂醇合酶的第323亮氨酸、328亮氨酸、331苯丙氨酸、341位半胱氨酸分别替换为丙氨酸，经PCR和测序验证成功得到突变质粒pYLC323，pYLC328，pYLC331，pYLC341(验证图如图3)。

其中，突变质粒pYLC323上含有Leu323Ala突变体编码基因；突变质粒pYLC328上含有Leu328Ala突变体编码基因；突变质粒pYLC331上含有Phe331Ala突变体编码基因；突变质粒pYLC341上含有Cys341Ala突变体编码基因。

4)将原始质粒pYLEX1-CrMAS和测序正确的突变质粒pYLC323，pYLC328，pYLC331，pYLC341分别转化Po1gΔKU70菌株感受态细胞，待亮氨酸缺陷板上长出可见菌落后，随机挑取单个菌落，过夜培养后提基因组进行PCR验证。经验证，如图4所示，成功得到Po1gKCrMAS菌株和突变菌株Po1g KC323A，Po1g KC328A，Po1g KC331A，Po1g KC341A。

其中，突变菌株Po1g KC323A含有突变质粒pYLC323；突变菌株Po1g KC328A含有突变质粒pYLC328；突变菌株Po1g KC331A突变质粒pYLC331；突变菌株Po1g KC341A含有突变质粒pYLC341。

所述pYLEX1质粒购于Yeastern Biotech Co.,Ltd.，所述质粒携带有营养缺陷筛选基因亮氨酸表达盒、标记基因Amp、启动子hp4d以及终止子XPR2 term。

所述解脂耶氏酵母Po1gΔKU70是在解脂耶氏酵母Po1g菌株中敲除KU70基因获得的，其构建方法参考下述文献：Genetic engineering of an unconventional yeast forrenewable biofuel and biochemical production.Journal of VisualizedExperiments,2016,115,e54371。

实施例3香树脂醇合酶定点突变菌株对香树脂醇产量的影响

1)将实施例2构建的菌株：Po1g KCrMAS菌株和突变菌株Po1g KC323A，Po1gKC328A，Po1g KC331A，Po1g KC341A，分别经两次活化后按照接种量1％接种于YPD培养基中，于30℃，250r/min，摇瓶发酵5天。

2)所述基因工程菌的活化步骤为：取斜面菌体一环，接种于含25mL种子培养基中，30℃，225r/min，振荡培养24h后(完成一次活化)，以1％的接种量接种于25mL种子培养基，30℃，225r/min，继续振荡培养16h(完成二次活化)。

种子培养基为YPD培养基，具体组成为：蛋白胨20g/L，酵母浸粉10g/L，葡萄糖20g/L，余量为水，pH5.7-5.8，115℃，灭菌20min。

3)发酵结束后，取10mL菌液，6000r/min，4℃离心5min，弃废液，通过将发酵菌体加等量石英砂和正己烷涡旋震荡20min以萃取香树脂醇。

4)将震荡后的菌体、石英砂和正己烷混合物6000r/min，4℃离心5min，取上层有机相过有机膜，气相色谱-质谱联用测定胞内中的香树脂醇含量。

其中，上述气相色谱-质谱联用检测条件为：

色谱柱HP-5MS(30m×0.25mm×0.25μm，岛津)，载气：高纯氦气，流速为1mL/min，进样口温度280℃，升温程序：100℃，10℃/min升到280℃保持20min，溶剂延迟3min，进样量为1μL。

5)经过气相色谱检测，具有不同突变位点的香树脂醇合酶的重组菌催化合成香树脂醇的产量变化如下表及图5：

菌株名	α-香树脂醇(mg/L)	β-香树脂醇(mg/L)
			Po1g ΔKU70	0	0
Po1g KCrMAS	2.391	1.424
			Po1g KC323A	29.602	8.280
Po1g KC328A	10.348	11.379
			Po1g KC331A	7.206	6.022
Po1g KC341A	17.375	10.546

经测定，突变后的香树脂醇合酶在不同程度上催化合成α-香树脂醇和β-香树脂醇的产量均有提高，说明了突变后酶活和与底物的结合能力有一定程度的提高。其中Po1gKC323A对α-香树脂醇作用最大，提高产量达12.38倍，Po1g KC328A对β-香树脂醇作用最大，提高产量达7.99倍。同时和解脂耶氏酵母出发菌株Po1gΔKU70相比，基因工程菌Po1gKC323A、Po1g KC328A、Po1g KC331A、Po1g KC341A菌株在菌种的其他正常功能上未受明显影响。

