CN114807107A

CN114807107A - 嵌合三萜合酶和三萜化合物

Info

Publication number: CN114807107A
Application number: CN202210378582.1A
Authority: CN
Inventors: 刘天罡
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-07-29

Abstract

本发明公开了嵌合三萜合酶和三萜化合物，属于生物技术领域。本发明的嵌合三萜合酶为氨基酸序列如SEQ ID NO.1所示的CgCS、氨基酸序列如SEQ ID NO.2所示的TvTS或氨基酸序列如SEQ ID NO.3所示的MpMS。本发明的嵌合三萜合酶能够产生结构新颖的三萜化合物。

Description

嵌合三萜合酶和三萜化合物

技术领域

本发明属于生物技术领域，涉及三个真菌来源的嵌合三萜合酶和嵌合三萜合酶产生的结构新颖的三萜化合物。

背景技术

萜类化合物是自然界中数量最多的一类天然产物，主要分布在真菌、植物以及细菌中。真菌来源的萜类化合物因其新颖且多样化的结构以及显著的药理活性，成为天然产物生物合成领域的研究热点。其生物合成首先是通过甲羟戊酸(mevalonate，MVA)途径合成萜类前体物质，异戊烯焦磷酸(isopentenyl diphosphate，IPP)和烯丙基焦磷酸(dimethylallyl diphosphate，DMAPP)；然后异戊烯转移酶(prenyltransferases，PT)催化DMAPP和IPP缩合形成不同链长的异戊烯基前体物质(C10-C25)；随后在萜类合酶(terpenesynthase，TS)作用下形成单萜、倍半萜、二萜、二倍半萜、三萜以及多萜等一系列结构多样的萜类骨架化合物；最后这些骨架化合物在各种修饰酶(如P450酶、脱水酶、甲基转移酶)的作用下形成结构复杂多样且具有生理活性的萜类化合物。

PT和TS是萜类化合物的生物合成中至关重要的两个酶。通常情况下，PT和TS是连续且独立发挥作用的两种酶，然而，有一些真菌来源萜类合酶同时具有PT结构域和I型TS结构域，属于嵌合类萜类合酶。这类型的酶通常被报道主要产生二倍半萜以及少量的二萜化合物，从未见三萜产物的报道。

发明内容

本发明的目的在于提供同时具有异戊烯转移酶活性和萜类合酶活性的可产生三萜化合物的嵌合三萜合酶及其应用。

本发明的目的还在于提供通过所述嵌合三萜合酶产生的结构新颖的三萜化合物。

本发明的目的通过下述技术方案实现：

本发明所提供的嵌合三萜合酶，为Colletotrichum gloeosporioides来源的CgCS、Talaromyces verruculosus来源的TvTS或Macrophomina phaseolina来源的MpMS；其中，CgCS的氨基酸序列如SEQ ID NO.1所示，TvTS的氨基酸序列如SEQ ID NO.2所示，MpMS的氨基酸序列如SEQ ID NO.3所示。

编码上述嵌合三萜合酶CgCS、TvTS、MpMS的核酸分子的核苷酸序列分别优选如SEQID NO.4、5、6所示。

一种可表达上述嵌合三萜合酶的重组质粒，含有上述核酸分子。

一种可表达上述嵌合三萜合酶的重组细胞，含有上述核酸分子或重组质粒。在一些实施方案中，所述的重组细胞以酿酒酵母作为宿主。

上述嵌合三萜合酶、核酸分子、重组质粒或重组细胞可用于制备三萜化合物，所述的三萜化合物为结构如式I所示的化合物2colleterpenol(2)或结构如式II所示的化合物1talaropentaene(1)。其中，化合物1talaropentaene(1)由TvTS合成，化合物2colleterpenol(2)由CgCS合成。

