CN116478972A - 梅片树倍半萜合酶CbTPS4及其相关生物材料与应用 - Google Patents
梅片树倍半萜合酶CbTPS4及其相关生物材料与应用 Download PDFInfo
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Abstract
本发明公开了一种梅片树倍半萜合酶CbTPS4及其相关生物材料与应用。本发明首先公开了如下任一所示蛋白质:A1)由序列2所示的氨基酸序列组成的蛋白质;A2)序列2所示的蛋白质的N端或/和C端连接蛋白标签得到的融合蛋白;A3)将序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有90%以上的同一性且功能相同的蛋白质。本发明进一步公开了上述蛋白质相关生物材料及其应用。本发明首次从梅片树中得到单萜类成分合成关键酶基因,并证明其蛋白CbTPS4能够催化FPP生产(‑)‑β‑榄香烯,对于调节和生产植物萜类化合物及培育高品质的梅片树具有重要的理论及实际意义。
Description
技术领域
本发明涉及药用植物基因工程领域,具体涉及一种梅片树萜类合酶CbTPS4及其相关生物材料与应用。
背景技术
梅片树(physiolgical type of Cinnamomum burmannii)是樟科樟属常绿乔木,富含右旋龙脑((+)-borneol),是珍稀药材,也是一种高级香料、日化美妆用品原料、化工用品,被广泛应用于医疗、美容、香料等多个行业。中国科学院华南植物园在广东境内发现的一种具有生产利用价值的天然右旋龙脑新资源,填补了我国生产天然右旋龙脑的空白。其挥发油中除右旋等单萜类成分外,还有大量倍半萜成分。其中部分倍半萜类化合物具有显著的药理学活性,例如,梅片树挥发性成分中含有微量的β-榄香烯。β-榄香烯是具有榄香烷骨架的倍半萜,从温莪术的挥发油中分离出的β-榄香烯是我国自行开发研制的抗肿瘤药物榄香烯乳的主要成分。
萜类合酶(Terpene synthase,TPS)是梅片树基因组中一类重要的萜类次生代谢产物合成相关的基因家族。CbTPS4是梅片树萜类合酶基因家族中的一员。目前,尚未发现从梅片树可以得到具有β-榄香烯合成能力的关键酶基因的相关研究。
发明内容
本发明所要解决的技术问题为得到新的参与倍半萜化合物合成的梅片树萜类合酶,以合成或制备β-榄香烯。
为解决上述问题,本发明首先提供了一种蛋白质,所述蛋白质为CbTPS4,来源于梅片树(physiolgical type of Cinnamomum burmannii),命名为梅片树萜类合酶CbTPS4,是如下a)或b)或c)的蛋白质:
a)氨基酸序列是序列2所示的蛋白质;
b)在序列2所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质;
c)将序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质。
其中,序列2由561个氨基酸残基组成。
上述蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
上述蛋白质中,蛋白标签(protein-tag)是指利用DNA体外重组技术,与目的蛋白一起融合表达的一种多肽或者蛋白,以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中,同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
上述蛋白质中,所述90%以上的同一性可为至少91%、92%、95%、96%、98%、99%或100%的同一性。
与CbTPS4的相关生物材料也在本发明的保护范围之内。
本发明提供的与CbTPS4的相关生物材料为下述A1)至A12)中的任一种:
A1)编码CbTPS4的核酸分子;
A2)含有A1)所述核酸分子的表达盒;
A3)含有A1)所述核酸分子的重组载体;
A4)含有A2)所述表达盒的重组载体;
A5)含有A1)所述核酸分子的重组微生物;
A6)含有A2)所述表达盒的重组微生物;
A7)含有A3)所述重组载体的重组微生物;
A8)含有A4)所述重组载体的重组微生物;
A9)含有A1)所述核酸分子的转基因植物细胞系;
A10)含有A2)所述表达盒的转基因植物细胞系;
A11)含有A3)所述重组载体的转基因植物细胞系;
A12)含有A4)所述重组载体的转基因植物细胞系。
上述生物材料中,A1)所述核酸分子为如下B1)-B5)任一所示:
B1)序列表中序列1所示的DNA分子;