以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

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725 730 735

Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr

740 745 750

Arg Lys Arg Val Trp Pro Thr Lys Ala Leu

755 760

<210> 2

<211> 762

<212> PRT

<213> 人工序列

<400> 2

Met Trp Lys Leu Lys Ile Ala Lys Gly Lys Gly Pro Tyr Leu Tyr Ser

1 5 10 15

Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Tyr Asp Pro Asn Ala

20 25 30

Gly Thr Pro Gln Glu Arg Glu Ala Phe Glu Lys Ala Arg Glu Gln Phe

35 40 45

Arg Asn Asn Arg Lys Lys Gly Val His Asn Pro Cys Ala Asp Leu Phe

50 55 60

Met Arg Met Gln Leu Ile Lys Glu Asn Gly Ile Asp Leu Met Ser Ile

65 70 75 80

Pro Pro Val Arg Val Glu Glu Lys Glu Glu Leu Thr Phe Glu Lys Thr

85 90 95

Thr Ile Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala

100 105 110

Thr Asp Gly His Trp Pro Ala Glu Asn Ala Gly Pro Met Phe Phe Thr

115 120 125

Pro Pro Leu Leu Ile Ala Leu Tyr Ile Ser Gly Ala Ile Asn Thr Ile

130 135 140

Leu Thr Ser Glu His Lys Lys Glu Leu Val Arg Tyr Ile Tyr Asn His

145 150 155 160

Gln Asn Glu Asp Gly Gly Trp Gly Phe Tyr Ile Glu Gly His Ser Thr

165 170 175

Met Ile Gly Ser Ala Leu Ser Tyr Val Ala Leu Arg Leu Leu Gly Glu

180 185 190

Gly Pro Asp Asp Gly Asp Gly Ala Val Gly Arg Gly Arg Gln Trp Ile

195 200 205

Leu Asp His Gly Gly Ala Thr Gly Ile Pro Ser Trp Gly Lys Thr Tyr

210 215 220

Leu Ser Val Leu Gly Val Tyr Asp Trp Asp Gly Cys Asn Pro Leu Pro

225 230 235 240

Pro Glu Phe Trp Leu Phe Pro Ser Phe Phe Pro Tyr His Pro Ala Lys

245 250 255

Met Trp Cys Tyr Cys Arg Thr Thr Tyr Met Pro Met Ser Tyr Leu Tyr

260 265 270

Gly Lys Lys Tyr His Gly Pro Leu Thr His Leu Val Met Gln Leu Arg

275 280 285

Gln Glu Ile His Val Lys Pro Tyr Asp Gln Ile Asp Trp Asn Lys Ala

290 295 300

Arg His Asp Cys Cys Lys Asp Asp Leu Tyr Tyr Pro His Ser Phe Ile

305 310 315 320

Gln Asp Ala Leu Trp Asp Thr Leu Asn Tyr Phe Ser Glu Pro Val Met

325 330 335

Arg Arg Trp Pro Cys Asn Lys Ile Arg Glu Lys Ala Met Arg Lys Cys

340 345 350

Ile Lys Tyr Met Arg Tyr Gly Ala Glu Glu Ser Arg Tyr Ile Thr Ile

355 360 365

Gly Cys Val Glu Lys Ser Leu Gln Met Met Cys Trp Cys Ala His Asp

370 375 380

Pro Asn Cys Asp Glu Phe Lys Tyr His Leu Ala Arg Val Pro Asp Tyr

385 390 395 400

Leu Trp Leu Ala Glu Asp Gly Met Lys Met Gln Ser Phe Gly Ser Gln

405 410 415

Leu Trp Asp Cys Thr Leu Ala Thr Gln Ala Ile Ile Ala Thr Gly Met

420 425 430

Val Glu Glu Tyr Gly Asp Thr Ile Lys Lys Ala His Phe Tyr Ile Lys

435 440 445

Glu Ser Gln Val Lys Glu Asn Pro Lys Glu Asp Phe Lys Ala Met Tyr

450 455 460

Arg His Phe Thr Lys Gly Ser Trp Thr Phe Ser Asp Gln Asp Gln Gly

465 470 475 480

Trp Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys Leu Leu Val

485 490 495

Cys Ser Gln Met Pro Gln Asp Leu Ala Gly Glu Lys Ala Asp Val Glu

500 505 510

Arg Leu Tyr Asp Ala Val Asn Val Leu Leu Tyr Leu Gln Ser Pro Glu

515 520 525

Ser Gly Gly Phe Ala Ile Trp Glu Pro Pro Val Pro Gln Pro Tyr Leu

530 535 540

Gln Val Leu Asn Pro Ser Glu Leu Phe Ala Asp Ile Val Val Glu Gln

545 550 555 560

Glu His Val Glu Asn Thr Ala Ser Ile Val Gln Ala Leu Val Leu Phe

565 570 575

Lys Arg Leu His Pro Gly His Arg Glu Lys Glu Ile Glu Val Ser Val

580 585 590

Ser Lys Ala Val Arg Phe Leu Glu Gly Arg Gln Trp Pro Asp Gly Ser

595 600 605

Trp Tyr Gly Tyr Trp Gly Ile Cys Phe Leu Tyr Gly Thr Met Phe Val

610 615 620

Leu Gly Gly Leu Thr Ala Ala Gly Lys Thr Tyr Lys Asn Ser Glu Ala

625 630 635 640

Ile Arg Lys Ala Val Lys Phe Tyr Leu Ser Thr Gln Asn Glu Glu Gly

645 650 655

Gly Trp Gly Glu Cys Leu Glu Ser Cys Pro Ser Met Lys Tyr Ile Pro

660 665 670

Leu Glu Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp Ala Met Leu

675 680 685

Gly Leu Met Tyr Gly Gly Gln Ala Glu Arg Asp Pro Thr Pro Leu His

690 695 700

Lys Ala Ala Lys Leu Leu Ile Asn Ala Gln Met Asp Asp Gly Asp Phe

705 710 715 720

Pro Gln Gln Glu Ile Thr Gly Val Tyr Met Lys Asn Cys Met Leu His

725 730 735

Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr

740 745 750

Arg Lys Arg Val Trp Pro Thr Lys Ala Leu

755 760

<210> 3

<211> 762

<212> PRT

<213> 人工序列

<400> 3

Met Trp Lys Leu Lys Ile Ala Lys Gly Lys Gly Pro Tyr Leu Tyr Ser

1 5 10 15

Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Tyr Asp Pro Asn Ala

20 25 30

