CN109762837B - 利用丹参长链异戊烯基转移酶基因SmPPS2调控辅酶Q合成的方法 - Google Patents
利用丹参长链异戊烯基转移酶基因SmPPS2调控辅酶Q合成的方法 Download PDFInfo
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- CN109762837B CN109762837B CN201811584009.6A CN201811584009A CN109762837B CN 109762837 B CN109762837 B CN 109762837B CN 201811584009 A CN201811584009 A CN 201811584009A CN 109762837 B CN109762837 B CN 109762837B
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Abstract
本发明涉及利用丹参长链异戊烯基转移酶基因SmPPS2调控辅酶Q合成的方法。本发明通过克隆获得了药用植物丹参中的长链异戊烯基转移酶SmPPS2的编码基因,并发现SmPPS2具有合成辅酶Q的功能。本发明构建了转SmPPS2基因的酿酒酵母,在酿酒酵母中实现了辅酶Q9和辅酶Q10的同时积累;同时,通过在丹参中过表达SmPPS2基因,提高了丹参的辅酶Q的含量。本发明提供的丹参长链异戊烯基转移酶SmPPS2及其编码基因能够促进生物体内辅酶Q的合成,可以在实践中用于辅酶Q的生产。
Description
技术领域
本发明涉及基因工程及分子生物学领域,具体涉及利用丹参长链异戊烯基转移酶基因SmPPS2调控辅酶Q合成的方法。
背景技术
辅酶Q,亦称为泛醌、CoQ或者UQ,是生物体内的脂溶性醌类化合物,广泛的存在于线粒体内膜,主要参与细胞氧化磷酸化和ATP的生成。辅酶Q的存在形式多样,且具有物种特异性,例如大肠杆菌中主要为辅酶Q8,酿酒酵母中为辅酶Q6,裂殖酵母和人类体内主要是辅酶Q10。辅酶Q分子由苯醌环和聚异戊二烯基侧链两部分组成,其类型因催化聚异戊二烯基侧链的酶(异戊烯基转移酶,PPS)的不同而不同。异戊烯基转移酶(PPS)广泛存在于生物体内,催化多种异戊烯基类化合物的合成,其中包括萜类、维生素E、植物激素以及辅酶Q等。异戊烯基类化合物的生物合成从异戊烯基焦磷酸(IPP)和其异构体二甲基烯丙基焦磷酸(DMAPP)的缩合反应开始,根据其催化的IPP和DMAPP合成的最终产物的链长长短,异戊烯基转移酶可以分为三种,短链(C15-C25)、中链(C30-C35)和长链(C40-C50)异戊烯基转移酶(Kellogg et al,1997,Current Opinion in Chemical Biology,1,570-578)。其中,辅酶Q是由长链异戊烯基转移酶(LPPS)催化的。
研究发现,辅酶Q除了作为呼吸链的必需成分之外,还参与细胞抗氧化、分支链氨基酸的代谢、细胞信号转导与基因表达等功能。其中,辅酶Q10与人体健康密切相关:大量的临床试验研究表明,辅酶Q10对心脏病、高血压、脑血管障碍、急性肝炎等疾病有很好的疗效,可作为机体的非特异免疫增强剂、细胞代谢及细胞呼吸激活剂(万艳娟等,2014,食品工作科技,35:390-395)。人体内缺乏辅酶Q10可导致神经***疾病和代谢紊乱,而适量补充辅酶Q10可以对抗帕金森综合症、埃莫森综合症的发展(刘菲等,2016,中国实用神经疾病杂志,1:139-140)。除此之外,辅酶Q10可阻止癌细胞的转移,对抗癌症的发展(杨敬源等,2018,海南医学,2:242-245)。饮食中适当添加辅酶Q10可以减缓多种慢性疾病的发生。目前辅酶Q10已经应用于化妆品和保健品行业,从基础的科学研究走向了市场。