一种制备上述三萜化合物的方法，包括如下步骤：培养上述重组细胞得到含所述三萜化合物的培养物。

本发明所提供的三萜化合物为结构如式I所示的colleterpenol或结构如式II所示的talaropentaene。

本发明的优点和有益效果：本发明发现了同时具有异戊烯转移酶活性和萜类合酶活性的嵌合三萜合酶，其可用于制备三萜化合物。

附图说明

图1是化合物1talaropentaene的GC检测图。

图2是化合物2colleterpenol的GC检测图。

图3是化合物3的GC检测图。

图4是化合物1talaropentaene的MS图。

图5是化合物2colleterpenol的MS图。

图6是化合物3的MS图。

图7是化合物的结构鉴定，化合物1和2关键的HMBC、¹H-¹H COSY和NOESY相关。

图8是化合物1的氢谱图(C₆D₆,600MHz)。

图9是化合物1的¹³C-NMR谱图(C₆D₆,150MHz)。

图10是化合物1的¹³C-DEPT谱图(C₆D₆,150MHz)。

图11是化合物1的¹H-¹H-COSY谱图(C₆D₆)。

图12是化合物1的HSQC谱图(C₆D₆)。

图13是化合物1的HMBC谱图(C₆D₆)。

图14是化合物1的NOESY谱图(C₆D₆)。

图15是化合物2的氢谱图(C₆D₆,600MHz)。

图16是化合物2的¹³C-NMR谱图(C₆D₆,150MHz)。

图17是化合物2的¹³C-DEPT谱图(C₆D₆,150MHz)。

图18是化合物2的¹H-¹H-COSY谱图(C₆D₆)。

图19是化合物2的HSQC谱图(C₆D₆)。

图20是化合物2的HMBC谱图(C₆D₆)。

图21是化合物2的NOESY谱图(C₆D₆)。

具体实施方式

以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。

实施例1三个嵌合三萜合酶(TvTS、CgCS和MpMS)表达质粒的构建

根据菌株Colletotrichum gloeosporioides ES026基因组测序结果，以及嵌合萜类合酶的具有的保守结构域3个DDXXD/E和1个NSE/DTE序列，筛选得到CgCS基因，以菌株Colletotrichum gloeosporioides ES026的cDNA为模板扩增CgCS基因(CgCS基因序列见SEQ ID NO.4)，以pGB315为模板(序列见SEQ ID NO.7)扩增质粒骨架。然后再利用酵母组装或者Gibson将两个片段组装起来。

在NCBI和Uniprot数据库中搜索，限定氨基酸长度为700-900，下载所有符合的核苷酸/蛋白质序列，然后将所下载的序列进行分析，筛选出具有嵌合萜类合酶保守结构域DDXXD/E和NSE/DTE的序列，进行基因合成，获得pUC57-PTTS139(萜类合酶的基因序列见SEQID NO.5)。以基因公司合成回来的质粒为模板扩增TvTS基因，以pGB315为模板扩增质粒骨架。然后再利用酵母组装或者Gibson将两个片段组装起来。

在NCBI和Uniprot数据库中搜索，限定氨基酸长度为700-900，下载所有符合的核苷酸/蛋白质序列，然后将所下载的序列进行分析，筛选出具有嵌合萜类合酶保守结构域DDXXD/E和NSE/DTE的序列，进行基因合成，获得pUC57-PTTS018(萜类合酶的基因序列见SEQID NO.6)。以基因公司合成回来的质粒为模板扩增MpMS基因，以pGB315为模板扩增质粒骨架。然后再利用酵母组装或者Gibson将两个片段组装起来。

(1)构建质粒如下表

表1.嵌合三萜合酶表达质粒特征

pRC180	pGB315 derived,URA,P<sub>GAL1</sub>-P<sub>GAL10</sub>-PTTS139-FDS-T<sub>ADH1</sub>
		pRC181	pGB315 derived,URA,P<sub>GAL1</sub>-P<sub>GAL10</sub>-Cgl13855-FDS-T<sub>ADH1</sub>
pRC182	pGB315 derived,URA,P<sub>GAL1</sub>-P<sub>GAL10</sub>-PTTS018-FDS-T<sub>ADH1</sub>

质粒pRC180构建过程：以引物PTTS139-F/R对基因PTTS139进行扩增获得TvTS，以引物pRC180V-F/R对pGB315进行扩增获得pRC180-Vector。获得的片段经过Gibson或酵母组装的方式将片段重组，获得质粒pRC180。

质粒pRC181构建过程：以引物Cgl13855-F/R对基因Cgl13855进行扩增获得CgCS，以引物pRC181V-F/R对pGB315进行扩增获得pRC181-Vector。获得的片段经过Gibson或酵母组装的方式将片段重组，获得质粒pRC181。

质粒pRC182构建过程：以引物PTTS018-F/R对基因PTTS018进行扩增获得MpMS，以引物pRC182V-F/R对pGB315进行扩增获得pRC182-Vector。获得的片段经过Gibson或酵母组装的方式将片段重组，获得质粒pRC182。

表2.构建质粒所用引物

PTTS139-F	CAATTGAGCTGATCCaGTaCTGCCACCAACAACCAACAATTCAACC
		PTTS139-R	CAATATAAGCTTgccaccATGGATTTCAAATATTCAAGAGAATTG
pRC180V-R	GAATTGTTGGTTGTTGGTGGCAGtACtGGATCAGCTCAATTGTCTG
		pRC180V-F	CTCTTGAATATTTGAAATCCATggtggcAAGCTTATATTGAATTTTC
Cgl13855-F	TTGAGCTGATCCaGTaCTGCCTTAGGAAATCCACAACCTTCTCAAC
		Cgl13855-R	CAATATAAGCTTgccaccATGCCTACCTTCGCACAACCAGTTCCGG
pRC181V-R	GAAGGTTGTGGATTTCCTAAGGCAGtACtGGATCAGCTCAATTGTC
		pRC181V-F	CTGGTTGTGCGAAGGTAGGCATggtggcAAGCTTATATTGAATTTTC
PTTS018-F	GAGCTGATCCaGTaCTGCCTGCTTTAATTCTTAATCTTTCTAAAG
		PTTS018-R	CAATATAAGCTTgccaccATGTGTAACACTAAGTGTTACAACAC
pRC182V-R	GAAAGATTAAGAATTAAAGCAGGCAGtACtGGATCAGCTCAATTG
		pRC182V-F	GTAACACTTAGTGTTACACATggtggcAAGCTTATATTGAATTTTC

实施例2潜在嵌合萜类合酶表达菌株的构建

将实施例3中获得的质粒pRC180、pRC181、pRC182分别转入基因挖掘底盘酵母菌株YZL141(菌株YZL141的构建见本申请人在先专利申请：申请号CN202110618456.4，名称一种嵌合萜类合酶及其应用，公开号CN113480660A)，筛选标记为尿嘧啶(URA3)，筛选平板为Sc-ura(合成酵母氮源YNB 6.7g/L，葡萄糖20g/L，缺尿嘧啶的混合氨基酸粉末1.3g/L，2％琼脂粉)，获得对应的嵌合萜类合酶表达菌株YZL141-pRC180、YZL141-pRC181、YZL141-pRC182。