Gly Thr Pro Gln Glu Arg Glu Ala Phe Glu Lys Ala Arg Glu Gln Phe

35 40 45

Arg Asn Asn Arg Lys Lys Gly Val His Asn Pro Cys Ala Asp Leu Phe

50 55 60

Met Arg Met Gln Leu Ile Lys Glu Asn Gly Ile Asp Leu Met Ser Ile

65 70 75 80

Pro Pro Val Arg Val Glu Glu Lys Glu Glu Leu Thr Phe Glu Lys Thr

85 90 95

Thr Ile Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala

100 105 110

Thr Asp Gly His Trp Pro Ala Glu Asn Ala Gly Pro Met Phe Phe Thr

115 120 125

Pro Pro Leu Leu Ile Ala Leu Tyr Ile Ser Gly Ala Ile Asn Thr Ile

130 135 140

Leu Thr Ser Glu His Lys Lys Glu Leu Val Arg Tyr Ile Tyr Asn His

145 150 155 160

Gln Asn Glu Asp Gly Gly Trp Gly Phe Tyr Ile Glu Gly His Ser Thr

165 170 175

Met Ile Gly Ser Ala Leu Ser Tyr Val Ala Leu Arg Leu Leu Gly Glu

180 185 190

Gly Pro Asp Asp Gly Asp Gly Ala Val Gly Arg Gly Arg Gln Trp Ile

195 200 205

Leu Asp His Gly Gly Ala Thr Gly Ile Pro Ser Trp Gly Lys Thr Tyr

210 215 220

Leu Ser Val Leu Gly Val Tyr Asp Trp Asp Gly Cys Asn Pro Leu Pro

225 230 235 240

Pro Glu Phe Trp Leu Phe Pro Ser Phe Phe Pro Tyr His Pro Ala Lys

245 250 255

Met Trp Cys Tyr Cys Arg Thr Thr Tyr Met Pro Met Ser Tyr Leu Tyr

260 265 270

Gly Lys Lys Tyr His Gly Pro Leu Thr His Leu Val Met Gln Leu Arg

275 280 285

Gln Glu Ile His Val Lys Pro Tyr Asp Gln Ile Asp Trp Asn Lys Ala

290 295 300

Arg His Asp Cys Cys Lys Asp Asp Leu Tyr Tyr Pro His Ser Phe Ile

305 310 315 320

Gln Asp Leu Leu Trp Asp Thr Ala Asn Tyr Phe Ser Glu Pro Val Met

325 330 335

Arg Arg Trp Pro Cys Asn Lys Ile Arg Glu Lys Ala Met Arg Lys Cys

340 345 350

Ile Lys Tyr Met Arg Tyr Gly Ala Glu Glu Ser Arg Tyr Ile Thr Ile

355 360 365

Gly Cys Val Glu Lys Ser Leu Gln Met Met Cys Trp Cys Ala His Asp

370 375 380

Pro Asn Cys Asp Glu Phe Lys Tyr His Leu Ala Arg Val Pro Asp Tyr

385 390 395 400

Leu Trp Leu Ala Glu Asp Gly Met Lys Met Gln Ser Phe Gly Ser Gln

405 410 415

Leu Trp Asp Cys Thr Leu Ala Thr Gln Ala Ile Ile Ala Thr Gly Met

420 425 430

Val Glu Glu Tyr Gly Asp Thr Ile Lys Lys Ala His Phe Tyr Ile Lys

435 440 445

Glu Ser Gln Val Lys Glu Asn Pro Lys Glu Asp Phe Lys Ala Met Tyr

450 455 460

Arg His Phe Thr Lys Gly Ser Trp Thr Phe Ser Asp Gln Asp Gln Gly

465 470 475 480

Trp Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys Leu Leu Val

485 490 495

Cys Ser Gln Met Pro Gln Asp Leu Ala Gly Glu Lys Ala Asp Val Glu

500 505 510

Arg Leu Tyr Asp Ala Val Asn Val Leu Leu Tyr Leu Gln Ser Pro Glu

515 520 525

Ser Gly Gly Phe Ala Ile Trp Glu Pro Pro Val Pro Gln Pro Tyr Leu

530 535 540

Gln Val Leu Asn Pro Ser Glu Leu Phe Ala Asp Ile Val Val Glu Gln

545 550 555 560

Glu His Val Glu Asn Thr Ala Ser Ile Val Gln Ala Leu Val Leu Phe

565 570 575

Lys Arg Leu His Pro Gly His Arg Glu Lys Glu Ile Glu Val Ser Val

580 585 590

Ser Lys Ala Val Arg Phe Leu Glu Gly Arg Gln Trp Pro Asp Gly Ser

595 600 605

Trp Tyr Gly Tyr Trp Gly Ile Cys Phe Leu Tyr Gly Thr Met Phe Val

610 615 620

Leu Gly Gly Leu Thr Ala Ala Gly Lys Thr Tyr Lys Asn Ser Glu Ala

625 630 635 640

Ile Arg Lys Ala Val Lys Phe Tyr Leu Ser Thr Gln Asn Glu Glu Gly

645 650 655

Gly Trp Gly Glu Cys Leu Glu Ser Cys Pro Ser Met Lys Tyr Ile Pro

660 665 670

Leu Glu Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp Ala Met Leu

675 680 685

Gly Leu Met Tyr Gly Gly Gln Ala Glu Arg Asp Pro Thr Pro Leu His

690 695 700

Lys Ala Ala Lys Leu Leu Ile Asn Ala Gln Met Asp Asp Gly Asp Phe

705 710 715 720

Pro Gln Gln Glu Ile Thr Gly Val Tyr Met Lys Asn Cys Met Leu His

725 730 735

Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr

740 745 750

Arg Lys Arg Val Trp Pro Thr Lys Ala Leu

755 760

<210> 4

<211> 762

<212> PRT

<213> 人工序列

<400> 4

Met Trp Lys Leu Lys Ile Ala Lys Gly Lys Gly Pro Tyr Leu Tyr Ser

1 5 10 15

Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Tyr Asp Pro Asn Ala

20 25 30

Gly Thr Pro Gln Glu Arg Glu Ala Phe Glu Lys Ala Arg Glu Gln Phe

35 40 45

Arg Asn Asn Arg Lys Lys Gly Val His Asn Pro Cys Ala Asp Leu Phe

50 55 60

Met Arg Met Gln Leu Ile Lys Glu Asn Gly Ile Asp Leu Met Ser Ile

65 70 75 80

Pro Pro Val Arg Val Glu Glu Lys Glu Glu Leu Thr Phe Glu Lys Thr

85 90 95

Thr Ile Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala

100 105 110

Thr Asp Gly His Trp Pro Ala Glu Asn Ala Gly Pro Met Phe Phe Thr

115 120 125

