CN114107240B - 苦荞来源的大黄素糖基转移酶及其编码基因和应用 - Google Patents
苦荞来源的大黄素糖基转移酶及其编码基因和应用 Download PDFInfo
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- CN114107240B CN114107240B CN202111232676.XA CN202111232676A CN114107240B CN 114107240 B CN114107240 B CN 114107240B CN 202111232676 A CN202111232676 A CN 202111232676A CN 114107240 B CN114107240 B CN 114107240B
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Abstract
本发明公开了苦荞来源的大黄素糖基转移酶及其编码基因和应用。本发明从苦荞中克隆了一个苦荞蒽醌类合成途径中的新的糖基转移酶基因;进一步利用基因工程手段,将该糖基转移酶编码基因序列遗传转化荞麦外植体获得过表达的转基因荞麦毛状根;通过原核表达获得重组大黄素糖基转移酶蛋白,通过体外酶活检测和催化验证表明该糖基转移酶可高效地将蒽醌类化合物大黄素转化成对应的糖苷。应用本发明提供的葡萄糖转移酶可以通过体外生物工程方法大量合成大黄素对应糖苷,为商业化生产利用大黄素及其糖苷类化合物提供了新的方法,该方法效果可靠、成本低廉,生产过程高效、绿色、安全、无环境污染。
Description
技术领域
本发明涉及糖基转移酶及其编码基因,尤其涉及从苦荞中分离得到的大黄素糖基转移酶及其编码基因,本发明进一步涉及它们在蒽醌类物质大黄素的8-OH位的糖苷催化中的应用,属于大黄素糖基转移酶及其编码基因和应用领域。
背景技术
苦荞(Fagopyrum tataricum)属于双子叶蓼科(Polygonaceae)荞麦属作物,苦荞含有丰富的次生代谢产物,如黄酮类(芦丁、槲皮素和异槲皮素等)、肌醇类和蒽醌类物质。其中,蒽醌类化合物具有抗肿瘤、抗菌、抗氧化、利尿和止血等功能。蒽醌类化合物在天然药物中主要以游离态形式和与糖成苷的结合态形式共同存在,在体内常以与葡萄糖成苷的形式存在。
植物分子糖基化是对体内天然化合物常见的一类修饰反应,会形成结构更加稳定的天然糖苷化合物。糖基化反应中糖基转移酶在植物重要天然活性产物合成及生理活动过程中发挥重要作用,是植物次生代谢物质合成途径中的常见且重要的下游修饰反应。药用植物中有很多重要活性成分都是糖基化的产物,比如大黄的活性成分大黄素苷,红景天活性成分红景天苷,荞麦中的槲皮苷、烟花苷和紫云英苷等。但是,目前在苦荞中各蒽醌类化合物如大黄素的代谢机理尚不清晰,尤其是关于荞麦属植物的大黄素等蒽醌类物质的糖基化反应几乎仍属空白。此外,迄今为止还没有关于荞麦中大黄素等蒽醌类化合物的特异性8-OH位的葡萄糖基化反应及其功能应用的报道。
发明内容
本发明的目的之一是提供一种苦荞来源的大黄素糖基转移酶及其编码基因;
本发明的目的之二将所述苦荞来源的大黄素糖基转移酶应用于催化蒽醌类物质的糖基化反应。
为实现上述目的,本发明所采取的主要技术方案包括:
本发明一方面公开了一种苦荞来源的大黄素糖基转移酶,其氨基酸序列为SEQ IDNo.2所示。
本发明另一方面公开了苦荞来源的大黄素糖基转移酶的编码基因 (FtUGT74L2),其CDS的多核苷酸序列为(a)、(b)或(c)所示:
(a)SEQ ID No.1所示的多核苷酸序列;或
(b)与SEQ ID No.1的互补序列在严谨杂交条件能够进行杂交的多核苷酸序列,该多核苷酸编码的蛋白仍具有糖基转移酶的功能或活性;或