实施例3潜在嵌合萜类合酶表达菌株的摇瓶发酵与产物检测

对实施例2中获得的菌株通过摇瓶发酵培养，具体方法如下：从转化平板挑取单克隆到含有5mL种子培养基的PA瓶中，种子培养基配方为：YNB(6.7g/L)，酵母粉(10g/L)，葡萄糖(20g/L)，缺尿嘧啶的混合氨基酸粉末(1.3g/L)。种子液30℃摇床培养过夜后进行转接，按照初始OD₆₀₀＝0.1转接，置于30℃摇床开始摇瓶发酵，发酵培养基配方为：蛋白胨(20g/L)，酵母粉(10g/L)，葡萄糖(10g/L)，半乳糖(10g/L)。发酵结束后3500rpm离心10min收集菌体部分，用丙酮浸泡菌体并超声40min，以上操作重复三次。将三次丙酮提取液用旋转蒸发仪浓缩除去大部分丙酮后，再用适量的乙酸乙酯萃取三次。将三次乙酸乙酯萃取液合并并浓缩，最后离心取少量上清液进行GCMS检测。

检测结果(图1-6)显示三个嵌合萜类合酶表达菌株中分别检测到了三萜化合物。TvTS可以合成化合物1talaropentaene(1)，CgCS可以合成化合物2colleterpenol(2)，MpMS可以合成分子量为408的三萜骨架化合物(3)。

talaropentaene(1)和colleterpenol(2)的结构鉴定见图7，两者关键的HMBC、¹H-¹H COSY和NOESY相关(图7)，talaropentaene(1)和colleterpenol(2)的核磁谱图、数据见图8-21和下表3。

表3.talaropentaene(1)和colleterpenol(2)的核磁数据

[a]NMR data of 1recorded in CDCl3(600MHz,298K).[b]NMR data of3recorded in CDCl3(400MHz,298K).Chemical shiftsδin ppm,coupling constants Jin Hertz,s＝singlet,d＝doublet,m＝multiplet,br＝broad.