植物体内最常见的是辅酶Q9和辅酶Q10。拟南芥和大多数农作物(如小麦和水稻)都含有辅酶Q9。另外还有一些蔬菜、水果和一些浆果类的植物中同时含有辅酶Q9和辅酶Q10。辅酶Q10在动物心脏、肝脏和肉中含量比较高,相比而言,大多数植物体内的辅酶Q10含量较少。辅酶Q的合成过程在植物中的研究比较少,以辅酶Q10为例,催化辅酶Q10合成的长链异戊烯基转移酶目前只在烟草和番茄中发现。在辅酶Q10的生产上,利用烟草提取纯植物来源的辅酶Q10的方法获得的辅酶Q10含量较低,生产成本较高;而新型的半合成法合成的辅酶Q10不仅人体吸收差,而且会造成不同程度的环境污染;在生物法合成中,辅酶Q10的合成多是通过异源表达微生物相关基因实现,如通过在水稻中表达葡萄杆菌DdsA基因获得含有辅酶Q10的水稻。然而,尽管含辅酶Q10的水稻满足了人类对同步摄取碳水化合物摄取和辅酶Q10的需求,但水稻种子中的辅酶Q10含量比较低,而且也存在转微生物基因食品安全问题。因此,植物来源的辅酶Q基因成为较好的候选基因,通过发掘植物来源的相关辅酶Q合成基因并将其应用于辅酶Q的生产具有重要的意义和良好的应用前景。
发明内容
为了解决现有技术中存在的问题,本发明的目的是提供丹参长链异戊烯基转移酶基因SmPPS2在调控辅酶Q合成中的应用以及利用丹参长链异戊烯基转移酶基因SmPPS2调控辅酶Q合成的方法。
本发明通过克隆获得了丹参中的长链异戊烯基转移酶SmPPS2的编码基因(开放阅读框序列如SEQ ID NO.2所示),并发现SmPPS2酶(氨基酸序列如SEQ ID NO.1所示)具有催化合成异戊烯基类化合物的功能。SmPPS2属于异戊烯基转移酶家族,具有异戊烯基转移酶典型的七个(I-VII)保守结构,其中DDXXDD为异戊烯基焦磷酸结合位点。
本发明通过在丹参及酵母等宿主中进行实验验证,证明本发明提供的丹参长链异戊烯基转移酶SmPPS2参与异戊烯基类化合物辅酶Q的合成,通过表达丹参长链异戊烯基转移酶基因SmPPS2,能够显著提高宿主的异戊烯基类化合物的合成效率。
第一方面,本发明提供丹参长链异戊烯基转移酶基因SmPPS2在调控异戊烯基类化合物合成中的应用。
第二方面,本发明提供丹参长链异戊烯基转移酶基因SmPPS2在提高异戊烯基类化合物产量中的应用。
第三方面,本发明提供丹参长链异戊烯基转移酶基因SmPPS2在制备转基因植物、植物遗传育种或品种改良中的应用。
第四方面,本发明提供丹参长链异戊烯基转移酶SmPPS2或含有丹参长链异戊烯基转移酶基因SmPPS2的生物材料在调控异戊烯基类化合物合成、提高异戊烯基类化合物产量或制备转基因植物中的应用。
所述生物材料包括表达盒、载体、宿主细胞或工程菌。
优选地,所述宿主细胞包括微生物细胞、非繁殖性动物或植物细胞、非繁殖性动物或植物细胞系。
本发明中,所述载体可以为任何包含所述长链异戊烯基转移酶基因SmPPS2的复制型载体(如克隆载体或表达载体)或非复制型载体。
本发明中,所述丹参长链异戊烯基转移酶基因SmPPS2具有如下任意一种核苷酸序列:
(1)如SEQ ID NO.2所示的核苷酸序列;
(2)如SEQ ID NO.2所示的核苷酸序列经一个或多个碱基的替换、缺失或***得到的编码与所述SmPPS2具有相同功能蛋白的核苷酸序列;
(3)在严格条件下,能够与如SEQ ID NO.2所示的核苷酸序列杂交且编码与所述SmPPS2具有相同功能蛋白的核苷酸序列。
本发明中,所述丹参长链异戊烯基转移酶SmPPS2具有如下任意一种氨基酸序列:
(1)如SEQ ID NO.1所示的氨基酸序列;
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、缺失或***得到的与所述SmPPS2具有相同功能蛋白的氨基酸序列。
优选地,本发明所述的异戊烯基类化合物为辅酶Q。
进一步地,本发明提供一种调控生物体内辅酶Q的合成的方法,其为调控生物体内丹参长链异戊烯基转移酶基因SmPPS2的表达量。