(c)与SEQ ID No.1的多核苷酸序列至少有80%以上同源性的多核苷酸序列,且该多核苷酸编码的蛋白仍具有糖基转移酶的功能或活性;优选的,与SEQ ID No.1的多核苷酸序列至少有85%以上同源性的多核苷酸序列,且该多核苷酸编码的蛋白仍具有糖基转移酶的功能或活性;更优选的,与SEQ ID No.1的多核苷酸序列至少有90%以上同源性的多核苷酸序列,且该多核苷酸编码的蛋白仍具有糖基转移酶的功能或活性。
本发明再一方面提供了将大黄素糖基转移酶应用于催化蒽醌类化合物转化为对应的糖苷的糖基化反应;其中,所述的蒽醌类物质优选是大黄素。
作为本发明的一种优选的具体实施方案,所述的大黄素糖基转移酶酶在催化蒽醌类物质转化为对应的糖苷包括:以大黄素糖基转移酶为催化酶催化大黄素进行8-OH位的特异性糖基化反应生成大黄素-8-O-葡萄糖苷。
由此,本发明提供了一种体外将蒽醌类化合物转化为对应的糖苷的方法,包括:以大黄素糖基转移酶为催化酶,催化蒽醌类化合物的糖基化反应得到蒽醌类化合物对应的糖苷化合物。
此外,本发明所分离的大黄素糖基转移酶酶编码基因FtUGT74L2也能应用于促进植物中蒽醌类物质的糖基化反应,譬如,将大黄素糖基转移酶编码基因FtUGT74L2可操作的与植物表达载体连接构建得到重组植物表达载体,将所构建的重组植物表达载体转化到植物中得到过表达植物。
可以采用本领域的常规技术手段,将大黄素糖基转移酶编码基因 FtUGT74L2通过常规的原核表达或真核表达的方法获得重组大黄素糖基转移酶,采用该重组大黄素糖基转移酶就可通过体外转化或催化的方法,催化蒽醌类化合物的糖基化反应得到蒽醌类化合物对应的糖苷化合物。
本发明还公开了含有所述大黄素糖基转移酶编码基因的重组表达载体;优选的,所述重组表达载体可以为重组植物表达载体或重组原核表达载体。
本发明进一步公开了含有所述大黄素糖基转移糖酶编码基因 FtUGT74L2的重组宿主细胞或重组菌;其中,所述重组菌包括但不限于重组大肠杆菌或重组植物细胞。
在本发明中,可以采用任何植物转化方法将本发明所构建的重组植物表达载体引入到目标植物的细胞、组织或器官中,得到转化体;再由转化体通过植物组织培养方法再生得到完整的植株及其无性系或其后代;所述的转化方法包括:农杆菌介导的转化、原生质体转化、Ti质粒、Ri质粒、植物病毒载体、显微注射、电穿孔法、微粒轰击等。
将本发明的SEQ ID No.1所示的基因与其他基因相嵌合或连接得到的嵌合基因或表达盒均属于本发明的保护范畴;含有所述的嵌合基因或表达盒的重组表达载体同样也属于本发明的保护范围之内。
通过本发明所披露的方法获得的转基因植物细胞和植物也可以进一步用于后续的转化程序中,例如用来引入其它的嵌合基因。
本发明从苦荞克隆了一个苦荞蒽醌类(大黄素)合成途径中的新的葡萄糖转移酶基因,将其命名为FtUGT74L2;本发明利用基因工程手段进一步将糖基转移酶FtUGT74L2基因编码序列遗传转化荞麦外植体获得过表达的转基因荞麦毛状根;进一步创建了将MBP-FtUGT74L2融合载体,转化至宿主菌进行原核表达以快速获得重组MBP-FtUGT74L2蛋白,完成了重组蛋白的体外酶活检测和催化验证,结果表明该酶可在体外高效地将蒽醌类化合物大黄素转化成对应的糖苷,即将大黄素8-OH位与UDP-糖中一分子葡萄糖结合并特异性加成修饰,生成大黄素-8-O-葡萄糖苷。因此,应用本发明提供的葡萄糖转移酶可以通过体外生物工程方法大量合成大黄素对应糖苷,为商业化生产利用大黄素及其糖苷类化合物提供了新型优质药源,该方法效果可靠、成本低廉低;生产过程高效、绿色、安全、无环境污染。
本发明明确了荞麦蒽醌类物质生物合成过程中所涉及的糖基化酶的功能,为后续深入研究糖基转移酶调控蒽醌类大黄素化合物代谢机制奠定了理论基础。
本发明所涉及到的术语定义
除非另外定义,否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。虽然在本发明的实践或测试中可使用与本文所述者类似或等效的任何方法、装置和材料,但现在描述优选方法、装置和材料。