Pro Pro Leu Leu Ile Ala Leu Tyr Ile Ser Gly Ala Ile Asn Thr Ile

130 135 140

Leu Thr Ser Glu His Lys Lys Glu Leu Val Arg Tyr Ile Tyr Asn His

145 150 155 160

Gln Asn Glu Asp Gly Gly Trp Gly Phe Tyr Ile Glu Gly His Ser Thr

165 170 175

Met Ile Gly Ser Ala Leu Ser Tyr Val Ala Leu Arg Leu Leu Gly Glu

180 185 190

Gly Pro Asp Asp Gly Asp Gly Ala Val Gly Arg Gly Arg Gln Trp Ile

195 200 205

Leu Asp His Gly Gly Ala Thr Gly Ile Pro Ser Trp Gly Lys Thr Tyr

210 215 220

Leu Ser Val Leu Gly Val Tyr Asp Trp Asp Gly Cys Asn Pro Leu Pro

225 230 235 240

Pro Glu Phe Trp Leu Phe Pro Ser Phe Phe Pro Tyr His Pro Ala Lys

245 250 255

Met Trp Cys Tyr Cys Arg Thr Thr Tyr Met Pro Met Ser Tyr Leu Tyr

260 265 270

Gly Lys Lys Tyr His Gly Pro Leu Thr His Leu Val Met Gln Leu Arg

275 280 285

Gln Glu Ile His Val Lys Pro Tyr Asp Gln Ile Asp Trp Asn Lys Ala

290 295 300

Arg His Asp Cys Cys Lys Asp Asp Leu Tyr Tyr Pro His Ser Phe Ile

305 310 315 320

Gln Asp Leu Leu Trp Asp Thr Leu Asn Tyr Ala Ser Glu Pro Val Met

325 330 335

Arg Arg Trp Pro Cys Asn Lys Ile Arg Glu Lys Ala Met Arg Lys Cys

340 345 350

Ile Lys Tyr Met Arg Tyr Gly Ala Glu Glu Ser Arg Tyr Ile Thr Ile

355 360 365

Gly Cys Val Glu Lys Ser Leu Gln Met Met Cys Trp Cys Ala His Asp

370 375 380

Pro Asn Cys Asp Glu Phe Lys Tyr His Leu Ala Arg Val Pro Asp Tyr

385 390 395 400

Leu Trp Leu Ala Glu Asp Gly Met Lys Met Gln Ser Phe Gly Ser Gln

405 410 415

Leu Trp Asp Cys Thr Leu Ala Thr Gln Ala Ile Ile Ala Thr Gly Met

420 425 430

Val Glu Glu Tyr Gly Asp Thr Ile Lys Lys Ala His Phe Tyr Ile Lys

435 440 445

Glu Ser Gln Val Lys Glu Asn Pro Lys Glu Asp Phe Lys Ala Met Tyr

450 455 460

Arg His Phe Thr Lys Gly Ser Trp Thr Phe Ser Asp Gln Asp Gln Gly

465 470 475 480

Trp Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys Leu Leu Val

485 490 495

Cys Ser Gln Met Pro Gln Asp Leu Ala Gly Glu Lys Ala Asp Val Glu

500 505 510

Arg Leu Tyr Asp Ala Val Asn Val Leu Leu Tyr Leu Gln Ser Pro Glu

515 520 525

Ser Gly Gly Phe Ala Ile Trp Glu Pro Pro Val Pro Gln Pro Tyr Leu

530 535 540

Gln Val Leu Asn Pro Ser Glu Leu Phe Ala Asp Ile Val Val Glu Gln

545 550 555 560

Glu His Val Glu Asn Thr Ala Ser Ile Val Gln Ala Leu Val Leu Phe

565 570 575

Lys Arg Leu His Pro Gly His Arg Glu Lys Glu Ile Glu Val Ser Val

580 585 590

Ser Lys Ala Val Arg Phe Leu Glu Gly Arg Gln Trp Pro Asp Gly Ser

595 600 605

Trp Tyr Gly Tyr Trp Gly Ile Cys Phe Leu Tyr Gly Thr Met Phe Val

610 615 620

Leu Gly Gly Leu Thr Ala Ala Gly Lys Thr Tyr Lys Asn Ser Glu Ala

625 630 635 640

Ile Arg Lys Ala Val Lys Phe Tyr Leu Ser Thr Gln Asn Glu Glu Gly

645 650 655

Gly Trp Gly Glu Cys Leu Glu Ser Cys Pro Ser Met Lys Tyr Ile Pro

660 665 670

Leu Glu Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp Ala Met Leu

675 680 685

Gly Leu Met Tyr Gly Gly Gln Ala Glu Arg Asp Pro Thr Pro Leu His

690 695 700

Lys Ala Ala Lys Leu Leu Ile Asn Ala Gln Met Asp Asp Gly Asp Phe

705 710 715 720

Pro Gln Gln Glu Ile Thr Gly Val Tyr Met Lys Asn Cys Met Leu His

725 730 735

Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr

740 745 750

Arg Lys Arg Val Trp Pro Thr Lys Ala Leu

755 760

<210> 5

<211> 762

<212> PRT

<213> 人工序列

<400> 5

Met Trp Lys Leu Lys Ile Ala Lys Gly Lys Gly Pro Tyr Leu Tyr Ser

1 5 10 15

Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Tyr Asp Pro Asn Ala

20 25 30

Gly Thr Pro Gln Glu Arg Glu Ala Phe Glu Lys Ala Arg Glu Gln Phe

35 40 45

Arg Asn Asn Arg Lys Lys Gly Val His Asn Pro Cys Ala Asp Leu Phe

50 55 60

Met Arg Met Gln Leu Ile Lys Glu Asn Gly Ile Asp Leu Met Ser Ile

65 70 75 80

Pro Pro Val Arg Val Glu Glu Lys Glu Glu Leu Thr Phe Glu Lys Thr

85 90 95

Thr Ile Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala

100 105 110

Thr Asp Gly His Trp Pro Ala Glu Asn Ala Gly Pro Met Phe Phe Thr

115 120 125

Pro Pro Leu Leu Ile Ala Leu Tyr Ile Ser Gly Ala Ile Asn Thr Ile

130 135 140

Leu Thr Ser Glu His Lys Lys Glu Leu Val Arg Tyr Ile Tyr Asn His

145 150 155 160

Gln Asn Glu Asp Gly Gly Trp Gly Phe Tyr Ile Glu Gly His Ser Thr

165 170 175

Met Ile Gly Ser Ala Leu Ser Tyr Val Ala Leu Arg Leu Leu Gly Glu

180 185 190

Gly Pro Asp Asp Gly Asp Gly Ala Val Gly Arg Gly Arg Gln Trp Ile

195 200 205

Leu Asp His Gly Gly Ala Thr Gly Ile Pro Ser Trp Gly Lys Thr Tyr

210 215 220