尽管上面已经示出和描述了本发明的实施例，但应理解本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

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1 5 10 15

Thr Glu Pro Ala Met Glu Tyr Met Tyr Ser Val Pro Leu Asp Glu Ser

20 25 30

Glu Tyr Asp Lys Cys Gly Phe Cys Gln Asp Pro Arg Tyr Arg Pro Arg

35 40 45

Arg His Lys Asp Gln His Leu Ala Arg Ala Gly Ser Ala Lys Ala Lys

50 55 60

Glu Leu Cys Glu Ala Leu Ile Gly Val Tyr Pro Arg Pro Thr Cys Glu

65 70 75 80

Ser Ala Val Gly His Ser Ile Ala Leu Val Met Pro Glu Cys Met Pro

85 90 95

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

100 105 110

Asp Asn Ile Ala Glu Ser Gly Ser Gln Gln Asp Thr Gly Asn Leu Gly

115 120 125

Thr Glu Trp Ala Asn Asp Met Glu Thr Gly Pro Thr Thr Ser Val Asn

130 135 140

Ser Asn Thr Gly Ala Lys Gln Val Met Ala Lys Leu Ala Leu Gln Leu

145 150 155 160

Leu Ser Ile Asp Pro Val Cys Ala Gly Asn Val Met Lys Ala Trp Lys

165 170 175

Glu Trp Ala Ala Gly Phe Ala Lys Pro Arg Arg Phe Asp Ser Ile Glu

180 185 190

Gln Tyr Ile Asp Tyr Arg Leu Val Asp Ser Gly Ala Ile Val Ala Val

195 200 205

His Leu Met Asn Phe Gly Met Gly Leu Asp Ile Ser Val Glu Glu Leu

210 215 220

Arg Glu Val Ser Asp Ile Val Asn His Ala Gly Lys Ala Leu Ser Tyr

225 230 235 240

Gln Asn Asp Phe Phe Ser Phe Asn Tyr Glu His Asp Met Phe Val Lys

245 250 255

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

260 265 270

Thr Glu Gly Leu Ser Leu Ala Glu Ala Lys Glu Arg Val Lys Glu Leu

275 280 285

Ala Lys Glu His Glu Asp Ala Val Leu Arg Leu Lys Asp Glu Val Glu

290 295 300

Ser Lys Val Ser Tyr Lys Leu Arg Ile Cys Leu Glu Gly Leu Val Asp

305 310 315 320

Met Val Val Gly Asn Leu Val Trp Ser Ala Ser Cys Asp Arg Tyr Ser

325 330 335

Ser Tyr Arg Arg Glu Lys His Gln Met Glu Leu Pro Ile Arg Ile Gln

340 345 350

Gly Pro Pro Thr Pro Pro Gln Glu Pro Val Tyr Glu Lys Ala Thr Leu

355 360 365

Pro Asn Gly Lys Gln Leu Asp Ala Pro Thr Glu Ser Ser Gly Lys Asp

370 375 380

Leu Ser Asp Gly Val Ala Thr Leu Ser Gly Asp Glu Pro Val Leu Gly

385 390 395 400

Asp Glu Ile Val Ser Ala Pro Ile Lys Tyr Leu Glu Ser Leu Pro Ser

405 410 415

Lys Gly Phe Arg Glu Ala Ile Ile Asp Gly Met Asn Gly Trp Leu Asn

420 425 430

Leu Pro Ala Arg Ser Val Ser Ile Ile Lys Asp Val Val Lys His Ile

435 440 445

His Thr Ala Ser Leu Leu Cys Asp Asp Ile Glu Asp Ser Ser Pro Leu

450 455 460

Arg Arg Gly Gln Pro Ser Ala His Ile Ile Phe Gly Val Ser Gln Thr

465 470 475 480

Val Asn Ser Thr Ser Tyr Leu Trp Thr Leu Ala Ile Asp Arg Leu Ser

485 490 495

Glu Leu Ser Ser Pro Lys Ser Leu Arg Ile Phe Ile Asp Glu Val Arg

500 505 510

Lys Met Gln Ile Gly Gln Ser Phe Asp Leu His Trp Thr Ala Ala Leu

515 520 525

Gln Cys Pro Ser Glu Glu Glu Tyr Leu Ser Met Ile Asp Met Lys Thr

530 535 540

Gly Gly Leu Phe His Leu Leu Ile Arg Leu Met Ile Ala Glu Ser Pro

545 550 555 560

Arg Lys Val Asp Met Asp Phe Ser Gly Leu Val Ser Met Thr Gly Arg

565 570 575

Tyr Phe Gln Ile Arg Asp Asp Leu Ser Asn Leu Thr Ser Glu Glu Tyr

580 585 590

Glu Asn Gln Lys Gly Tyr Cys Glu Asp Leu Asp Glu Gly Lys Tyr Ser

595 600 605

Leu Pro Leu Ile His Ala Leu Lys His Thr Lys Asn Lys Val Gln Leu

610 615 620

Glu Ser Leu Leu Ile Gln Arg Lys Thr Gln Gly Gly Met Thr Leu Glu

625 630 635 640

Met Lys Arg Leu Ala Ile Gln Ile Met Lys Glu Ala Gly Ser Leu Glu

645 650 655

His Thr Arg Lys Val Val Leu Glu Leu Gln Asp Ala Val His Arg Glu

660 665 670

Leu Ala Lys Leu Glu Glu Ala Phe Gly Gln Glu Asn Tyr Val Ile Gln

675 680 685

Leu Ala Leu Glu Arg Leu Arg Ile Lys Ala

690 695

<210> 4

<211> 2274

<212> DNA

<213> Colletotrichum gloeosporioides

<400> 4

atgcctacct tcgcacaacc agttccggac gacatcgtcg cctcgtcggg gctgcggtca 60

aagttccggc ctcatgtgca cggcaactac caaaactgcg tggagccgtc aaaggggatg 120

gagactttct acaacgacgc catgtcgaca cagcttgagt ccaagacgct ggcagacatt 180

ccagggctcg ggctggtgca ccccatggcg ctggccatgg ccaactgtct tccggagcgg 240

ctgccggcca tcacgcggtt tgcggacttc accattctga atgatgatta ttatgatatt 300

gccaagagag acgagatcga gaaagtcaac agtgatatcc aagatgcgct gcaagacgct 360

tcagcgcctg gcaacaagac gaagagctcg ggtggttctg atatcgactt caagccaaaa 420

cagatgcagg ctgcccttgt tcttgagctg atcatgctgg atcagcaact cgcgatggac 480

atcatgtcat catacagcca gggccttgat gtcgccacct ttgctccgga caatctgagg 540

acgttggatg agtacttgcc cgtgcgcaag gtcaactcgg gtcttgacgt caccgctgaa 600

atggtctgct tcggcatggg tctccgaatc gccaaggaag acaaggccaa gctccgcccg 660

gtagtcgatc tcgccaactt cgccatcacc gtcgtcaacg acctctacag ctggcccaag 720

gagatcaagt gccacctcga gacccccggc tccgagctgc ccttcaacgc cgtggccgtg 780

ctcatgcggc acggcgggta ctccgagccc gaggccttcc gcatcctgta cgccaagcag 840

gccgagctgg aggcggagca cctccgccag ctcgacgcgc tcagggcgca ggagggcggc 900

cggctgcctg agaaccagga gctgtacgtc gagaacgcgc agcgcgcggt ctgcggctcg 960

gagctgtgga gcgtgtatac cacgcggtat ccgtccaagg cggacttgca gcagccggag 1020

gtagagtttg tggacgggtc gttcaggtat gtggctgaca gcgagggcgc cggtgaggag 1080