当所述调控生物体内辅酶Q的合成为提高生物体内辅酶Q的合成时,所述提高生物体内辅酶Q的合成为通过提高丹参长链异戊烯基转移酶基因SmPPS2的表达量实现。
所述提高丹参长链异戊烯基转移酶基因SmPPS2的表达量可以通过本领域常规方法实现,优选通过将携带SmPPS2的表达载体转化至生物体内实现;所述基因SmPPS2的核苷酸序列如SEQ ID NO.2所示。
本发明中,所述生物体包括微生物和植物。
所述微生物包括但不限于酵母、霉菌、蕈菌等真菌以及大肠杆菌、农杆菌等细菌。
所述植物包括但不限于番茄、烟草、拟南芥等模式植物,丹参、人参等药用植物,水稻、玉米、小麦、小米等农作物,以及蔬菜、水果、浆果等。
当所述调控生物体内辅酶Q的合成为降低生物体内辅酶Q的合成时,所述降低生物体内辅酶Q的合成为通过降低丹参长链异戊烯基转移酶基因SmPPS2的表达量实现。
所述降低丹参长链异戊烯基转移酶基因SmPPS2的表达量可以通过本领域常规方法实现,例如,通过RNA干扰降低SmPPS2的表达量。
本发明的有益效果在于:
(1)本发明通过克隆获得了药用植物丹参中的长链异戊烯基转移酶SmPPS2的编码基因,通过丹参体内的缺失和互补实验以及酵母突变体的互补实验证明了长链异戊烯基转移酶SmPPS2参与辅酶Q的合成,发现SmPPS2的表达能够提高辅酶Q的合成,尤其能够促进辅酶Q9和Q10的合成,为辅酶Q合成的研究提供了新的基因和思路。
(2)本发明通过构建转SmPPS2基因的酵母,在酵母中实现了辅酶Q9和辅酶Q10的同时积累。
(3)本发明通过在丹参中过表达SmPPS2基因,提高了丹参的辅酶Q9和辅酶Q10的合成和积累。
附图说明
图1为本发明实施例3中SmPPS2的亚细胞定位结果图,其中,A为1302-SmPPS2:GFP亚细胞定位载体中关键元件的结构示意图;B为SmPPS2:GFP烟草叶片瞬时表达的荧光显微镜观察结果图。
图2为本发明实施例4中酵母诱导表达载体pYES2-SmPPS2的结构示意图。
图3为本发明实施例5中pYES2-SmPPS2转化酵母coq1突变体的功能互补实验结果,其中,A为酵母coq1突变体功能互补的生长实验结果图;B为野生型和pYES2-SmPPS2转化酵母中辅酶Q的分析;WT代表野生型酵母BY4741;Coq1-代表酵母Coq1突变体3138;pYES2代表转空载体的Coq1-酵母突变体3138;PPS2代表转pYES2-SmPPS2酵母Coq1突变体3138;RS为标准品。
图4为本发明实施例6和实施例7中过表达和敲减SmPPS2的丹参的载体构建、SmPPS2表达量分析和辅酶Q9和辅酶Q10的UQ总量的检测结果,其中A为SmPPS2过表达和敲减载体中关键元件的结构示意图,LB代表T-DNA区左边界;RB代表T-DNA区右边界;HYG代表潮霉素基因;POLY代表poly(A)信号;B为过表达SmPPS2丹参株系的PCR鉴定电泳图;C为SmPPS2在过表达株系中的相对表达量分析;D为SmPPS2过表达株系的辅酶Q9和辅酶Q10的UQ总量分析;E为SmPPS2敲减丹参株系的PCR鉴定电泳图;F为SmPPS2在敲减丹参株系中的相对表达量分析;G为SmPPS2敲减株系的辅酶Q9和辅酶Q10的UQ总量分析;WT代表野生型丹参;5、13、16分别代表3个不同的SmPPS2过表达丹参株系;3、6、22分别代表3个不同的SmPPS2敲减丹参株系。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用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材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1丹参叶片总RNA的提取和RACE cDNA文库的制备