Leu Ser Val Leu Gly Val Tyr Asp Trp Asp Gly Cys Asn Pro Leu Pro

225 230 235 240

Pro Glu Phe Trp Leu Phe Pro Ser Phe Phe Pro Tyr His Pro Ala Lys

245 250 255

Met Trp Cys Tyr Cys Arg Thr Thr Tyr Met Pro Met Ser Tyr Leu Tyr

260 265 270

Gly Lys Lys Tyr His Gly Pro Leu Thr His Leu Val Met Gln Leu Arg

275 280 285

Gln Glu Ile His Val Lys Pro Tyr Asp Gln Ile Asp Trp Asn Lys Ala

290 295 300

Arg His Asp Cys Cys Lys Asp Asp Leu Tyr Tyr Pro His Ser Phe Ile

305 310 315 320

Gln Asp Leu Leu Trp Asp Thr Leu Asn Tyr Phe Ser Glu Pro Val Met

325 330 335

Arg Arg Trp Pro Ala Asn Lys Ile Arg Glu Lys Ala Met Arg Lys Cys

340 345 350

Ile Lys Tyr Met Arg Tyr Gly Ala Glu Glu Ser Arg Tyr Ile Thr Ile

355 360 365

Gly Cys Val Glu Lys Ser Leu Gln Met Met Cys Trp Cys Ala His Asp

370 375 380

Pro Asn Cys Asp Glu Phe Lys Tyr His Leu Ala Arg Val Pro Asp Tyr

385 390 395 400

Leu Trp Leu Ala Glu Asp Gly Met Lys Met Gln Ser Phe Gly Ser Gln

405 410 415

Leu Trp Asp Cys Thr Leu Ala Thr Gln Ala Ile Ile Ala Thr Gly Met

420 425 430

Val Glu Glu Tyr Gly Asp Thr Ile Lys Lys Ala His Phe Tyr Ile Lys

435 440 445

Glu Ser Gln Val Lys Glu Asn Pro Lys Glu Asp Phe Lys Ala Met Tyr

450 455 460

Arg His Phe Thr Lys Gly Ser Trp Thr Phe Ser Asp Gln Asp Gln Gly

465 470 475 480

Trp Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys Leu Leu Val

485 490 495

Cys Ser Gln Met Pro Gln Asp Leu Ala Gly Glu Lys Ala Asp Val Glu

500 505 510

Arg Leu Tyr Asp Ala Val Asn Val Leu Leu Tyr Leu Gln Ser Pro Glu

515 520 525

Ser Gly Gly Phe Ala Ile Trp Glu Pro Pro Val Pro Gln Pro Tyr Leu

530 535 540

Gln Val Leu Asn Pro Ser Glu Leu Phe Ala Asp Ile Val Val Glu Gln

545 550 555 560

Glu His Val Glu Asn Thr Ala Ser Ile Val Gln Ala Leu Val Leu Phe

565 570 575

Lys Arg Leu His Pro Gly His Arg Glu Lys Glu Ile Glu Val Ser Val

580 585 590

Ser Lys Ala Val Arg Phe Leu Glu Gly Arg Gln Trp Pro Asp Gly Ser

595 600 605

Trp Tyr Gly Tyr Trp Gly Ile Cys Phe Leu Tyr Gly Thr Met Phe Val

610 615 620

Leu Gly Gly Leu Thr Ala Ala Gly Lys Thr Tyr Lys Asn Ser Glu Ala

625 630 635 640

Ile Arg Lys Ala Val Lys Phe Tyr Leu Ser Thr Gln Asn Glu Glu Gly

645 650 655

Gly Trp Gly Glu Cys Leu Glu Ser Cys Pro Ser Met Lys Tyr Ile Pro

660 665 670

Leu Glu Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp Ala Met Leu

675 680 685

Gly Leu Met Tyr Gly Gly Gln Ala Glu Arg Asp Pro Thr Pro Leu His

690 695 700

Lys Ala Ala Lys Leu Leu Ile Asn Ala Gln Met Asp Asp Gly Asp Phe

705 710 715 720

Pro Gln Gln Glu Ile Thr Gly Val Tyr Met Lys Asn Cys Met Leu His

725 730 735

Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr

740 745 750

Arg Lys Arg Val Trp Pro Thr Lys Ala Leu

755 760

<210> 6

<211> 2286

<212> DNA

<213> 长春花

<400> 6

atgtggaagc tgaagatcgc caagggcaag ggcccctatc tgtattctac taacaacttc 60

gtgggccgac agatttggga gtacgacccc aatgccggca ctccccaaga aagagaggct 120

tttgaaaagg ccagagagca gttcagaaac aaccgaaaga agggcgtgca caacccctgc 180

gccgatctgt ttatgcgaat gcagctgatc aaggagaacg gcatcgacct gatgtctatc 240

ccccccgtga gagtggaaga gaaggaggaa ctgaccttcg agaagaccac catcgccgtg 300

aagaaggccc tgcgactgaa tcgagctatc caggccactg atggccactg gcctgctgaa 360

aatgctggac ctatgttctt tactcccccc ctgctgatcg ccctgtacat ctctggagcc 420

atcaacacca tcctgacctc tgagcacaag aaggagctgg tgcgatacat ctacaaccac 480

cagaacgagg acggcggctg gggattttat atcgagggac attctactat gatcggctct 540

gccctgtctt acgtggccct gagactgctg ggagaaggcc ctgatgatgg agatggcgct 600

gtgggaagag gacgacagtg gattctggat cacggcggcg ctactggtat tccttcttgg 660

ggaaaaactt acctgtctgt gctgggcgtg tatgactggg acggatgcaa tcctctgcct 720

cccgagttct ggctgtttcc ctctttcttc ccctaccacc ccgccaagat gtggtgctat 780

tgtcgaacca cctacatgcc catgtcttac ctgtacggca agaagtacca cggccccctg 840

actcatctgg tgatgcaact gcgacaggag atccacgtca agccctacga ccagatcgac 900

tggaacaagg cccgacacga ctgctgcaaa gacgacctgt attaccccca ctctttcatc 960

caggacgccc tgtgggacac cctgaactac ttctctgagc ccgtgatgcg acgatggccc 1020

tgtaataaga tccgagagaa ggccatgcga aagtgcatca agtacatgcg atacggcgcc 1080

gaggagtctc gatacatcac catcggctgc gtggagaagt ctctgcagat gatgtgctgg 1140

tgcgcccacg accctaattg tgatgagttt aagtaccacc tggcccgagt gcccgattac 1200

ctgtggctgg ctgaagatgg aatgaagatg cagtctttcg gctctcagct gtgggactgc 1260

accctggcta ctcaggctat tatcgctacc ggaatggtgg aggagtacgg cgataccatc 1320