B2)编码序列是序列表中序列1所示的DNA分子;
B3)序列表中序列4所示的DNA分子;
B4)编码序列是序列表中序列4所示的DNA分子;
B5)在严格条件下与B1)或B2)或B3)或B4)限定的DNA分子杂交,且编码CbTPS4的DNA分子。
其中,序列表中的序列1由1686个核苷酸组成,编码序列2所示的蛋白质。
所述严格条件是在2×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次5min,又于0.5×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次15min。
其中,所述核酸分子可以为DNA,如cDNA、基因组DNA或重组DNA,所述核酸分子也可以为RNA,如mRNA或hnRNA。
上述生物材料中,A2)所述的含有编码CbTPS4的核酸分子的表达盒(CbTPS4基因表达盒),是指能够在宿主细胞中表达CbTPS4的DNA,该DNA不但可包括启动CbTPS4转录的启动子,还可包括终止CbTPS4转录的终止子。进一步,所述表达盒还可包括增强子序列。
上述生物材料中,所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中,所述微生物可为酵母、细菌、藻或真菌,如农杆菌。
上述生物材料中,所述转基因植物细胞系、转基因植物组织和转基因植物器官均不包括繁殖材料。
本发明进一步提供了上述蛋白质或其相关生物材料的应用。
所述应用具体如下所示:
1)上述蛋白质作为萜类合酶的应用;
2)上述相关生物材料在制备萜类合酶中的应用;
3)上述蛋白质或相关生物材料在制备或合成萜类化合物中的应用;
4)上述蛋白质或相关生物材料在催化法尼基焦磷酸形成(-)-β-榄香烯中的应用。
上述应用中,所述萜类化合物为(-)-β-榄香烯。
本发明还提供了制备CbTPS4的方法。
本发明制备CbTPS4的方法,包括将CbTPS4的编码基因导入到受体微生物中,得到表达CbTPS4的重组微生物,培养所述重组微生物,表达得到CbTPS4。
上述方法中,所述受体微生物为原核微生物。具体的,所述原核微生物为大肠杆菌。更具体的,所述大肠杆菌为大肠杆菌表达菌株Transetta(DE3)。
上述方法中,所述CbTPS4的编码基因可通过重组质粒pET32a::CbTPS4导入到大肠杆菌表达菌株Transetta(DE3)中;所述重组质粒pET32a::CbTPS4是用序列1所示的CbTPS4基因构建至pET32a(+)载体的BamHI酶切位点处,且保持pET32a(+)载体其他序列不变得到的重组表达载体。
本发明进一步提供了一种制备(-)-β-榄香烯的方法,所述方法包括用CbTPS4催化法尼基焦磷酸(FPP)的步骤。
上述方法中,在催化过程中还需要加入酶促缓冲液,所述酶促缓冲液由HEPES、MgCl2、DTT组成;
所述HEPES在所述酶促缓冲液中的浓度为50mM;
所述MgCl2在所述酶促缓冲液中的浓度为10mM;
所述DTT在所述酶促缓冲液中的浓度为5mM;
所述酶促缓冲液中的pH值为7.2。
本发明从梅片树cDNA中克隆得到CbTPS4基因,通过实验证明:本发明的CbTPS4蛋白能够催化FPP形成(-)-β-榄香烯,对梅片树中的(-)-β-榄香烯等单萜类化合物的生物合成具有重要作用,为利用基因工程技术提高梅片树中活性成分(-)-β-榄香烯含量或直接生产(-)-β-榄香烯提供重要基础,进一步对于调节和生产植物萜类化合物及培育高品质的梅片树树具有重要的理论及实际意义。
附图说明
图1为梅片树CbTPS4基因克隆琼脂糖凝胶电泳图;M表示Trans2K DNA Marker(核酸分子量标准,条带由上往下分别为2000、1000、750bp),CbTPS4表示CbTPS4基因。
图2为聚丙烯酰胺凝胶电泳(SDS-PAGE)分析在大肠杆菌中表达的CbTPS4蛋白。其中,M为Premixed Protein Marker(蛋白分子量标准,条带由上往下分别为170、130、95、72、55、43KDa),Vector为对照菌上清的电泳结果,CbTPS4为重组质粒pET32a::CbTPS4重组菌上清的电泳结果;箭头表示重组质粒pET32a::CbTPS4表达的目的蛋白(即重组蛋白CbTPS4)。
图3为CbTPS4酶促反应产物GC-MS分析。其中,A为标准品(-)-β-榄香烯的提取离子流图,B为pET32a::CbTPS4重组菌上清的目标化合物的提取离子流图;C为标准品(-)-β-榄香烯的质谱图;D为pET32a::CbTPS4重组菌上清的目标化合物的质谱图。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中的High-Fidelity DNA Polymerase、BamHI限制性内切酶是New England Biolabs公司的产品;