术语“同源性”,指与天然核酸序列的序列相似性。“同源性”包括与本发明的调控片段的核苷酸序列具有优选地85%或更高,更优选地90%或更高,以及最优选地95%或更高同一性的核苷酸序列。同源性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同源性可以用百分比(%)表示,其可以用来评价相关序列之间的同源性。
术语“互补的”在此指的是两种包括反向平行核苷酸序列的核苷酸序列,反向平行核苷酸序列能在反向平行核苷酸序列的互补碱基残基之间形成氢键后彼此相互配对。本领域已知的是,当都从5’到3’的方向看序列时,两种互补链的核苷酸序列是彼此反向互补的。本领域也已知的是,两种在给定的条件组下能彼此杂交的序列不必必须是100%完全互补的。
术语“严谨杂交条件”意指在所属领域中已知的低离子强度和高温的条件。通常,在严谨条件下,探针与其靶序列杂交的可检测程度比与其它序列杂交的可检测程度更高(例如超过本底至少2倍)。严谨杂交条件是序列依赖性的,在不同的环境条件下将会不同,较长的序列在较高温度下特异性杂交。通过控制杂交的严谨性或洗涤条件可鉴定与探针100%互补的靶序列。对于核酸杂交的详尽指导可参考有关文献(Tijssen,Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles of hybridization and the strategy of nucleic acidassays.1993)。更具体的,所述严谨条件通常被选择为低于特异序列在规定离子强度pH下的热熔点(Tm)约5-10℃。Tm为在平衡状态下50%与目标互补的探针杂交到目标序列时所处的温度(在指定离子强度、pH和核酸浓度下)(因为目标序列过量存在,所以在Tm下在平衡状态下50%的探针被占据)。严谨条件可为以下条件:其中在pH 7.0到8.3下盐浓度低于约1.0M钠离子浓度,通常为约0.01到1.0M钠离子浓度(或其它盐),并且温度对于短探针(包括(但不限于)10到50个核苷酸)而言为至少约30℃,而对于长探针(包括(但不限于)大于50个核苷酸)而言为至少约60℃。严谨条件也可通过加入诸如甲酰胺的去稳定剂来实现。对于选择性或特异性杂交而言,正信号可为至少两倍的背景杂交,视情况为10倍背景杂交。例示性严谨杂交条件可如下:50%甲酰胺,5×SSC和1%SDS,在42℃下培养;或5×SSC,1%SDS,在65℃下培养,在0.2×SSC中洗涤和在65℃下于 0.1%SDS中洗涤。所述洗涤可进行5、15、30、60、120分钟或更长时间。
术语“宿主细胞”或“重组宿主细胞”意指包含本发明多核苷酸的细胞,而不管使用何种方法进行插入以产生重组宿主细胞,例如直接摄取、转导、 f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。
术语“多核苷酸”或“核苷酸”意指单股或双股形式的脱氧核糖核苷酸、脱氧核糖核苷、核糖核苷或核糖核苷酸及其聚合物。除非特定限制,否则所述术语涵盖含有天然核苷酸的已知类似物的核酸,所述类似物具有类似于参考核酸的结合特性并以类似于天然产生的核苷酸的方式进行代谢。除非另外特定限制,否则所述术语也意指寡核苷酸类似物,其包括PNA(肽核酸)、在反义技术中所用的DNA类似物(硫代磷酸酯、磷酰胺酸酯等等)。除非另外指定,否则特定核酸序列也隐含地涵盖其保守修饰的变异体(包括(但不限于)简并密码子取代)和互补序列以及明确指定的序列。特定而言,可通过产生其中一个或一个以上所选(或所有)密码子的第3 位经混合碱基和/或脱氧肌苷残基取代的序列来实现简并密码子取代(Batzer等人,Nucleic Acid Res.19:5081(1991);Ohtsuka等人,J.Biol. Chem.260:2605-2608(1985);和Cassol等人,(1992);Rossolini等人,Mol Cell.Probes 8:91-98(1994))。