aagaaggccc acttctacat caaggagtct caggtgaagg agaaccccaa ggaggacttc 1380

aaggccatgt accgacactt caccaagggc tcttggacct tctctgacca ggaccagggc 1440

tgggtggttt ctgattgtac tgctgaggct ctgaaatgtc tgctggtgtg ctctcagatg 1500

ccccaggacc tggctggaga aaaagccgat gtggaacgac tgtacgatgc cgtgaacgtg 1560

ctgctgtacc tgcagtctcc cgagtctggc ggatttgcca tttgggagcc tcctgtgcct 1620

caaccctatc tgcaggtgct gaacccctct gagctgttcg ccgacattgt ggtggagcag 1680

gaacacgtcg agaataccgc ctctatcgtg caggccctgg tgctgttcaa acgactgcat 1740

cccggacacc gagagaagga gattgaagtg tctgtgtcta aggccgtgcg attcctggag 1800

ggcagacagt ggcccgatgg atcttggtat ggatattggg gcatctgctt cctgtacggc 1860

accatgttcg tgctgggcgg cctgactgct gctggaaaaa cttataaaaa ctctgaggcc 1920

atccgaaagg ccgtgaagtt ctacctgtct acccagaacg aggagggcgg ctggggagaa 1980

tgtctggaat cttgtccttc tatgaagtac atccccctgg agggcaaccg aaccaacctg 2040

gtgcaaacct cttgggccat gctgggcctg atgtacggcg gacaagctga gagagatccc 2100

actcctctgc ataaagctgc caagctgctg atcaatgccc agatggacga cggcgatttc 2160

ccccagcaag aaattaccgg cgtgtacatg aaaaactgca tgctgcacta cgccgagtac 2220

cgaaacatct tccccctgtg ggccctggcc gaatatcgaa aaagagtgtg gcccaccaag 2280

gccctg 2286

<210> 7

<211> 2286

<212> DNA

<213> 人工序列

<400> 7

atgtggaagc tgaagatcgc caagggcaag ggcccctatc tgtattctac taacaacttc 60

gtgggccgac agatttggga gtacgacccc aatgccggca ctccccaaga aagagaggct 120

tttgaaaagg ccagagagca gttcagaaac aaccgaaaga agggcgtgca caacccctgc 180

gccgatctgt ttatgcgaat gcagctgatc aaggagaacg gcatcgacct gatgtctatc 240

ccccccgtga gagtggaaga gaaggaggaa ctgaccttcg agaagaccac catcgccgtg 300

aagaaggccc tgcgactgaa tcgagctatc caggccactg atggccactg gcctgctgaa 360

aatgctggac ctatgttctt tactcccccc ctgctgatcg ccctgtacat ctctggagcc 420

atcaacacca tcctgacctc tgagcacaag aaggagctgg tgcgatacat ctacaaccac 480

cagaacgagg acggcggctg gggattttat atcgagggac attctactat gatcggctct 540

gccctgtctt acgtggccct gagactgctg ggagaaggcc ctgatgatgg agatggcgct 600

gtgggaagag gacgacagtg gattctggat cacggcggcg ctactggtat tccttcttgg 660

ggaaaaactt acctgtctgt gctgggcgtg tatgactggg acggatgcaa tcctctgcct 720

cccgagttct ggctgtttcc ctctttcttc ccctaccacc ccgccaagat gtggtgctat 780

tgtcgaacca cctacatgcc catgtcttac ctgtacggca agaagtacca cggccccctg 840

actcatctgg tgatgcaact gcgacaggag atccacgtca agccctacga ccagatcgac 900

tggaacaagg cccgacacga ctgctgcaaa gacgacctgt attaccccca ctctttcatc 960

caggacctgc tgtgggacac cgccaactac ttctctgagc ccgtgatgcg acgatggccc 1020

tgtaataaga tccgagagaa ggccatgcga aagtgcatca agtacatgcg atacggcgcc 1080

gaggagtctc gatacatcac catcggctgc gtggagaagt ctctgcagat gatgtgctgg 1140

tgcgcccacg accctaattg tgatgagttt aagtaccacc tggcccgagt gcccgattac 1200

ctgtggctgg ctgaagatgg aatgaagatg cagtctttcg gctctcagct gtgggactgc 1260

accctggcta ctcaggctat tatcgctacc ggaatggtgg aggagtacgg cgataccatc 1320

aagaaggccc acttctacat caaggagtct caggtgaagg agaaccccaa ggaggacttc 1380

aaggccatgt accgacactt caccaagggc tcttggacct tctctgacca ggaccagggc 1440

tgggtggttt ctgattgtac tgctgaggct ctgaaatgtc tgctggtgtg ctctcagatg 1500

ccccaggacc tggctggaga aaaagccgat gtggaacgac tgtacgatgc cgtgaacgtg 1560

ctgctgtacc tgcagtctcc cgagtctggc ggatttgcca tttgggagcc tcctgtgcct 1620

caaccctatc tgcaggtgct gaacccctct gagctgttcg ccgacattgt ggtggagcag 1680

gaacacgtcg agaataccgc ctctatcgtg caggccctgg tgctgttcaa acgactgcat 1740

cccggacacc gagagaagga gattgaagtg tctgtgtcta aggccgtgcg attcctggag 1800

ggcagacagt ggcccgatgg atcttggtat ggatattggg gcatctgctt cctgtacggc 1860

accatgttcg tgctgggcgg cctgactgct gctggaaaaa cttataaaaa ctctgaggcc 1920

atccgaaagg ccgtgaagtt ctacctgtct acccagaacg aggagggcgg ctggggagaa 1980

tgtctggaat cttgtccttc tatgaagtac atccccctgg agggcaaccg aaccaacctg 2040

gtgcaaacct cttgggccat gctgggcctg atgtacggcg gacaagctga gagagatccc 2100

actcctctgc ataaagctgc caagctgctg atcaatgccc agatggacga cggcgatttc 2160

ccccagcaag aaattaccgg cgtgtacatg aaaaactgca tgctgcacta cgccgagtac 2220

cgaaacatct tccccctgtg ggccctggcc gaatatcgaa aaagagtgtg gcccaccaag 2280

gccctg 2286

<210> 8

<211> 2286

<212> DNA

<213> 人工序列

<400> 8

atgtggaagc tgaagatcgc caagggcaag ggcccctatc tgtattctac taacaacttc 60

gtgggccgac agatttggga gtacgacccc aatgccggca ctccccaaga aagagaggct 120

tttgaaaagg ccagagagca gttcagaaac aaccgaaaga agggcgtgca caacccctgc 180

gccgatctgt ttatgcgaat gcagctgatc aaggagaacg gcatcgacct gatgtctatc 240

