CN106434703A - 参与丹参酮类化合物生物合成的细胞色素p450基因cyp71d410及其编码产物与应用 - Google Patents
参与丹参酮类化合物生物合成的细胞色素p450基因cyp71d410及其编码产物与应用 Download PDFInfo
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Abstract
本发明涉及一个参与丹参酮生物合成的CYP450基因CYP71D410及其编码产物与应用,属于药用植物基因工程领域。该基因从丹参中克隆得到,其编码蛋白可以催化铁锈醇及其衍生物在3位进行羟基化,是丹参酮生物合成途径中的关键酶。CYP71D410基因具有SEQ ID No.1所示的核苷酸序列,其编码蛋白具有SEQ ID No.2所示的氨基酸序列。本发明提供的CYP71D410基因可应用于通过合成生物学或代谢工程技术异源合成和生产二萜类化合物,也可用于调节植物二萜类化合物的生物合成,及通过生物技术提高丹参中丹参酮类化合物的含量。同时该基因也可以用于丹参的杂交育种和优良品种选育。
Description
技术领域
本发明属于药用植物基因工程领域,主要涉及丹参酮代谢途径相关细胞色素P450基因CYP71D410的筛选,鉴定及其应用。
背景技术
次生代谢产物是药用植物生物活性成分的主要来源。近年来,药用植物基因组学研究的快速发展为次生代谢产物合成相关基因的发掘与鉴定奠定了前期研究基础。次生代谢产物合成相关基因的克隆及功能验证将为天然产物的代谢工程和合成生物学研究提供必需的生物元件,同时也将为药用植物的选种育种及品质改良提供指导。
丹参(Salvia miltiorrhiza Bunge)为唇形科鼠尾草属多年生草本植物,具有活血通经,祛疲止痛,清心除烦等功效。丹参酮为二萜醌类化合物,是丹参的主要脂溶性活性成分,主要包括丹参酮IIA、丹参酮IIB、丹参酮I、隐丹参酮、二氢丹参酮、异隐丹参酮等,具有扩张血管、抗肿瘤、抗菌消炎以及抗血栓、抗氧化等多种药理作用。目前研究认为丹参酮的合成途径可分为三步:第一步合成萜类的前体物质,第二步形成丹参酮骨架结构,第三步对丹参酮骨架结构进行修饰,包括氧化、甲基化、芳香化等修饰。根据丹参酮合成途径推测,细胞色素P450是参与丹参酮骨架结构修饰的主要酶类。
细胞色素P450在生物界中广泛存在,为含血红素的膜蛋白,具有单加氧酶活性。细胞色素P450可催化多种类型的反应,主要包括羟基化、环氧化、异构化、脱烷基化、脱硫、脱卤、脱氢作用等。尽管细胞色素P450可催化多种类型的反应,但却具有相同的催化机制,即通过NADPH或者NADH为细胞色素P450传递电子,激活氧分子,将其中的一个氧插入到底物上,同时生成一分子水。近年来利用异源表达及RNA干扰等技术相继在黄花蒿、长春花、人参等多种药用植物中鉴定出参与次生代谢途径的细胞色素P450。
本发明通过基因筛选,异源表达,酶活性检测等技术对丹参的一个P450进行了功能鉴定,该酶可以催化铁锈醇及其衍生物在3位进行羟基化,依据细胞色素P450基因命名法则命名为CYP71D410,该基因可应用于二萜类化合物的生物合成与调控及丹参育种。
发明内容
本发明的目的在于提供一个参与丹参酮合成途径的细胞色素P450基因CYP71D410,其编码的蛋白能够催化铁锈醇及其衍生物在3位进行羟基化。
本发明提供了一个与丹参酮类化合物合成代谢有关的基因:CYP71D410,它是下列核苷酸序列之一:
1)序列表中SEQ ID No.1所示的cDNA序列;或
2)SEQ ID No.1所示的核苷酸序列经取代、缺失或增加一个或多个核苷酸,且表达相同功能蛋白质的核苷酸序列:或
3)在严格条件下与SEQ ID No.1所示序列杂交的核苷酸序列;所述严格条件为:在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜。
一种由上述基因编码的蛋白质,其特征为:
i)具有序列表中SEQ ID No.2的氨基酸残基序列;或
ii)SEQ ID No.2所示氨基酸序列经取代、缺失或添加一个或几个氨基酸产生的具有相同功能的蛋白。
本发明SEQ ID No.1的DNA序列由1515个碱基组成,编码序列表中SEQ ID No.2的蛋白质由504个氨基酸残基组成。
含有本发明基因的表达载体、细胞系、转基因植株及宿主菌和使用该基因在调节和生产植物二萜类化合物及丹参育种中的应用也在本发明的保护范围之内。将SEQ IDNo.1所示基因克隆到表达载体pYES2-URA(pYES2)的HindIII和EcoRI两个限制性内切酶之间,构建带有CYP71D410基因的重组表达载体pYES2-CYP71D410;转化酿酒酵母菌株WAT11,半乳糖诱导基因表达,并在培养液中加入底物铁锈醇或其衍生物。诱导24小时后用正己烷对其表达产物进行提取并进行GC-MS分析。分析结果表明CYP71D410可以催化铁锈醇及其衍生物在3位进行羟基化。
本发明还提供用于PCR扩增所述CYP71D410编码基因cDNA序列的特异性引物对,包括:
正向引物:ATGGATCCCGAGTTCCCATC
反向引物:TCACTTGAGGAGGCGCAACG