aaggttgtgt acgaaagtgt cgactcggtg ccgacggagg ttgaggacga gttctcctcg 1140

tcggatgcgt ctccgggctc tgttgaccag gccatctcaa caccgccatc gacaacgttc 1200

tgcagctgcg aagacgagga tgagcacatc gccaacgtta aggaagtatc cgaggacgaa 1260

gccaacggcg tccgagacat gtcggatatc tcccaaaagt ccaagaagaa gatcgacacc 1320

ctgcttccgg aaggaccagg actcaccacc tacgcatccc gcctggaggc tgcgcctgac 1380

cacgccgtca tcaccccaat caagtacctc gcctcactgc cctccaaggg cgtccgggac 1440

accttcatcg acgcgctcaa ctggtggctc gaggtccccg aagactcgct gcgcaccatc 1500

aagaccatca tcagcatgct ccacgactca tctctgatct tggacgacat cgaggatgac 1560

tctaccctcc ggagaggcag ccccgcggca cacatgatat tcggaacagc gcagtgcatc 1620

aacgcggcga accacatctt cgtcatggtg ctcgccgagc tgcagaagct tcgcagcccg 1680

ttgaagactg ctatcctcat tgaggagctg gagagtcttt tcgtcggaca ggccgatgac 1740

ctgcactgga agtaccacgt cgactgcccc tcgacggagg actacatgga gatgatcgac 1800

aacaaaaccg gcggcctctt ccgtctatgt gtcagactcc tccaagcaga atcaacaaga 1860

accgacgtcc tcgacctcga cccgcgcccc ttcgtccgcc aactaagcct cttcttccaa 1920

atccgcgacg actaccagaa cctcgtctca gacgcctacg ccaagcaaaa gggcttcgcc 1980

gaggacctcg acgagggcaa gatctccctc cccatcatcc tcaccctgca gcgcgcgcgc 2040

acgcgtcccg agatcatggg cgtgctcaag cacaagcagc ccggcccgat ggcgctcgag 2100

atgaagcagt acatcgtcaa ggagatggaa aagtgcggcg cgctcgagtc cacgcgggaa 2160

ctgctgcagg ggatgcagga ggacttgatt gcggagctga ggaggctgga gggcgacttt 2220

ggggcgaaga atgcgacgtt ggagttggtg ttgagaaggt tgtggatttc ctaa 2274

<210> 5

<211> 2016

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 5

atggatttta aatactctcg ggaactaaag cttgaatccc tggatgcttt gaaccttact 60

gaaggcatcc cattgcgagt caatgaaaac attgaccttg aattccgggg aattgaacga 120

gcacatagcg actgggagcg ctatgtggga aaactcaacg gctttcacgg aggacgaggt 180

ccgcagtttg gattcgttag cgcctgcata ccagagtgtt tgccggaacg tatggagact 240

gtctcctacg caaatgaatt cgcctttctt catgacgata tgacagacgc cgcatcaaag 300

gatcaggtaa atggcctaaa tgatgatctg cttgggggcc ttgatttcac aacagaagca 360

cgttcctcag cttccggaaa gcagcagatg caggcaaagc tgttacttga aatgctttcc 420

attgataggg aacgaacaat ggtcaccatt aaggcatggg cggactttat gcgcggagcc 480

gctggacgtg atcaccatag gggcttctct tccttagatg agtatattcc ttacagatgc 540

gcagactgcg gcgagaaatt ctggtttggc ctggtcacgt ttgcgatggc tctatcaatc 600

cctgagcaag agctagagct ggtgcaacgc cttgcacaaa acgcatacct ggccgcaggc 660

ttaaccaatg acctctactc ttacgagaaa gaacaacttg tggcagagag atcaggaaca 720

ggtcaggtct tcaatgctat tgcggtgatc atgcaggaac attccgtgtc aatcagtgag 780

gcggaggaca tctgccgtgg cagaattcgc gaatacgcgg caaagtatgt cagggatgtg 840

gcggatttga gagctaaaaa tgagctttct cgtgattcct tggcatatct cgagactggg 900

ctttacggaa tcagtggtag tacggcatgg aatcttgact gtccccgcta tcaggtgtcg 960

acatttgttg attttaaaac cccagaagat gagacagcta aagaggagtt tatacatgtt 1020

cccgagcaga aacagttcgt tggcgatggc tctatagagg accaaacgac agaaggaaac 1080

caagaaatcg tgctaaggaa actgccacag atgagtactg aggtgatcga agcaccatat 1140

acatatgtca aatcgctacc gtccaagggc gtacggcaac gcgcgatgca tgcaatcaat 1200

acttggctgc aagtgccgat ggcaaagatg aagcttatcg aggatgttgt tgagaggatt 1260

cataactcct cactcatgct tgatgatatt gaggattctt caccccttcg tcgagagtat 1320

ccggcggcac atatgatatt tggagtcccg cagacgatta attccgccaa ttatgagctt 1380

gttctcgcct tgaatgcagc ccatcaactg ggtaatccaa cttgcctaca gatattcatc 1440

gaggaactac aacgtttaaa tgtcggccag agttatgatc tgtattggac acacaacatg 1500

ataactcctt cgatgaatga ttacttgcgc atgattgaca gcaagactgg cggtctattt 1560

agtatgctta gccggctcat ggtggcctgc tccccgcgca cagtctctgc agatttcgat 1620

tctctttcca gacttgtcgg tcgatttttt caaattcggg atgattatca gaacctagtc 1680

tcagctgagt actccaaaca gaagggcttc tgcgaagacc tggatgaggg gaaatactca 1740

ctgccattga ttcatgcgtt ggagacctgt gtcaatagcg accgcgatat gctgcgcagt 1800

ctactcgtac aacgacgtgt agctggacat ctgaccttcg agcagaaaaa attggtgctg 1860

caaataatgc aacgctgtga aagcttggaa ttcaccaaga gccaactgtg tgtgcttcaa 1920

acacggatcc aggaagagat cgacaaactt gtggcggaat ttggtgacga aaacttttca 1980

ttaaggcttc tcgttgagct gctggtagtt ggttga 2016

<210> 6

<211> 2097

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

atgtgcaata ccaaatgcta caacacctta gcgaaaatga cggtaatcac agagccagcc 60

atggaatata tgtactctgt acctctggat gagtccgaat acgacaaatg cggcttctgc 120

caggatccgc gctaccgtcc gagacgccac aaggatcaac acctcgcccg cgccggttct 180

gcgaaggcga aagagctgtg tgaagcgctc attggtgttt accccaggcc aacatgcgag 240

tctgcagtgg gccactccat cgctttagtg atgccagagt gcatgcctgg gcgagttgag 300

gcgatgggcg agttcatgga atccatcttc tacatggaca acatcgccga gtctggatct 360

cagcaggata cgggcaacct tggaacggag tgggccaacg atatggagac tggcccgacc 420

acttccgtga acagcaatac cggcgcaaag caagtcatgg caaagttagc ccttcagctt 480

ctgtctattg atccggtatg cgcgggcaat gtcatgaagg cgtggaagga gtgggcagcg 540

ggcttcgcca aaccccgtcg tttcgacagc atcgaacaat acatcgacta ccggctcgtc 600

gactccgggg ctattgtggc cgtccatctc atgaacttcg gcatgggcct cgatatttct 660

gtggaggagc tccgtgaagt ctccgacata gtcaaccacg ccggcaaggc gctatcttac 720