ccccccgtga gagtggaaga gaaggaggaa ctgaccttcg agaagaccac catcgccgtg 300

aagaaggccc tgcgactgaa tcgagctatc caggccactg atggccactg gcctgctgaa 360

aatgctggac ctatgttctt tactcccccc ctgctgatcg ccctgtacat ctctggagcc 420

atcaacacca tcctgacctc tgagcacaag aaggagctgg tgcgatacat ctacaaccac 480

cagaacgagg acggcggctg gggattttat atcgagggac attctactat gatcggctct 540

gccctgtctt acgtggccct gagactgctg ggagaaggcc ctgatgatgg agatggcgct 600

gtgggaagag gacgacagtg gattctggat cacggcggcg ctactggtat tccttcttgg 660

ggaaaaactt acctgtctgt gctgggcgtg tatgactggg acggatgcaa tcctctgcct 720

cccgagttct ggctgtttcc ctctttcttc ccctaccacc ccgccaagat gtggtgctat 780

tgtcgaacca cctacatgcc catgtcttac ctgtacggca agaagtacca cggccccctg 840

actcatctgg tgatgcaact gcgacaggag atccacgtca agccctacga ccagatcgac 900

tggaacaagg cccgacacga ctgctgcaaa gacgacctgt attaccccca ctctttcatc 960

caggacctgc tgtgggacac cctgaactac gcctctgagc ccgtgatgcg acgatggccc 1020

tgtaataaga tccgagagaa ggccatgcga aagtgcatca agtacatgcg atacggcgcc 1080

gaggagtctc gatacatcac catcggctgc gtggagaagt ctctgcagat gatgtgctgg 1140

tgcgcccacg accctaattg tgatgagttt aagtaccacc tggcccgagt gcccgattac 1200

ctgtggctgg ctgaagatgg aatgaagatg cagtctttcg gctctcagct gtgggactgc 1260

accctggcta ctcaggctat tatcgctacc ggaatggtgg aggagtacgg cgataccatc 1320

aagaaggccc acttctacat caaggagtct caggtgaagg agaaccccaa ggaggacttc 1380

aaggccatgt accgacactt caccaagggc tcttggacct tctctgacca ggaccagggc 1440

tgggtggttt ctgattgtac tgctgaggct ctgaaatgtc tgctggtgtg ctctcagatg 1500

ccccaggacc tggctggaga aaaagccgat gtggaacgac tgtacgatgc cgtgaacgtg 1560

ctgctgtacc tgcagtctcc cgagtctggc ggatttgcca tttgggagcc tcctgtgcct 1620

caaccctatc tgcaggtgct gaacccctct gagctgttcg ccgacattgt ggtggagcag 1680

gaacacgtcg agaataccgc ctctatcgtg caggccctgg tgctgttcaa acgactgcat 1740

cccggacacc gagagaagga gattgaagtg tctgtgtcta aggccgtgcg attcctggag 1800

ggcagacagt ggcccgatgg atcttggtat ggatattggg gcatctgctt cctgtacggc 1860

accatgttcg tgctgggcgg cctgactgct gctggaaaaa cttataaaaa ctctgaggcc 1920

atccgaaagg ccgtgaagtt ctacctgtct acccagaacg aggagggcgg ctggggagaa 1980

tgtctggaat cttgtccttc tatgaagtac atccccctgg agggcaaccg aaccaacctg 2040

gtgcaaacct cttgggccat gctgggcctg atgtacggcg gacaagctga gagagatccc 2100

actcctctgc ataaagctgc caagctgctg atcaatgccc agatggacga cggcgatttc 2160

ccccagcaag aaattaccgg cgtgtacatg aaaaactgca tgctgcacta cgccgagtac 2220

cgaaacatct tccccctgtg ggccctggcc gaatatcgaa aaagagtgtg gcccaccaag 2280

gccctg 2286

<210> 9

<211> 2286

<212> DNA

<213> 人工序列

<400> 9

atgtggaagc tgaagatcgc caagggcaag ggcccctatc tgtattctac taacaacttc 60

gtgggccgac agatttggga gtacgacccc aatgccggca ctccccaaga aagagaggct 120

tttgaaaagg ccagagagca gttcagaaac aaccgaaaga agggcgtgca caacccctgc 180

gccgatctgt ttatgcgaat gcagctgatc aaggagaacg gcatcgacct gatgtctatc 240

ccccccgtga gagtggaaga gaaggaggaa ctgaccttcg agaagaccac catcgccgtg 300

aagaaggccc tgcgactgaa tcgagctatc caggccactg atggccactg gcctgctgaa 360

aatgctggac ctatgttctt tactcccccc ctgctgatcg ccctgtacat ctctggagcc 420

atcaacacca tcctgacctc tgagcacaag aaggagctgg tgcgatacat ctacaaccac 480

cagaacgagg acggcggctg gggattttat atcgagggac attctactat gatcggctct 540

gccctgtctt acgtggccct gagactgctg ggagaaggcc ctgatgatgg agatggcgct 600

gtgggaagag gacgacagtg gattctggat cacggcggcg ctactggtat tccttcttgg 660

ggaaaaactt acctgtctgt gctgggcgtg tatgactggg acggatgcaa tcctctgcct 720

cccgagttct ggctgtttcc ctctttcttc ccctaccacc ccgccaagat gtggtgctat 780

tgtcgaacca cctacatgcc catgtcttac ctgtacggca agaagtacca cggccccctg 840

actcatctgg tgatgcaact gcgacaggag atccacgtca agccctacga ccagatcgac 900

tggaacaagg cccgacacga ctgctgcaaa gacgacctgt attaccccca ctctttcatc 960

caggacctgc tgtgggacac cctgaactac ttctctgagc ccgtgatgcg acgatggccc 1020

gccaataaga tccgagagaa ggccatgcga aagtgcatca agtacatgcg atacggcgcc 1080

gaggagtctc gatacatcac catcggctgc gtggagaagt ctctgcagat gatgtgctgg 1140

tgcgcccacg accctaattg tgatgagttt aagtaccacc tggcccgagt gcccgattac 1200

ctgtggctgg ctgaagatgg aatgaagatg cagtctttcg gctctcagct gtgggactgc 1260

accctggcta ctcaggctat tatcgctacc ggaatggtgg aggagtacgg cgataccatc 1320

aagaaggccc acttctacat caaggagtct caggtgaagg agaaccccaa ggaggacttc 1380

aaggccatgt accgacactt caccaagggc tcttggacct tctctgacca ggaccagggc 1440