术语“可操作地连接”指两个或更多个核酸区域或核酸序列的功能性空间排列。例如,启动子区可以相对于编码感兴趣表达产物的核酸序列如此安置,从而所述核酸序列的转录由该启动子区指导。因此,启动子区“与该核酸序列可操作地连接”。
术语“转化”在此指的是用于将异源DNA引入到植物细胞、植物组织、或植物中的过程。转化植物细胞、植物组织、或植物被理解为不仅包括转化过程的终末产物,也包括其子代。
术语“转化”、“转基因”、和“重组体”在此指的是其中已经被引入异源核酸分子的宿主细胞或生物体例如细菌或植物细胞(例如植物)。核酸分子可以被稳定地整合到宿主的基因组中,或者核酸分子也可以以染色体外分子的形式存在。这样一种染色体外分子可以是自我复制的。转化细胞、组织或植物被理解为不仅包括转化过程的终末产物,还包括其转基因子代。“未转化”、“未转基因”、或“未重组的”宿主指的是野生型生物体例如细菌或植物,它不包含异源核酸分子。
术语“启动子”指下述的任何核酸序列(如DNA序列):这种序列在转录起始期间被DNA依赖性RNA聚合酶识别并(直接或间接)结合,导致生成与转录的DNA互补的RNA分子;这种区域也可以称作“5'调节区”。启动子通常位于在待转录的编码序列前方存在的5'非翻译区(UTR)的上游并且具有多个区域,这些区域充当RNA聚合酶II和其他蛋白质如转录因子的结合位点以引发可操作地连接的基因的转录。启动子本身可以含有调节可操作地连接的基因的转录的子元件(即启动子基序)如顺式元件或增强子结构域。该启动子和连接的5'UTR也称作“启动子区”。
附图说明
图1: FtUGT74L2 CDS的克隆。
图2:转基因毛状根和空载体毛状根中FtUGT74L2的qPCR检测相对表达量。
图3 :FtUGT74L2糖基转移酶的序列分析和比对结果;(a)FtUGT74L2 的糖基转移酶(PSPG)区的保守序列;(b)FtUGT74L2与其他GTs蛋白序列多重比对。
图4 :FtUGT74L2糖基转移酶原核诱导表达蛋白western鉴定结果。
图5 :FtUGT74L2糖基转移酶的催化反应模式。
图6:大黄素在FtUGT74L2糖基转移酶下合成大黄素-8-O-葡萄糖苷的高效液相色谱法谱图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的,不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改或替换均落入本发明的保护范围。
实施例1 FtUGT74L2 CDS的克隆
本实施例采用基因克隆的方法从苦荞(品苦1号)无菌苗中克隆获得糖基转移酶FtUGT74L2基因并进行基因序列信息分析,具体如下:
选取两周龄品苦1号的幼苗,取幼苗50-100mg,加液氮充分研磨后,使用Trizol法提取总RNA。以此RNA为模板,使用III 1st Strand cDNA Synthesis Kit(+gDNAwiper)试剂盒(南京诺唯赞生物技术有限公司)进行反转录获得幼苗的cDNA。
根据FtUGT74L2的ORF设计特异引物:
FtUGT74L2-F:5'-ATGAAGGTAGTTCCCGGG-3',
FtUGT74L2-R:5'-AGCCCTTAACTCCTTCACAA-3';
以品苦1号cDNA为模板进行PCR扩增,获得目的基因的CDS序列:
PCR程序为95℃3min;95℃30s,58℃30s,72℃90s,31个循环。PCR纯化产物连接到pTOPO-Blunt Simple平末端克隆载体上,获得FtUGT74L2-T载体质粒。
最终本发明克隆得到FtUGT74L2基因的CDS序列,其核苷酸序列为 SEQ ID No.1所示,其编码蛋白的氨基酸序列为SEQ ID No.2所示。
实施例2 FtUGT74L2基因在苦荞毛状根中表达以及表达量的检测
将FtUGT74L2基因可操作性地构建于表达调控序列得到含 FtUGT74L2基因的植物表达载体pCAMBIA 1302-FtUGT74L2,具体的,植物表达载体pCAMBIA 1302-FtUGT74L2的构建方法包括:
设计同源重组引物,以FtUGT74L2-T载体为模板,OE-FtUGT74L2- F/R为引物,PCR扩增FtUGT74L2的全长序列。
上游引物:1302-FtUGT74L2-NcoI-F:
5'-ccatggATGAAGGTAGTTCCCGGG-3'
下游引物:1302-FtUGT74L2-BglII-R:
5'-agatctAGCCCTTAACTCCTTCACAA-3'。
随后经酶切、回收和连接转化后,将FtUGT74L2全长序列正向插入 pCAMBIA-1302载体的CaMV35S启动子下游,经测序完全后得到过表达载体pCAMBIA1302-FtUGT74L2。
将所构建的含FtUGT74L2基因的过表达载体pCAMBIA1302- FtUGT74L2转化发根农杆菌,获得用于转化苦荞的含FtUGT74L2基因植物表达载体的发根农杆菌菌株,具体包括:测序验证正确的pCAMBIA1302-FtUGT74L2重组质粒以及pCAMBIA1302-空载体质粒用热激法分别转化发根农杆菌A4感受态细胞。经菌落PCR鉴定后,得到pCAMBIA 1302-FtUGT74L2重组质粒阳性菌,pCAMBIA1302-空载体阳性菌。
将pCAMBIA 1302-FtUGT74L2重组质粒阳性菌侵染苦荞,经培养得转基因和空载体毛状根,经PCR检测阳性结果见图1(引物为 FtUGT74L2-F/R)。挑选12份苦荞毛状根进行PCR检测。其中,编号1, 2,3,5,6,7,8,9,10,11,12呈现阳性结果。
将经PCR检测为阳性的苦荞转基因毛状根克隆进行PCR检测表达:将侵染后的苦荞下胚轴和子叶于MS固体培养基上,待毛状根的量足够后,取适量于MS液体培养基中,室温震荡(120r/min)处理(与此同时,转pCAMBIA1302-FtUFGT3基因毛状根作为阳性对照,转pCAMBIA1302- 空载体毛状根作为阴性对照)。利用FtUGT74L2-F/R检测基因表达,PCR 程序与实施例1中所述一致。
PCR检测结果显示(图2),过表达的FtUGT74L2-OE毛状根中的该基因的表达量比pCAMBIA-1302空载转化毛状根中更高。
实施例3大黄素糖基转移酶的氨基酸序列分析及比对
将大黄素糖基转移酶FtUGT74L2基因氨基酸序列在NCBI数据库中 Blast比对,获得其他物种GTs类蛋白序列,使用Bioxm软件翻译氨基酸序列,预测其蛋白质大小以及等电点信息。
大黄素糖基转移酶FtUGT74L2基因的CDS长度为897bp(SEQ ID No.1),编码蛋白分子量33.3KDa的298个氨基酸(SEQ ID No.2),所编码蛋白的等电点(pI)为5.18。用NCBI的blast在线分析FtUGT74L2 氨基酸的保守域,发现具备糖基转移酶(PSPG)结构域,所编码的蛋白属于UGT型糖基转移酶(图3a)。在NCBI数据库中Blast比对,获得其他物种UGT蛋白序列,使用MEGA6.0软件构建了聚类树(图3b)。
实施例4大黄素糖基转移酶的原核诱导表达以及鉴定
将FtUGT74L2基因通过表达载体转化至宿主菌进行表达以快速获得重组MBP-FtUGT74L2蛋白,具体包括:将PCR产物用TaKaRa MiniBEST Plasmid Purification KitVer 4.0进行切胶回收纯化后,连接到MBP(麦芽糖结合蛋白)标签载体,获得MBP-FtUGT74L2重组质粒。