快速通用植物RNA提取试剂盒是北京华越洋生物科技有限公司的产品;
TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix、Trans2KDNA Marker、pEASY-Uni Seamless Cloning and Assembly Kit、大肠杆菌感受态细胞Transetta(DE3)是北京全式金生物技术有限公司的产品;
Premixed Protein Marker(Low)是Takara公司的产品;
PageRulerTMPrestained Protein Ladder是ThermoFisher Scientific公司的产品;
pET32a(+)载体是Novagen公司的产品;
Ni-NTA Agarose是Qiagen公司的产品(产品目录号:30210);
蛋白纯化及SDA-PAGENa2HPO4、NaCl、DTT、PMSF、Imidazole、丙烯酰胺/甲叉双丙烯酰胺(30%溶液)是生工生物工程(上海)股份有限公司的产品;
蛋白超滤管(Amicon-Ultra-15)是Millipore公司的产品(产品目录号:UFC903024);
pESC-Leu载体是Agilent公司的产品;
SD-Ura、SD-Ura-Leu是北京泛基诺科技有限公司的产品;
ZYMO RESEARCH Frozen-EZ Yeast Transformation II试剂盒Zymo Research公司的产品;
BY4741酵母菌株(基因型:MATa his3Δ1leu2Δ0met15Δ0ura3Δ0)是北京华越洋生物科技有限公司的产品;
法尼基焦磷酸(FPP)是Sigma公司的产品,产品目录号为G6772,CAS号为763-10-0;
(-)-β-榄香烯是中检院对照品,批号:100268-201903。
实施例1、梅片树CbTPS4基因全长cDNA序列克隆
1、总RNA的提取
根据北京华越洋生物科技有限公司快速通用植物RNA提取试剂盒说明书进行操作,提取梅片树树叶片的总RNA。
2、第一链cDNA的合成
根据北京全式金生物技术有限公司第一链cDNA合成试剂盒TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix说明书进行操作,最终反转录获得cDNA。
其中,反转录反应体系如表1所示。
表1
反转录的步骤如下:
(1)为获得更高的合成效率,取Total RNA、Anchored Oligo(dT)18Primer、RNase-free Water于PCR管中,混匀,65℃,5min;
(2)在上述PCR管中加入10.0μL 2×TS Reaction Mix、1.0μLRT/RIEnzyme Mix、1.0μL gDNA Remover,轻轻混匀;
(3)以“42℃ 30min,85℃ 5s”条件进行反转录反应,获得第一链cDNA;
(4)第一链cDNA于-20℃保存。
3、引物的设计
根据梅片树叶片转录组数据,获得开放阅读框(ORF)序列,并基于此设计克隆引物CbTPS4-F1和CbTPS4-R1,引物序列如下:
CbTPS4-F1:5’-ATGGCTCTTGTTTCTGGTTCTGG-3’;
CbTPS4-R1:5’-TTAAACTGGAATAGGATTAACAAGC-3’。
4、PCR扩增
以步骤2获得的第一链cDNA为模板,采用高保真酶High-Fidelity DNAPolymerase、CbTPS4-F1和CbTPS4-R1引物进行PCR扩增,得到PCR扩增产物,基因克隆琼脂糖凝胶电泳结果如图1所示,并对PCR扩增产物进行测序。
其中,PCR扩增的程序如下:
98℃预变性3min;98℃ 20s,55℃ 20s,72℃ 1min,35个循环;72℃延伸5min。
测序结果表明:PCR扩增产物的序列如序列1所示,将序列1所示的基因命名为CbTPS4,编码由561个氨基酸残基组成的蛋白质,该蛋白命名为CbTPS4,该蛋白的氨基酸序列为序列2。
实施例2、梅片树CbTPS4蛋白的获得及其功能分析
一、梅片树CbTPS4蛋白的获得
1、重组载体的构建
采用北京全式金生物技术有限公司pEASY-Uni Seamless Cloning and AssemblyKit,将序列1所示的CbTPS4基因构建至pET32a(+)载体(Novagen公司)的BamHI酶切位点处,且保持pET32a(+)载体其他序列不变,得到重组质粒pET32a::CbTPS4(已经测序验证)。