cagaacgact tcttcagctt caattacgag catgacatgt tcgtgaagct gccggactca 780

atcggaattg ccaatgccgt ctttgtgctt gccgaaactg aggggctcag cctcgccgaa 840

gcgaaagaaa gggtcaagga gctcgccaaa gaacatgaag atgccgttct gcgtctaaag 900

gatgaggtcg agtccaaggt cagctacaag ctccgtatct gcttggaagg cctggtagat 960

atggtagttg gaaacttggt ttggagcgca tcctgcgacc gttactcatc gtatcgtcgg 1020

gagaagcacc agatggagtt gccgattcga atccagggcc cgcctacacc tccccaggaa 1080

cctgtctatg agaaggctac cctacccaac ggaaaacagt tggatgcacc cacagaatca 1140

tcagggaaag atctgagcga cggcgtagca accttgtccg gagatgagcc ggtgttgggt 1200

gacgagattg tttctgctcc tatcaaatac ctcgaatcat tgccttcgaa agggtttaga 1260

gaagcaatca ttgacggcat gaatggctgg ctgaacctgc ccgctcgctc tgtctccatc 1320

atcaaggacg ttgtcaagca catccatacc gcatcacttc tatgtgatga catcgaagac 1380

tcctcaccac ttcgtcgcgg gcagccttcg gcccacatca tcttcggcgt ctctcaaaca 1440

gtgaactcaa caagttacct atggacgctg gccattgacc gcttatcaga actctcaagt 1500

ccaaaatccc tccgtatatt catagatgag gttcgcaaaa tgcaaattgg ccaaagcttt 1560

gacctgcact ggacggcagc cctgcaatgc ccctcggagg aagagtacct cagcatgata 1620

gatatgaaga cgggtggtct cttccacctg cttatccgcc tgatgattgc tgaaagtcca 1680

cgaaaagttg atatggactt cagtggtctc gtgagcatga ccggtcgtta ctttcagatc 1740

cgtgacgacc tttccaattt gacatccgag gagtacgaaa accaaaaggg atactgcgaa 1800

gaccttgacg agggcaaata ctcactaccc ctcattcatg ctctcaagca tacgaaaaac 1860

aaggtgcagc tggagagcct tctcatccaa cggaagacac agggcggaat gactctcgag 1920

atgaagcgtc tcgctatcca aatcatgaag gaggccggga gcctggaaca tacacgaaag 1980

gtggtgctgg agctacaaga cgccgtccac agagaactcg caaagctcga agaggcattc 2040

ggacaggaaa actacgtcat ccagcttgcg ctcgagaggt taaggatcaa ggcgtag 2097

<210> 7

<211> 10833

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 7

ggtggcaagc ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag 60

aagtttaata atcatattac atggcattac caccatatac atatccatat acatatccat 120

atctaatctt acttatatgt tgtggaaatg taaagagccc cattatctta gcctaaaaaa 180

accttctctt tggaactttc agtaatacgc ttaactgctc attgctatat tgaagtacgg 240

attagaagcc gccgagcggg tgacagccct ccgaaggaag actctcctcc gtgcgtcctc 300

gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa 360

taaagattct acaatactag cttttatggt tatgaagagg aaaaattggc agtaacctgg 420

ccccacaaac cttcaaatga acgaatcaaa ttaacaacca taggatgata atgcgattag 480

ttttttagcc ttatttctgg ggtaattaat cagcgaagcg atgatttttg atctattaac 540

agatatataa atgcaaaaac tgcataacca ctttaactaa tactttcaac attttcggtt 600

tgtattactt cttattcaaa tgtaataaaa gtatcaacaa aaaattgtta atatacctct 660

atactttaac gtcaaggaga aaaaactata gtcgactcat gtaattagtt atgtcacgct 720

tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg 780

aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat 840

ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa 900

ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcggccgg tacccaattc 960

gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg 1020

ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 1080

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 1140

cgaatggcgc gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 1200

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 1260

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 1320

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 1380

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 1440

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 1500

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 1560

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcct gatgcggtat 1620

tttctcctta cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta 1680

aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta 1740

gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc 1800

ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga 1860

tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc 1920

atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat 1980

gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt 2040

acccttagta tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa 2100

aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc 2160

tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc 2220

cgcagaatac tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag 2280

taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa 2340

atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac 2400

taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt 2460

ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc 2520

cttatatgta gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag 2580

ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac 2640

caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa 2700

aaaatttcaa agaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaattg 2760

aaaagctgtg gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 2820

gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 2880

cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 2940

atcaccgaaa cgcgcgaaag agatggagga acgggaaaaa gttagttgtg gtgataggtg 3000

gcaagtggta ttccgtaaga acaacaagaa aagcatttca tattatggct gaactgagcg 3060

aacaagtgca aaatttaagc atcaacgaca acaacgagaa tggttatgtt cctcctcact 3120

taagaggaaa accaagaagt gccagaaata acagtagcaa ctacaataac aacaacggcg 3180

gctacaacgg tggccgtggc ggtggcagct tctttagcaa caaccgtcgt ggtggttacg 3240

gcaacggtgg tttcttcggt ggaaacaacg gtggcagcag atctaacggc cgttctggtg 3300

gtagatggat cgatggcaaa catgtcccag ctccaagaaa cgaaaaggcc gagatcgcca 3360

tatttggtgt ccccgaggat ccaaatttcc aatcttctgg tattaacttc gataactacg 3420

atgatattcc agtggacgcc tctggtaagg atgttcctga accaatcaca gaatttacct 3480

cacctccatt ggacggattg ttattggaaa acatcaaatt ggcccgtttc accaagccaa 3540

cacctgtgca aaaatactcc gtccctatcg ttgccaacgg cagagatttg atggcctgtg 3600

cgcagaccgg ttctggtaag actggtgggt ttttattccc agtgttgtcc gaatcattta 3660

agactggacc atctcctcaa ccagagtctc aaggctcctt ttaccaaaga aaggcctacc 3720

caactgctgt cattatggct ccaactagag agttggccac ccaaattttc gatgaagcca 3780

agaaatttac ttatagatcc tgggtcaagg cctgcgtcgt ctacggtggt tctccaattg 3840

gtaaccaact aagagaaatt gaacgtggtt gcgatctttt agtcgctact ccaggtcgtt 3900

tgaatgactt gttggaacgt ggtaaaattt ctttggccaa cgtcaagtat ttggttctag 3960

atgaagctga tagaatgttg gatatgggtt tcgaacctca aattagacat attgtcgaag 4020

actgtgatat gactcctgtt ggtgaaagac aaactctgat gttctcagct acttttcccg 4080

ctgatatcca acatttggcc cgtgatttct taagtgacta catctttttg tctgttggta 4140

gagtcggttc tacttcagaa aacattactc aaaaagtctt atacgttgaa aatcaagata 4200

agaagtcagc cttattggat ctattgtctg catccactgc tcgaggcttt tgttcccttt 4260

agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 4320

gttatccgct cacaattcca cacaacatag gagccggaag cataaagtgt aaagcctggg 4380

gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 4440

cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 4500

tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 4560

tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 4620

ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 4680

ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 4740

gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 4800

gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 4860

ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 4920

tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 4980

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 5040

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 5100

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 5160

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 5220

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 5280

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 5340

gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 5400

aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 5460

aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 5520

cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 5580

ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 5640

cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 5700

ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 5760

ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 5820

ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 5880

gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 5940

ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 6000

ctggtgaata ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 6060

gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 6120

ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 6180

cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 6240

ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 6300

aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 6360

gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 6420

gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt tgtagaacaa 6480

aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa 6540

cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc atttttgtaa 6600

aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta 6660

cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat acttcttttt 6720

tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct tagattactt 6780

tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca ctgtaggtcc 6840

gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa aaagcctgac 6900

tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt caagataaag 6960

gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa cagaaagtga 7020

tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta ttttgtctct 7080

atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac tctatgaata 7140

gttcttacta caattttttt gtctaaagag taatactaga gataaacata aaaaatgtag 7200

aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt atatagggat 7260

atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga agcggtattc 7320

gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa agtgcgtctt 7380

cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag agaataggaa 7440

cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa tgcaacgcga 7500

gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg cctgtatata 7560

tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac ttatatgcgt 7620

ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc attccatgcg 7680

gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg ccactcctca 7740

attggattag tctcatcctt caatgctatc atttcctttg atattggatc atactaagaa 7800

accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc 7860

gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 7920

gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 7980

ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact cgagtagtga 8040

gtcaacaatg ggttctgggg cccgattgcc tttctcaatg ccaccaaagg gaatttcgac 8100

gaagaagtca ctcctcatct tcaaattcgt tcttacgccc tggctttcgt tccccaccac 8160

tagaacaaca ggcagctcgt tacataatcc gttcaaatcg tgcatgctaa tagtttttcc 8220

aacagtgtat ttttctgacg tggcattagc taagtggctt gtaataaacg tccagccacc 8280

catttcttgt gatttagtaa aaaactctaa cggtttatca acgtaaaata tgggcagaag 8340

ttcgagggcc ccactgcttg tcttggacac cacaggcgtc aaaggagagc agtttcttct 8400

cgacatcaca atgaagtcaa cccccaggaa gtaagcgctt ctaataatgg caccgatatt 8460

gtgagggtca gttatttcat ccagatataa cccgagagga aacttcttag cgtctgtttt 8520

cgtaccataa ggcagttcat gaggtatatt ttcgttattg aagcccagct cgtgaatgct 8580

taatgctgct gaactggtgt ccatgtcgcc taggtacgca atctccacag gctgcaaagg 8640

ttttgtctca agagcaatgt tattgtgcac cccgtaattg gtcaacaagt ttaatctgtg 8700

cttgtccacc agctctgtcg taaccttcag ttcatcgact atctgaagaa atttactagg 8760

aatagtgcca tggtacagca accgagaatg gcaatttcta ctcgggttca gcaacgctgc 8820

ataaacgctg ttggtgccgt agacatattc gaagatagga ttatcattca taagtttcag 8880

agcaatgtcc ttattctgga acttggattt atggctcttt tggtttaatt tcgcctgatt 8940

cttgatctcc tttagcttct cgacgtgggc ctttttcttg ccatatggat ccgctgcacg 9000

gtcctgttcc ctagcatgta cgtgagcgta tttcctttta aaccacgacg ctttgtcttc 9060

attcaacgtt tcccattgtt tttttctact attgctttgc tgtgggaaaa acttatcgaa 9120

agatgacgac tttttcttaa ttctcgtttt aagagcttgg tgagcgctag gagtcactgc 9180

caggtatcgt ttgaacacgg cattagtcag ggaagtcata acacagtcct ttcccgcaat 9240

tttctttttc tattactctt ggcctcctct agtacactct atattttttt atgcctcggt 9300

aatgattttc attttttttt ttccacctag cggatgactc tttttttttc ttagcgattg 9360

gcattatcac ataatggagg gagcagtaaa ggggcgatca gagttgaaaa aaaaatgctg 9420

ccgtaaaaaa agcatgcacg tatacacttg agtaaatata tttatgtatt catatctcaa 9480

gataagatca tgatacataa aagcgatata acgtcttgaa gctcaggtaa ggggctagta 9540

gatgcaatga atgacgatga agatagagcc caactgaagg ctaggctgtg gatccgtgtg 9600

gaagaacgat tacaacaggt gttgtcctct gaggacataa aatacacacc gagattcatc 9660

aactcattgc tggagttagc atatctacaa ttgggtgaaa tggggagcga tttgcaggca 9720

tttgctcggc atgccggtag aggtgtggtc aataagagcg acctcatgct atacctgaga 9780

aagcaacctg acctacagga aagagttact caagaataag aattttcgtt ttaaaaccta 9840

agagtcactt taaaatttgt atacacttat tttttttata acttatttaa taataaaaat 9900

cataaatcat aagaaattcg cgaattctta atgatctctt gcagcaacta attcacaaat 9960

gtatgccaat gcagcaccat caacatcagc atttctcaaa tgtctttgtg cagcttcgat 10020

atggaaagct aatttttcct ttgcaccagc caatgataac aatgctggat atgtagcctt 10080

attgtttgat tgatcagaac caactggttt accaatcttt tcttctgcac cttcaatatc 10140

taaaatatca tctctgattt ggaaagctaa acccaaatgt gcagcaaatt catccaattc 10200

tcttgtttgt cttgcatcag caccaccaat taatgcacca gcatgaactg agtattgcaa 10260

cattttacca gtcttatgtc tatgaatata ttctaattca gacaatgtca aagttttacc 10320

ttcaccttcc atatctgcag cttgacctgc agccataccc tctggacctg cagctttagc 10380

caatctttcg atcaatctca atctaacaga tggtggtatt ctttcatcat cgatttcagt 10440

gatcaattgg aatgcataag ctaacaaacc gtcaccagcc aaaattgcca tagcttcacc 10500

gaaaacctta tggtttgttg gtttacctct tctcaaatca tcattatcca ttgatggcaa 10560

atcatcatga atcaaagaag cagtatggat catttcgatt gcacaagcaa ctggtaaacc 10620

aactgctgga tctttaccca aagctctaac tgttgataac aacaacaatg gtctaattct 10680

tttaccacct gcttctaaag agtatgccat agctttcttc aatttagctg gaccttccaa 10740

tctttcgatg tatcttgaca atgcagtttc aacagcttgc ttttgttcgt tcaagaattg 10800

ttcaacagac aattgagctg atccagtact gcc 10833

Claims

1.一种嵌合三萜合酶，其特征在于：为氨基酸序列如SEQ ID NO.1所示的CgCS、氨基酸序列如SEQ ID NO.2所示的TvTS或氨基酸序列如SEQ ID NO.3所示的MpMS。

2.编码权利要求1所述的嵌合三萜合酶的核酸分子，其特征在于：编码CgCS、TvTS、MpMS的核酸分子的核苷酸序列分别如SEQ ID NO.4、5、6所示。

3.一种重组质粒，其特征在于：含有权利要求2所述的核酸分子。

4.一种重组细胞，其特征在于：含有权利要求2所述的核酸分子或权利要求3所述的重组质粒。

5.权利要求1所述的嵌合三萜合酶、权利要求2所述的核酸分子、权利要求3所述的重组质粒或权利要求4所述的重组细胞在制备三萜化合物中的应用。

6.根据权利要求5所述的应用，其特征在于：所述的三萜化合物为结构如式I或式II所示的化合物，

7.一种制备权利要求6中所述的三萜化合物的方法，其特征在于：培养权利要求4所述的重组细胞得到含所述三萜化合物的培养物。

8.一种三萜化合物，其特征在于：所述的三萜化合物的结构如式I或式II所示，