纯化方法采用NEB公司的amylase resin(E8021S)产品说明书上的方法,具体操作如下:取含已目的载体的单克隆在5mL液体LB(含50μg/mL 氨苄青霉素)培养基中,37℃,220rpm培养8-12h;将实施例2中菌液全部转入250mL无抗LB液体培养基中,37℃,220rpm培养1-3h使OD600达到0.8左右;将摇床温度调至20℃,转速调至150rpm,待培养基的温度降低至20℃后加入IPTG至终浓度为0.2mM,诱导培养8h;4℃,5000 rpm离心15min收集菌体;加入平衡缓冲液,重悬菌体,超声破碎菌体,至菌液澄清;4℃,12000rpm离心15min,将上清用0.4μm滤膜过滤后,加入预先用平衡缓冲液平衡的amylase resin柱子中,使蛋白与填料结合;用5-10倍柱体积的平衡缓冲液洗涤层析柱,以除去没有结合的杂蛋白;用5mL的洗脱缓冲液(含10mM麦芽糖)洗脱,收集流出液;SDS-PAGE、 Western blot检测纯化的蛋白,BCA蛋白定量试剂盒(康为世纪)测定蛋白浓度。在蛋白样品中加入5×Loading Buffer使其终浓度为1×,沸水浴煮 10min,12000rpm离心10min后吸取适量的上清加入点样孔中,80V恒压电泳,待溴酚蓝进入分离胶后将电压设置为120V继续电泳直至完成;电泳结束后小心剥取凝胶放入考马斯亮蓝R-250染液中,在水平摇床上缓慢摇动室温染色3h以上;染色结束后,将凝胶转移至考马斯亮蓝染色脱色液中,在水平摇床上缓慢摇动4-8h脱色,期间更换脱色液2-3次。 SDS-PAGE电泳结束后,小心取出凝胶并放入预冷的转膜缓冲液中浸泡;夹好凝胶,冰浴中转膜90min;转膜完成后,小心剥取PVDF膜,放入 TBST中漂洗3次,每次5min,然后在膜正面均匀滴加TBST稀释好的丽春红染色,观察转膜效果;将膜先在TBST中漂洗3-5次,每次5-10min,然后转入5%(W/V)脱脂奶粉的TBST溶液中,室温平缓摇动2-3h或4℃过夜;加一抗孵育:用TBST稀释一抗(根据抗体效价按一定比例稀释),然后将封闭好的PVDF膜放入稀释好的一抗中,室温下在摇床上缓慢摇动孵育3h左右或4℃过夜;加二抗孵育:在TBST中漂洗3-5次,每次5-10 min,用TBST按照比例稀释辣根过氧化物酶(HRP)标记的二抗,然后将一抗孵育完的PVDF膜放入稀释好的二抗中,室温下在摇床上缓慢摇动孵育 30-60min;TBST中漂洗3-5次,每次5-10min将膜平铺于透明塑料膜上,滴加ECL显色反应液至覆盖膜表面,然后盖上透明塑料膜,用化学发光成像仪检测。根据图4可看出,重组质粒转化到表达宿主大肠杆菌DH5α,经IPTG诱导后,有重组MBP-蛋白的表达,上清蛋白经Ni-NTA柱纯化后得到了较纯的重组蛋白,且该蛋白的条带大小与预测的一致,加上重组标签后在81.3kDa左右有明显的重组蛋白条带。
根据图4可看出,MBP蛋白为45KD左右,构建的MBP-FtUGT74L2 重组质粒转化到表达宿主大肠杆菌DH5α,经IPTG诱导后,有重组蛋白的表达,上清蛋白经Ni-NTA柱纯化后得到了较纯的重组蛋白,且该蛋白的条带大小与预测的一致,加上重组标签后在81.3kDa左右有明显的重组蛋白条带,纯化的蛋白可用于进一步的酶学分析。
实施例5大黄素在FtUGT74L2糖基转移酶下合成大黄素-8-O-葡萄糖苷及高效液相色谱检测
大黄素和大黄素-8-O-葡萄糖苷在FtUGT74L2糖基转移酶的催化反应模式:以大黄素为底物,以UDP-葡萄糖为糖基供体,在FtUGT74L2葡萄糖基转移酶的催化下,在大黄素的8-OH位置加一分子的葡萄糖基,生成大黄素-8-O-葡萄糖苷(图5)。
重组MBP-FtUGT74L2蛋白体外酶活检测:标准品为大黄素和大黄素 -8-O-葡萄糖苷。向200μL反应缓冲液(100mM Tris HCl(pH 8.0)、14mM β-巯基乙醇、10mM UDP-葡萄糖(RYON、RZ1070)、1mM大黄素) 中加入2μg纯化的重组蛋白MBP-FtUGT74L2,37℃孵育30分钟。加入 800μL乙酸乙酯终止反应,冷冻干燥成干粉。干燥后的反应产物在1mL 80%甲醇中重新溶解,溶解液利用高效液相色谱-质谱联用法测定产物。用大黄素、大黄素-8-O-葡萄糖苷标准品的保留时间值来鉴别反应产物。
高效液相色谱-质谱联用法测定产物:
甲醇溶液浓度为55-85%,温度为25-60℃、超声时间为15-40min、超声频率为30-60kHz。固定相是以十八烷基键合硅胶为填料的色谱柱;流动相0.1%甲酸/乙腈水溶液;流速:0.5mL/min;检测波长:230-280nm;进样量:5-20μL;柱温33-45℃。采用液相色谱-质谱联用仪(LC-MS/MS)建立了校正曲线, R2=0.9991、R2=0.9995,说明有良好的线性关系。提取程序:0.1g冻干粉中加入20毫升甲醇,超声提取3次,提取物通过0.22μm的过滤膜。色谱条件:C18 柱(2.1mm×75mm,2.7μm)。Em(大黄素)和EmG(大黄素-8-O-葡萄糖苷) 的流动相梯度洗脱程序为0~6min,10%~30%A;12min,80%A;15min,80%A; 15.1min,10%A;18min,10%A。用大黄素和大黄素-8-O-葡萄糖苷的峰面积定量反应产物。每个样本有三个重复。
大黄素在FtUGT74L2糖基转移酶下合成大黄素-8-O-葡萄糖苷的HPLC色谱图为图6所示,根据图6可见,大黄素标准品不受MBP蛋白的影响,而在 MBP-FtUGT74L2催化下高效转化生成大黄素-8-O-葡萄糖苷。
SEQUENCE LISTING
<110> 中国农业科学院作物科学研究所贵州省威宁县东方神谷有限责任公司
<120> 苦荞来源的大黄素糖基转移酶及其编码基因和应用
<130> BJ-2011-210702A
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 897
<212> DNA
<213> Fagopyrum tataricum
<400> 1
atgaaggtag ttcccggggg tgggatcacg tgggagacac gtgacctacc gagttttctc 60
acaaggcccg agagttaccc ggcgtacttg gagatgaagt tgagtcagtt tgggaactta 120
gactttgctc attgggtgtt ttgtaactct tttgacgatt tggagtctca ggtccttaaa 180
ggcatagtac ctgaccaatt ccctgcaaag ctaataggac caatggttcc atcagcctac 240
ctagacaatg caatcgaagg cgacaaaggt tacggtgcaa gcctatggaa gccgctaagc 300
caccaatgcc gaacatggct cgactccaag ccaataaaat ctgtcgtcta catctctttt 360
ggcagcatgg cgtcgctaac acgtgaacaa acatccgaaa ttgcaaccac tctgatagaa 420
cacgacttcc cattcttgtg gatcgtacgt gagtcacaac tcgacacatt gccggatgga 480
tttttcgagt caactaagga gaaggggatg gtggcaacat ggtgtgacca actcgaggtg 540
ttggtacacc cgtcgctagg gtgttttgtg acgcattgtg gttggaactc aacactagaa 600
ggactttgcc ttggtgttcc gatggtgggg gtcccacaat ggacggatca attcacggac 660
gcgaagtttg tgtcggatgt ttggagggtt ggggtgcgag cgagggtgga cgaggtgggg 720
atggttagga aggcggagct cgcggcgtgt ttggcagaag tgatggtgga tgggaagagt 780
agggaggaga tgtatgggaa tgggattaag tggaaagagt tggcaaagaa agctcttagt 840
aaagaaggga gctcaagtaa gaatattgat gaatttgtga aggagttaag ggcttga 897