tgggtggttt ctgattgtac tgctgaggct ctgaaatgtc tgctggtgtg ctctcagatg 1500

ccccaggacc tggctggaga aaaagccgat gtggaacgac tgtacgatgc cgtgaacgtg 1560

ctgctgtacc tgcagtctcc cgagtctggc ggatttgcca tttgggagcc tcctgtgcct 1620

caaccctatc tgcaggtgct gaacccctct gagctgttcg ccgacattgt ggtggagcag 1680

gaacacgtcg agaataccgc ctctatcgtg caggccctgg tgctgttcaa acgactgcat 1740

cccggacacc gagagaagga gattgaagtg tctgtgtcta aggccgtgcg attcctggag 1800

ggcagacagt ggcccgatgg atcttggtat ggatattggg gcatctgctt cctgtacggc 1860

accatgttcg tgctgggcgg cctgactgct gctggaaaaa cttataaaaa ctctgaggcc 1920

atccgaaagg ccgtgaagtt ctacctgtct acccagaacg aggagggcgg ctggggagaa 1980

tgtctggaat cttgtccttc tatgaagtac atccccctgg agggcaaccg aaccaacctg 2040

gtgcaaacct cttgggccat gctgggcctg atgtacggcg gacaagctga gagagatccc 2100

actcctctgc ataaagctgc caagctgctg atcaatgccc agatggacga cggcgatttc 2160

ccccagcaag aaattaccgg cgtgtacatg aaaaactgca tgctgcacta cgccgagtac 2220

cgaaacatct tccccctgtg ggccctggcc gaatatcgaa aaagagtgtg gcccaccaag 2280

gccctg 2286

<210> 10

<211> 2286

<212> DNA

<213> 人工序列

<400> 10

atgtggaagc tgaagatcgc caagggcaag ggcccctatc tgtattctac taacaacttc 60

gtgggccgac agatttggga gtacgacccc aatgccggca ctccccaaga aagagaggct 120

tttgaaaagg ccagagagca gttcagaaac aaccgaaaga agggcgtgca caacccctgc 180

gccgatctgt ttatgcgaat gcagctgatc aaggagaacg gcatcgacct gatgtctatc 240

ccccccgtga gagtggaaga gaaggaggaa ctgaccttcg agaagaccac catcgccgtg 300

aagaaggccc tgcgactgaa tcgagctatc caggccactg atggccactg gcctgctgaa 360

aatgctggac ctatgttctt tactcccccc ctgctgatcg ccctgtacat ctctggagcc 420

atcaacacca tcctgacctc tgagcacaag aaggagctgg tgcgatacat ctacaaccac 480

cagaacgagg acggcggctg gggattttat atcgagggac attctactat gatcggctct 540

gccctgtctt acgtggccct gagactgctg ggagaaggcc ctgatgatgg agatggcgct 600

gtgggaagag gacgacagtg gattctggat cacggcggcg ctactggtat tccttcttgg 660

ggaaaaactt acctgtctgt gctgggcgtg tatgactggg acggatgcaa tcctctgcct 720

cccgagttct ggctgtttcc ctctttcttc ccctaccacc ccgccaagat gtggtgctat 780

tgtcgaacca cctacatgcc catgtcttac ctgtacggca agaagtacca cggccccctg 840

actcatctgg tgatgcaact gcgacaggag atccacgtca agccctacga ccagatcgac 900

tggaacaagg cccgacacga ctgctgcaaa gacgacctgt attaccccca ctctttcatc 960

caggacctgc tgtgggacac cctgaactac ttctctgagc ccgtgatgcg acgatggccc 1020

tgtaataaga tccgagagaa ggccatgcga aagtgcatca agtacatgcg atacggcgcc 1080

gaggagtctc gatacatcac catcggctgc gtggagaagt ctctgcagat gatgtgctgg 1140

tgcgcccacg accctaattg tgatgagttt aagtaccacc tggcccgagt gcccgattac 1200

ctgtggctgg ctgaagatgg aatgaagatg cagtctttcg gctctcagct gtgggactgc 1260

accctggcta ctcaggctat tatcgctacc ggaatggtgg aggagtacgg cgataccatc 1320

aagaaggccc acttctacat caaggagtct caggtgaagg agaaccccaa ggaggacttc 1380

aaggccatgt accgacactt caccaagggc tcttggacct tctctgacca ggaccagggc 1440

tgggtggttt ctgattgtac tgctgaggct ctgaaatgtc tgctggtgtg ctctcagatg 1500

ccccaggacc tggctggaga aaaagccgat gtggaacgac tgtacgatgc cgtgaacgtg 1560

ctgctgtacc tgcagtctcc cgagtctggc ggatttgcca tttgggagcc tcctgtgcct 1620

caaccctatc tgcaggtgct gaacccctct gagctgttcg ccgacattgt ggtggagcag 1680

gaacacgtcg agaataccgc ctctatcgtg caggccctgg tgctgttcaa acgactgcat 1740

cccggacacc gagagaagga gattgaagtg tctgtgtcta aggccgtgcg attcctggag 1800

ggcagacagt ggcccgatgg atcttggtat ggatattggg gcatctgctt cctgtacggc 1860

accatgttcg tgctgggcgg cctgactgct gctggaaaaa cttataaaaa ctctgaggcc 1920

atccgaaagg ccgtgaagtt ctacctgtct acccagaacg aggagggcgg ctggggagaa 1980

tgtctggaat cttgtccttc tatgaagtac atccccctgg agggcaaccg aaccaacctg 2040

gtgcaaacct cttgggccat gctgggcctg atgtacggcg gacaagctga gagagatccc 2100

actcctctgc ataaagctgc caagctgctg atcaatgccc agatggacga cggcgatttc 2160

ccccagcaag aaattaccgg cgtgtacatg aaaaactgca tgctgcacta cgccgagtac 2220

cgaaacatct tccccctgtg ggccctggcc gaatatcgaa aaagagtgtg gcccaccaag 2280

gccctg 2286

Claims

1.一种香树脂醇合酶突变体，其特征在于，所述的香树脂醇合酶突变体，是在SEQ IDNO.1所示的野生型香树脂醇合酶基础上，将第323位的亮氨酸替换为丙氨酸所得。

2.如权利要求1所述的香树脂醇合酶突变体，其特征在于，所述突变体氨基酸序列如SEQ ID NO.2所示。

3.权利要求1所述的香树脂醇合酶突变体的编码基因。

4.如权利要求3所述的编码基因，其特征在于，核苷酸序列如SEQ ID NO.7所示。

5.含有权利要求3所述编码基因的重组载体或重组菌株。

6.如权利要求5所述的重组载体，其特征在于，采用的表达质粒为pYLEX1或pUG6。

7.如权利要求5所述的重组菌株，其特征在于，采用的宿主细胞为解脂耶氏酵母、酿酒酵母、乳酸克鲁维酵母或大肠杆菌。

8.如权利要求5所述的重组菌株，其特征在于，所述重组菌以解脂耶氏酵母Po1gΔKU70为宿主，采用pYLEX1质粒对SEQ ID NO.7所示基因进行表达。

9.权利要求5所述重组载体或重组菌株在生产香树脂醇中的应用。

10.权利要求1所述香树脂醇合酶突变体在生产香树脂醇中的应用。