具体步骤如下:
1)以实施例1中获得的PCR扩增产物为模板,利用引物CbTPS4-F2和CbTPS4-R2进行PCR扩增,回收并进行纯化获得纯化PCR产物。其中,所述引物序列如下(下划线所示序列为载体同源区):
引物序列如下(下划线所示序列为载体同源区):
CbTPS4-F2:
5’-CCATGGCTGATATCGGAATGTCTATTGTATTGACCTCTAGCC-3’;
CbTPS4-R2:
5’-ACGGAGCTCGAATTCGG TTAAACTGGAATAGGATTAACAAGC-3’。
2)取pET32a(+)载体(Novagen公司),用限制性内切酶BamHI进行酶切,回收线性化的载体骨架。
3)取步骤1)得到的纯化PCR产物,按北京全式金生物技术有限公司pEASY-UniSeamless Cloning and Assembly Kit说明书操作,将其克隆至步骤2)的线性化的载体骨架,得到重组质粒pET32a::CbTPS4。
2、重组菌的获得
将重组质粒pET32a::CbTPS4转化至大肠杆菌表达菌株Transetta(DE3)(购自北京全式金生物技术有限公司),得到pET32a::CbTPS4重组菌;同时用不含目的基因的pET32a(+)载体转化大肠杆菌表达菌株Transetta(DE3)作为对照菌。
3、诱导表达重组蛋白CbTPS4
挑取pET32a::CbTPS4重组菌和对照菌分别接种于2mL的LB液体培养基(含氨苄青霉素100mg/L)中,于37℃振荡培养过夜。次日按1:100稀释加入到200mL LB液体培养基中,37℃振荡培养至OD600为0.6-0.8时转入18℃振摇1小时,加入IPTG至终浓度0.5mM,继续于18℃摇床培养24小时诱导目标蛋白表达。将菌液用8000g离心5min,弃上清,收集pET32a::CbTPS4重组菌和对照菌菌体,保存在-80℃冰箱备用。
4、重组蛋白CbTPS4提取
提取pET32a::CbTPS4重组菌和对照菌菌体中的蛋白。
具体步骤如下:将pET32a::CbTPS4重组菌和对照菌菌体用预冷的5mL HEPES缓冲液(25mM HEPES,5M MgCl2,5M DTT,pH 7.0)重悬;置冰浴中超声破菌(30%功率,超声5s,间隔5s,持续5min,重复1次),12000g,4℃离心30min,分别得到pET32a::CbTPS4重组菌上清和对照菌上清,即为蛋白溶液。
将pET32a::CbTPS4重组菌上清和对照菌上清进行SDS-PAGE,结果如图2所示。从图中可以看出,pET32a::CbTPS4重组菌上清具有重组质粒pET32a::CbTPS4表达的重组蛋白CbTPS4,所述重组蛋白CbTPS4大小约为88kDa,大小与预期相符。对照菌上清无相应蛋白。
二、重组蛋白CbTPS4酶促活性分析
1、酶促活性
(1)取pET32a::CbTPS4重组菌上清进行酶促反应,获得酶促反应产物。其中,酶促反应具体步骤如下:
酶促反应总体系为0.2mL,取pET32a::CbTPS4重组菌上清190μL(所述pET32a::CbTPS4重组菌上清包含酶促缓冲液,即HEPES缓冲液(25mM HEPES,5M MgCl2,5M DTT,pH7.0)),加入底物法尼基焦磷酸(FPP)10μL,混匀,并将酶促反应总体系用200μL正己烷覆盖液封,在30℃放置2小时后,得到pET32a::CbTPS4重组菌上清酶促反应产物,,用于GC-MS分析。
3、GC-MS分析
利用气质联用GC-MS对pET32a::CbTPS4重组菌上清、纯化蛋白的目标化合物进行检测:GC-MS分析系统为Thermo TRACE 1310/TSQ 8000gas chromatograph,进样量1μL,splitless模式,气相色谱柱为Agilent J&W Cyclodex-B手性柱(30m×0.25mm×0.25μm),氦气流速1.0mL/min,进样口温度220℃,离子源温度200℃,升温程序为50℃保持2min,程序升温3℃·min-1到150℃,并保持5min,10℃·min-1到220℃,电子能量70eV,对样品进行50-500m/z范围扫描。
GC-MS分析结果如图3所示:说明CbTPS4重组蛋白能够催化FPP形成(-)-β-榄香烯((+)-elemene),表明重组蛋白CbTPS4为倍半萜合酶。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 四川弘合生物科技有限公司