取50-100mg丹参叶片,参考华越洋快速通用植物RNA提取试剂盒说明书的方法提取总RNA,利用Oligotex mRNA isolation kit(QIAGEN,Germany)纯化mRNA。得到质量良好的RNA,使用Nanodrop 2000(Thermo Scientific,USA)检测RNA的含量。利用SuperScriptIII reverse transcriptase(Invitrogen,USA)试剂盒合成first-strand cDNA;然后用GeneRacerTM Kit(Invitrogen,USA)试剂盒合成5'和3'RACE cDNA文库。
实施例2长链异戊烯基转移酶SmPPS2基因的克隆
以实施例1构建的丹参5'RACE cDNA文库为模板,克隆得到SmPPS2的5'端。以3'RACE cDNA文库为模板,克隆得到SmPPS2的3'端。以丹参的cDNA为模板,以SmPPS2的特异引物克隆得到SmPPS2的开放阅读框。将回收片段连接TaKaRa PMD-19T载体后,转入大肠杆菌Top10感受态中,经过筛选和PCR验证,挑取阳性克隆送至上海生工生物工程股份有限公司进行测序,经测序得到SmPPS2基因开放阅读框的核苷酸序列(如SEQ ID NO.2所示)和SmPPS2基因编码的氨基酸序列(如SEQ ID NO.1所示)。
实施例3长链异戊烯基转移酶SmPPS2基因的亚细胞定位分析
将实施例2克隆得到的SmPPS2基因的开放阅读框去掉终止密码子之后,连接至pCAMBIA1302植物双元表达载体上,构建载体1302-SmPPS2:GFP。1302-SmPPS2:GFP载体中关键元件的结构示意图如图1的A所示。将构建好的1302-SmPPS2:GFP载体通过冻融法转入农杆菌EHA105中。将农杆菌注射至烟草叶片分析SmPPS2基因表达的定位情况。亚细胞定位结果如图1的B所示,SmPPS2:GFP的绿色荧光与线粒体染料MitoTracker的红色荧光大部分重合,说明SmPPS2主要在线粒体中表达,这与辅酶Q的主要合成部位一致。
实施例4酵母诱导表达载体的构建、转化以及阳性克隆鉴定
根据实施例2克隆得到的SmPPS2的开放阅读框序列,设计特异引物,采用高保真Taq酶扩增SmPPS2的开放阅读框。将克隆得到的片段(1275bp,如SEQ ID NO.2所示)连接至酵母表达载体pYES2-CT上,连接产物转化大肠杆菌Top10,阳性克隆经PCR鉴定正确后,提取质粒pYES2-SmPPS2(质粒图谱如图2所示),将测序正确的质粒pYES2-SmPPS2转化至coq1-酿酒酵母突变体3138(Ducluzeau et al,Gene network reconstruction identifies theauthentic trans-prenyl diphosphate synthase that makes the solanesyl moietyof ubiquinone-9 in Arabidopsis,2012,The Plant Journal,69,366-375)中。酵母转化参考clontech网站的酵母转化方法进行,通过单缺陷Ura培养基以及G418抗性筛选得到阳性克隆。阳性转化子经过PCR和测序鉴定后用于后续诱导表达实验。
实施例5酵母互补实验以及SmPPS2功能分析
酵母coq1-突变体的互补试验参考文献(Ducluzeau et al,Gene networkreconstruction identifies the authentic trans-prenyl diphosphate synthasethat makes the solanesyl moiety of ubiquinone-9 in Arabidopsis,2012,The PlantJournal,69,366-375)进行。以野生型酵母BY4741和转化空载体pYES2-CT的酵母为对照。