<210> 2
<211> 298
<212> PRT
<213> Fagopyrum tataricum
<400> 2
Met Lys Val Val Pro Gly Gly Gly Ile Thr Trp Glu Thr Arg Asp Leu
1 5 10 15
Pro Ser Phe Leu Thr Arg Pro Glu Ser Tyr Pro Ala Tyr Leu Glu Met
20 25 30
Lys Leu Ser Gln Phe Gly Asn Leu Asp Phe Ala His Trp Val Phe Cys
35 40 45
Asn Ser Phe Asp Asp Leu Glu Ser Gln Val Leu Lys Gly Ile Val Pro
50 55 60
Asp Gln Phe Pro Ala Lys Leu Ile Gly Pro Met Val Pro Ser Ala Tyr
65 70 75 80
Leu Asp Asn Ala Ile Glu Gly Asp Lys Gly Tyr Gly Ala Ser Leu Trp
85 90 95
Lys Pro Leu Ser His Gln Cys Arg Thr Trp Leu Asp Ser Lys Pro Ile
100 105 110
Lys Ser Val Val Tyr Ile Ser Phe Gly Ser Met Ala Ser Leu Thr Arg
115 120 125
Glu Gln Thr Ser Glu Ile Ala Thr Thr Leu Ile Glu His Asp Phe Pro
130 135 140
Phe Leu Trp Ile Val Arg Glu Ser Gln Leu Asp Thr Leu Pro Asp Gly
145 150 155 160
Phe Phe Glu Ser Thr Lys Glu Lys Gly Met Val Ala Thr Trp Cys Asp
165 170 175
Gln Leu Glu Val Leu Val His Pro Ser Leu Gly Cys Phe Val Thr His
180 185 190
Cys Gly Trp Asn Ser Thr Leu Glu Gly Leu Cys Leu Gly Val Pro Met
195 200 205
Val Gly Val Pro Gln Trp Thr Asp Gln Phe Thr Asp Ala Lys Phe Val
210 215 220
Ser Asp Val Trp Arg Val Gly Val Arg Ala Arg Val Asp Glu Val Gly
225 230 235 240
Met Val Arg Lys Ala Glu Leu Ala Ala Cys Leu Ala Glu Val Met Val
245 250 255
Asp Gly Lys Ser Arg Glu Glu Met Tyr Gly Asn Gly Ile Lys Trp Lys
260 265 270
Glu Leu Ala Lys Lys Ala Leu Ser Lys Glu Gly Ser Ser Ser Lys Asn
275 280 285
Ile Asp Glu Phe Val Lys Glu Leu Arg Ala
290 295
Claims (1)
1.大黄素糖基转移酶在催化蒽醌类物质转化为对应的糖苷化合物中的应用;所述的应用包括:以大黄素糖基转移酶为催化酶催化大黄素进行8-OH位的特异性糖基化反应生成大黄素-8-O-葡萄糖苷;所述大黄素糖基转移酶的氨基酸序列为SEQ ID No.2所示,所述的蒽醌类物质是大黄素。
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