<120> 梅片树倍半萜合酶CbTPS4及其相关生物材料与应用
<130> 20220110
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1686
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtctattg tattgacctc tagcctttca gatgctccaa acaaacacca tccggtagga 60
aatagaactt ctgaggtagt tcgtagatca gtgaactatg ttccggagat atggggtgat 120
cgctttgttg catcctctcc ggataacttg aaacctgatg cacaaaccca acaaagagct 180
aatgagttga aggaagaagt gaggaaaatg ctaaggaatg tagaggatca tctgcaagaa 240
ttgaatctga tcgatgctgt ccaacgttta ggagtggcct accacttcga agaagagatt 300
gcacaagctc tactccggat gtacaaatca gggagggatt acggtgatga tttgcatgca 360
gttgctctcc aatttcgact tttaaggcaa gaaggttaca atgtgtcacc cgatgtattc 420
atgaagttta aagatgaaga aggtaaattc aagagaactc tagctggtga cacaagaagt 480
ttgcttagtt tatacgaagc agcacatatg ggaactcatg gagaaaacat attagatgaa 540
gccattgctt ttacaagaga gcatcttaac ttggcactcc cttgtcttaa cccacctttt 600
tcaaccctgg ttgagctcgc actagagcta cctcttcgta agcgcatgga aaggatacaa 660
acaaggtatt acatctctat ctaccaagaa gacgaggacc gaagcaatat cctactagag 720
tttgcaaagc gagatttcaa tcttttgcaa cttttgcatc aacaagagct aagagaagtc 780
tcaatgtggt ggaagagttg ggattttgct gcaaagctgc cattcatcag agatagaatt 840
gttgagtgct acttttggat attaggagtg tattttgaac cacaatactc tcgagccaga 900
aagatgatga ctaaaattgt atcattagga tcaatcatgg atgacatcta cgacttgcat 960
ggtacgctag aggaacttga accatacaca gatgcaatcc aaaggtggga tcgaagcgtc 1020
atcgatcagt ttcctgacta catgaagcta catttttccg cactcttaga cacttttgac 1080
aaatttggag aagagttggc tcaagaaggg aaatcctatc gtatacccta cttaaaacaa 1140
gtgttcaagg aagtctcaaa aggatacttg attgaagaac aatggtcaaa ctcaggtcat 1200
gtcccaacac tagaagagta tatgaccaat gccttgatta ctagtgcata ccctatgctt 1260
tctgtggctt catatgttgg catgggagat gttgcaacca aggaagcctt tgactgggga 1320
gtaaacatgc ctaagctcat tgaagctgct tctgtaattt gcagactcaa ggatgacatc 1380
acatcaaacc agcttgaaca agagagagga catgtggcgt caattatcca gatctacatg 1440
aatgaaaatg gaagcacata tgaagaggca tgtgaaaagt ttaaaaggat ggctgcagat 1500
gcatggaaag acataaacaa ggaatgcctg aagcccactc cagctcctat gcctctgctc 1560