转基因酵母平板上活化两次后,挑单菌落置于2mL的液体培养基中过夜培养;500μL液体培养酵母于20mL的液体培养基上扩大培养,直至酵母培养至OD600值为0.6左右;吸取100μL液体培养的酵母,分别涂布于含有2%葡萄糖+0.05%D-半乳糖固体培养基上或者3%甘油+2.5%乙醇+0.05%D-半乳糖固体培养基上;30℃倒置培养2天(葡萄糖作为碳源)或者7天(甘油和乙醇作为碳源)。结果如图3的A所示,结果表明,转SmPPS2的酵母coq1突变体可以利用非发酵型碳源甘油和乙醇,在酵母基本培养基上生长状况良好,与野生型BY4741的生长情况差别不大。而酵母coq1突变体和转空载体pYES2-CT的coq1-酵母突变体不能在只含有非发酵型碳源(甘油和乙醇)的培养基上生长。
进一步采用HPLC-UV方法分析转SmPPS2的酵母coq1突变体的辅酶Q的积累,检测结果如图3的B所示,在半乳糖诱导作用下,在野生型(WT)酵母提取物中只检测到辅酶Q6;而酵母中coq1基因突变后失去了合成辅酶Q6的能力,当转入pYES2-CT空载后仍不能合成辅酶Q6,并且检测不到辅酶Q9和辅酶Q10成分;但是在转SmPPS2的酵母coq1-突变体的诱导提取物中可以检测到辅酶Q9和辅酶Q10,表明SmPPS2具有催化合成辅酶Q9和辅酶Q10的功能。
实施例6 SmPPS2的过表达和敲减载体的构建和丹参的转化
1、载体构建:SmPPS2过表达载体采用植物双元表达载体pCAMBIA1301,采用SmPPS2的开放阅读框替换载体自身的GUS基因,构建表达载体pCAMBIA1301-SmPPS2(载体的关键元件结构示意图如图4的A所示)。SmPPS2敲减载体采用人工microRNA的方法,根据人工microRNA的工作原理设计了一条特异的amiRNA,命名为amiRPPS2(5′-TAGTACTCCATGCTGCAGCGA-3′)。在杨树ptc-MIR408载体(Shi et al,Specific down-regulation of PAL genes by artifical microRNAs in Populus trichocarpa,2010,Planta,232:1281-1288)基础上,通过重叠PCR的方法将ptc-MIR408载体上的杨树miR408替换为amiRPPS2,再将得到的载体连接至pCAMBIA1301载体上,构建用于SmPPS2敲减的载体pCAMBIA1301-amiRPPS2(载体的关键元件结构示意图如图4的A所示)。所有的载体都经过上海生工生物工程股份有限公司测序确定序列正确。
2、土壤农杆菌的转化:
采用冻融法分别将构建好的植物表达载体pCAMBIA1301-SmPPS2和pCAMBIA1301-amiRPPS2转化至农杆菌GV3101中,具体方法如下:
(1)取1-2μg目的质粒与土壤农杆菌感受态细胞轻轻混匀;
(2)冰浴30min,将质粒感受态细胞混合液在液氮中迅速冷冻1min;
(3)37℃水浴5min,迅速冰浴2min;
(4)加入800μL YEP液体培养基,轻轻地颠倒混匀,28℃缓慢振荡培养4-6h。
(5)常温3000g离心5min,弃去800μL上清液;
(6)用剩余150μL上清液重悬菌体,将菌液均匀涂布于含有抗生素的固体培养基,于28℃培养箱中倒置培养1-3天。
3、丹参的遗传转化
农杆菌侵染丹参的具体方法参考文献(Wang et al,ARGONSUTE Genes in Salviamiltiorrhiza:Identification,Characterization,and Genetic Transformation,2017,Method in molecular biology,1640,173-189)进行。将得到的丹参SmPPS2过表达和丹参SmPPS2敲减的抗性株系进行PCR鉴定,结果分别如图4的B和E所示。采用荧光定量PCR检测转pCAMBIA1301-SmPPS2和pCAMBIA1301-amiRPPS2的丹参转基因株系中SmPPS2的相对表达水平的变化,结果分别如图4的C和F所示,结果表明,与野生型丹参植株相比,转pCAMBIA1301-SmPPS2的过表达丹参植株5、13和16中SmPPS2的表达量显著提高;与野生型丹参植株相比,转pCAMBIA1301-amiRPPS2的敲减丹参植株3和6中SmPPS2的表达量显著降低。