附图说明
图1重组质粒pYES2-CYP71D410的PCR鉴定结果,其中M为DL5000DNA Maker,1为pYES2-CYP71D410的重组质粒。
图2 CYP71D410基因编码蛋白催化铁锈醇酶促反应产物的GC-MS分析结果。
图3 CYP71D410基因编码蛋白催化铁锈醇生成3-羟基铁锈醇的图示。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001),或按照制造厂商说明书建议的条件。
实施例1丹参中细胞色素P450基因的克隆
1、根据已测序的丹参BAC(bacterial artificial chromosome)数据,通过拼接、注释、筛选等操作,获得丹参酮合成途径中的候选细胞色素P450基因cDNA序列。
2、使用引物设计软件Lasergene PrimerSelect设计该候选细胞色素P450基因的引物,引物序列为:
正向引物:ATGGATCCCGAGTTCCCATC
反向引物:TCACTTGAGGAGGCGCAACG
引物由北京三博远志生物技术有限责任公司合成。
3、取生长旺盛的丹参植株叶片,使用QIAGENMini试剂盒提取总RNA,利用PROMEGA逆转录试剂盒进行逆转录获得cDNA,以cDNA为模板扩增CYP71D410的基因序列。
4、琼脂糖凝胶电泳在1500bp处出现特异性条带,对目标条带进行切胶回收,胶回收产物连接到pMD18T载体(TaKaRa),并转化大肠杆菌DH5a,挑取阳性克隆进行测序(北京农业科学院测序中心),选择和保存序列正确的CYP71D410基因克隆用于后续表达载体的构建。
实施例2、CYP71D410基因序列的生物信息学
本发明涉及的丹参二萜合成代谢途径细胞色素P450基因CYP71D410,该基因全长开放读码框(ORF)的长度为1515核苷酸,编码504个氨基酸,详细序列见序列表中的SEQ IDNo.1和SEQ ID No.2。将CYP71D410全长开放读码框用BLAST程序在NCBI数据库中进行同源性检索,该基因在氨基酸水平上比对分析显示,丹参CYP71D410基因编码的蛋白质氨基酸序列与其它物种的同源性较低。
实施例3、CYP71D410基因真核表达及功能分析
1、酵母表达载体的构建
根据基因CYP71D410的编码序列及酶切位点分析结果,对CYP71D410设计带有HindIII和EcoRI酶切位点的引物,用带酶切位点的引物对CYP71D410的ORF进行扩增,扩增产物连接到pMD18T后进行测序验证,最后通过酶切的方法将目的基因CYP71D410连接到酵母表达载体pYES2上,通过测序确定载体pYES2-CYP71D410的正确性。
2、酵母转化
利用醋酸锂转化法将pYES2-CYP71D410载体转入酿酒酵母菌株WAT11中,通过菌落PCR法挑选阳性克隆。
3、诱导表达
挑取阳性单克隆接种于5ml SD液体培养基中,30℃,200rmin-1培养24h;按照1∶50的接种量接入50ml SD液体培养基中,30℃,200rmin-1,培养2-4h至对数中期,2000g离心去上清,用去离子水洗涤菌体两次后转接入含半乳糖的SD诱导培养基中,30℃,200rmin-1,诱导两小时后加入铁锈醇作为底物,继续培养24h。
4、催化产物的提取与鉴定
加入等体积正己烷对催化产物进行涡旋提取。提取的产物进行GC-MS分析,结果发现含有CYP71D410基因重组表达载体pYES2-CYP71D410的催化组与含空载pYES2对照组相比有新物质产生,经分析显示该新产物为3-羟基铁锈醇。
Claims (8)
1.一个与丹参酮类化合物生物合成相关的CYP450基因CYP71D410,其特征在于:
1)如SEQ ID No.1所示的读码框为第1-1515位核苷酸序列;或
2)SEQ ID No.1所示的核苷酸序列经取代、缺失或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;或
3)在严格条件下与SEQ ID No.1所示序列杂交的核苷酸序列;所述严格条件为:在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜。
2.一个与丹参酮类化合物生物合成相关的萜类合酶CYP71D410,其特征在于:
i)如SEQ ID No.2所示的1-504位氨基酸序列构成的蛋白;或
ii)SEQ ID No.2所示氨基酸序列经取代、缺失或添加一个或几个氨基酸产生的具有相同功能的蛋白。
3.含有权利要求1所述基因的表达载体。
4.含有权利要求1所述基因的转基因细胞系和转基因植物。
5.含有权利要求1所述基因的工程菌。
6.权利要求1所述基因和权利要求2所述蛋白在调节和生产植物二萜类化合物中的应用。
7.权利要求1所述基因和权利要求2所述蛋白在丹参育种中的应用。
8.用于PCR扩增权利要求1所述编码基因cDNA序列的特异性引物对,包括:
正向引物:ATGGATCCCGAGTTCCCATC
反向引物:TCACTTGAGGAGGCGCAACG 。
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