atgcgaactg taaatctcac acgtgtgatt gaagtcctgt atcaacatag agatggatac 1620
accaatccca catatgagac caaagaacgt atcttgtcag tgcttgttaa tcctattcca 1680
gtttaa 1686
<210> 2
<211> 561
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Ile Val Leu Thr Ser Ser Leu Ser Asp Ala Pro Asn Lys His
1 5 10 15
His Pro Val Gly Asn Arg Thr Ser Glu Val Val Arg Arg Ser Val Asn
20 25 30
Tyr Val Pro Glu Ile Trp Gly Asp Arg Phe Val Ala Ser Ser Pro Asp
35 40 45
Asn Leu Lys Pro Asp Ala Gln Thr Gln Gln Arg Ala Asn Glu Leu Lys
50 55 60
Glu Glu Val Arg Lys Met Leu Arg Asn Val Glu Asp His Leu Gln Glu
65 70 75 80
Leu Asn Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr His Phe
85 90 95
Glu Glu Glu Ile Ala Gln Ala Leu Leu Arg Met Tyr Lys Ser Gly Arg
100 105 110
Asp Tyr Gly Asp Asp Leu His Ala Val Ala Leu Gln Phe Arg Leu Leu
115 120 125
Arg Gln Glu Gly Tyr Asn Val Ser Pro Asp Val Phe Met Lys Phe Lys
130 135 140
Asp Glu Glu Gly Lys Phe Lys Arg Thr Leu Ala Gly Asp Thr Arg Ser
145 150 155 160
Leu Leu Ser Leu Tyr Glu Ala Ala His Met Gly Thr His Gly Glu Asn
165 170 175
Ile Leu Asp Glu Ala Ile Ala Phe Thr Arg Glu His Leu Asn Leu Ala
180 185 190
Leu Pro Cys Leu Asn Pro Pro Phe Ser Thr Leu Val Glu Leu Ala Leu
195 200 205
Glu Leu Pro Leu Arg Lys Arg Met Glu Arg Ile Gln Thr Arg Tyr Tyr
210 215 220
Ile Ser Ile Tyr Gln Glu Asp Glu Asp Arg Ser Asn Ile Leu Leu Glu
225 230 235 240
Phe Ala Lys Gln Asp Phe Asn Leu Leu Gln Leu Leu His Gln Gln Glu
245 250 255
Leu Arg Glu Val Ser Met Trp Trp Lys Ser Trp Asp Phe Gly Ala Lys
260 265 270
Leu Pro Phe Ile Arg Asp Arg Ile Val Glu Cys Tyr Phe Trp Ile Leu
275 280 285
Gly Val Tyr Phe Glu Pro Gln Tyr Ser Arg Ala Arg Lys Met Met Thr
290 295 300
Lys Ile Val Ser Leu Gly Ser Ile Met Asp Asp Phe Tyr Asp Leu His
305 310 315 320
Gly Thr Leu Glu Glu Leu Glu Pro Tyr Thr Asp Ala Ile Gln Arg Trp
325 330 335
Asp Arg Ser Ile Ile Asp Gln Phe Pro Asp Tyr Met Lys Leu His Phe
340 345 350
Ser Ala Leu Leu Asp Thr Val Glu Lys Phe Glu Glu Glu Leu Ala Leu