实施例7过表达SmPPS2和敲减SmPPS2基因的丹参的辅酶Q9和Q10含量分析
1、丹参叶片中辅酶Q的提取:采用乙醇浸提法,料液比为1:50,液氮研粉末,涡旋仪涡旋均匀,室温120rpm浸提1h。上清挥发干,乙醇10倍体积浓缩复溶,冰箱4℃过夜,析出杂质,离心去除沉淀后,上清滤膜过滤后上样,上样量为10μL。
2、仪器和色谱条件
仪器:Waters 2695 Separations Module;检测器:Waters 2996 PhotodiodeArray delector
柱子:Eclipse Plus C18,4.6×250mm,5μm色谱柱(Agilent);流动相为乙醇:甲醇=75:25;流速为1mL/min;温度为25℃;检测波长为275nm。
检测结果如图4的D和G所示,与野生型丹参植株相比,转pCAMBIA1301-SmPPS2的过表达丹参株系5、13和16中辅酶Q9和辅酶Q10的总含量(UQ总量)显著提高;与野生型丹参植株相比,转pCAMBIA1301-amiRPPS2的敲减丹参株系3和6中辅酶Q9和辅酶Q10的总含量(UQ总量)显著降低。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国医学科学院药用植物研究所
<120> 利用丹参长链异戊烯基转移酶基因SmPPS2调控辅酶Q合成的方法
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Claims (6)
1.丹参长链异戊烯基转移酶基因SmPPS2在提高辅酶Q产量中的应用,所述丹参长链异戊烯基转移酶基因SmPPS2的核苷酸序列如SEQ ID NO.2所示,所述辅酶Q为辅酶Q9和/或辅酶Q10。
2.丹参长链异戊烯基转移酶基因SmPPS2在制备提高辅酶Q产量的转基因植物、提高辅酶Q产量的植物遗传育种或品种改良中的应用,所述丹参长链异戊烯基转移酶基因SmPPS2的核苷酸序列如SEQ ID NO.2所示,所述辅酶Q为辅酶Q9和/或辅酶Q10。
3.丹参长链异戊烯基转移酶SmPPS2或含有丹参长链异戊烯基转移酶基因SmPPS2的生物材料在提高辅酶Q产量或制备提高辅酶Q产量的转基因植物中的应用,所述丹参长链异戊烯基转移酶的氨基酸序列如SEQ ID NO.1所示,所述辅酶Q为辅酶Q9和/或辅酶Q10。
4.根据权利要求3所述的应用,其特征在于,所述生物材料包括表达盒、载体、宿主细胞或工程菌。
5.一种调控生物体内辅酶Q合成的方法,其特征在于,通过提高丹参长链异戊烯基转移酶基因SmPPS2的表达量,提高生物体内辅酶Q的产量,所述SmPPS2的核苷酸序列如SEQ IDNO.2所示,所述辅酶Q为辅酶Q9和/或辅酶Q10。
6.根据权利要求5所述的方法,其特征在于,所述提高丹参长链异戊烯基转移酶基因SmPPS2的表达量为通过将携带SmPPS2的表达载体转化至生物体内实现;所述生物体包括微生物和植物。
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WO2006034286A2 (en) * | 2004-09-17 | 2006-03-30 | Pioneer Hi-Bred International, Inc. | Isopentenyl transferase sequences and methods of use |
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