355 360 365
Glu Gly Lys Ser Tyr Arg Ile Pro Tyr Phe Lys Gln Ala Phe Lys Gly
370 375 380
Leu Ser Lys Ala Tyr Leu Val Glu Val Gln Trp Ser Asn Ser Ser His
385 390 395 400
Val Pro Thr Leu Asp Glu Tyr Met Thr Asn Ala Leu Met Ser Ser Glu
405 410 415
Tyr Pro Met Leu Ser Val Ala Ser Tyr Val Gly Met Gly Asp Val Ala
420 425 430
Thr Lys Glu Ala Phe Asp Trp Ala Val Ser Met Pro Lys Leu Ile Glu
435 440 445
Val Ala Ala Ala Asn Gly Arg Leu Lys Asn Asp Ile Thr Ser Asn Gln
450 455 460
Leu Glu Gln Glu Arg Asp His Val Ala Thr Ala Ile Gln Ile Tyr Met
465 470 475 480
Asn Glu Asn Gly Ser Thr Tyr Glu Glu Ala Cys Glu Lys Val Ser Arg
485 490 495
Met Ala Gly Asp Ala Trp Lys Asp Ile Asn Lys Glu Cys Leu Lys Pro
500 505 510
Pro Pro Ala Pro Met Pro Ile Leu Met Arg Ile Val Asn Leu Thr Arg
515 520 525
Ala Gly Glu Met Phe Tyr Gln His Arg Asp Gly Tyr Thr Asn Pro Thr
530 535 540
Tyr Glu Thr Lys Glu His Val Leu Ser Val Ile Val Asn Pro Ile Pro
545 550 555 560
Val
Claims (9)
1.蛋白质,是如下a)或b)或c)的蛋白质:
a)氨基酸序列是序列2所示的蛋白质;
b)在序列2所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质;
c)将序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质。
2.与权利要求1所述的蛋白质的相关生物材料,为下述任一种:
A1)编码权利要求1所述的蛋白质的核酸分子;
A2)含有A1)所述核酸分子的表达盒;
A3)含有A1)所述核酸分子的重组载体;
A4)含有A2)所述表达盒的重组载体;
A5)含有A1)所述核酸分子的重组微生物;
A6)含有A2)所述表达盒的重组微生物;
A7)含有A3)所述重组载体的重组微生物;
A8)含有A4)所述重组载体的重组微生物;
A9)含有A1)所述核酸分子的转基因植物细胞系;
A10)含有A2)所述表达盒的转基因植物细胞系;
A11)含有A3)所述重组载体的转基因植物细胞系;
A12)含有A4)所述重组载体的转基因植物细胞系。
3.根据权利要求2所述的相关生物材料,其特征在于A1)所述核酸分子为如下B1)-B5)任一所示:
B1)序列表中序列1所示的DNA分子;
B2)编码序列是序列表中序列1所示的DNA分子;
B3)序列表中序列4所示的DNA分子;
B4)编码序列是序列表中序列4所示的DNA分子;
B5)在严格条件下与B1)或B2)或B3)或B4)限定的DNA分子杂交,且编码权利要求1中所述蛋白质的DNA分子。
4.权利要求1所述的蛋白质作为倍半萜合酶的应用。
5.权利要求2所述的相关生物材料在制备倍半萜合酶中的应用。
6.权利要求1所述的蛋白质或权利要求2所述的相关生物材料在制备或合成单萜化合物的应用。
7.权利要求1所述的蛋白质或权利要求2所述的相关生物材料在催化法尼基焦磷酸形成(-)-β-榄香烯的应用。
8.制备权利要求1所述蛋白质的方法,其特征在于:所述方法包括将权利要求1所述的蛋白质的编码基因导入到受体微生物中,得到表达权利要求1所述蛋白质的重组微生物,培养所述重组微生物,表达得到权利要求1所述的蛋白质。
9.一种制备(-)-β-榄香烯的方法,其特征在于:所述方法包括用权利要求1所述的蛋白质催化法尼基焦磷酸的步骤。
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