CN116836953A - 一种艾叶氧甲基转移酶fomt4及其编码基因、载体与应用 - Google Patents
一种艾叶氧甲基转移酶fomt4及其编码基因、载体与应用 Download PDFInfo
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- CN116836953A CN116836953A CN202310995383.XA CN202310995383A CN116836953A CN 116836953 A CN116836953 A CN 116836953A CN 202310995383 A CN202310995383 A CN 202310995383A CN 116836953 A CN116836953 A CN 116836953A
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- fomt4
- oxymethyl
- transferase
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Abstract
本发明涉及基因工程技术和酶学领域,具体涉及一种艾叶氧甲基转移酶FOMT4及其编码基因、载体与应用。本发明中提供的艾叶氧甲基转移酶FOMT4,在艾中首次发现,属于I类氧甲基化转移酶,其甲基化反应过程不依赖于金属离子。酵母表达与底物饲喂体系试验表明FOMT4在体外可以催化类黄酮4’位,6位,7位的羟基发生甲基化,具体为催化野黄芩素生成高车前素、柳穿鱼黄素、鼠尾草素和催化棕矢车菊素生成异泽兰黄素。该酶活反应简单,且不需要额外添加金属离子,具有较高的应用价值和研究前景。
Description
技术领域
本发明涉及基因工程技术和酶学领域,具体涉及一种艾叶氧甲基转移酶FOMT4及其编码基因、载体与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
艾叶来源于菊科植物艾(Artemisia argyi Levl.et Vant.)的干燥叶,艾叶中除含有挥发油、三萜类、多糖等多种化学成分外,还含有丰富的黄酮类化合物。类黄酮的生物活性与其结构密切相关,其中O-甲基化修饰的黄酮可提高类黄酮结构稳定性、蛋白亲和力、转运力,降低其水溶性,更有利于发挥其生物活性,如抗氧化、抗肿瘤、抗病毒、抑菌等作用。
目前从艾叶中分离纯化氧甲基化黄酮工序主要有硅胶柱层析法和大孔树脂吸附法,复杂且得率较低。随着基因工程和酶学技术的发展,通过植物类黄酮氧甲基转移酶(Flavonoid O-methyltransferase,FOMT)酶法合成O-甲基化黄酮具有较高的应用前景。艾叶中富含多氧甲基黄酮(PMFs),且存在多种FOMT参与合成艾叶中多氧甲基黄酮。艾叶中FOMTs种类繁多,不同FOMT催化底物和催化位点均不相同,目前尚未有研究分离并鉴定艾叶中FOMTs,不利于利用特定FOMT催化特点产物的特定位点,生成目标O-甲基化黄酮产物。
发明内容
为了解决现有技术的不足,本发明的目的是提供一种艾叶氧甲基转移酶FOMT4及其编码基因、载体与应用。本发明中提供的艾叶氧甲基转移酶FOMT4,在艾中首次发现,属于I类氧甲基化转移酶,其甲基化反应过程不依赖于金属离子。酵母表达与底物饲喂体系试验表明FOMT4在体外可以催化类黄酮4’位,6位,7位的羟基发生甲基化,具体为催化野黄芩素生成高车前素、柳穿鱼黄素、鼠尾草素和催化棕矢车菊素生成异泽兰黄素。
为了实现上述目的,本发明是通过如下的技术方案来实现:
第一方面,本发明提供了一种艾叶氧甲基转移酶FOMT4,所述艾叶氧甲基转移酶FOMT4的氨基酸序列为以下之一:
(1)SEQ ID NO.1所示的氨基酸序列;
(2)将SEQ ID NO.1所示的氨基酸序列经过若干个氨基酸取代/添加/缺失且功能相同的蛋白质。
第二方面,本发明提供了第一方面所述的艾叶氧甲基转移酶FOMT4的编码基因,所述编码基因的核苷酸序列为以下之一:
(1)SEQ ID NO.2所示的核苷酸序列;
(2)与SEQ ID NO.2所示的核苷酸序列同源性大于90%且编码具有相同功能蛋白质的核苷酸序列。
第三方面,本发明提供了第二方面所述的编码基因的真核表达载体,其特征在于,所述表达载体为插入第二方面所述的编码基因的表达载体pPICZαA。
第四方面,本发明提供了第一方面所述的艾叶氧甲基转移酶FOMT4在催化类黄酮化合物特定位点的羟基发生甲基化中的应用。
上述本发明的一种或多种技术方案取得的有益效果如下:
本发明首次从艾基因组库中分离出一个能够催化黄酮4’位、6位、7位的羟基发生氧甲基化修饰的氧甲基转移酶FOMT4。通过构建酵母表达载体并转化至酵母表达菌株GS115,诱导表达得到目的蛋白。体外酶活试验表明FOMT4能够催化野黄芩素形成高车前素、柳穿鱼黄素、鼠尾草素,催化棕矢车菊素生成异泽兰黄素,该酶活反应简单,且不需要额外添加金属离子,具有较高的应用价值和研究前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中FOMT4基因扩增电泳图,其中Marker为DNA分子量标准Marker(100~5000bp);
图2为实施例2中FOMT4重组蛋白WB电泳图,其中泳道1为蛋白分子质量标准,泳道2、4为未使用甲醇诱导的酵母总蛋白,泳道3、5为甲醇分别诱导48h、72h时的酵母总蛋白;
图3为实施例3中LC-MS对野黄芩素(Scutellarein)的反应产物鉴定,其中a为FOMT4催化野黄芩素生成高车前素、鼠尾草素、柳穿鱼黄素的结构图,b为HPLC分析,Standards of Hispiduln代表标准的高车前素,Standards of Salvigenin代表标准的鼠尾草素,Standards of Pectolinarigenin代表标准的柳穿鱼黄素,Empty+Scutellarein代表携带空载体的酵母与野黄芩素为底物共孵育,FOMT4+Hispiduln代表表达FOMT4的酵母与野黄芩素为底物共孵育,1c为产物高车前素(Hispiduln)的质谱图,2c为产物鼠尾草素(Salvigeni)的质谱图,产物柳穿鱼黄素(Pectolinarigenin)的质谱图;
图4为实施例3中LC-MS对棕矢车菊素(Jaceosidin)的反应产物鉴定,其中a为FOMT4催化棕矢车菊素生成异泽兰黄素结构图,b为HPLC分析,Standards of Eupatilin代表标准的异泽兰黄素;Empty+Jaceosidin代表携带空载体的酵母与棕矢车菊素为底物共孵育,FOMT4+Eupatilin代表表达FOMT4的酵母与棕矢车菊素为底物共孵育,c为产物异泽兰黄素(Eupatilin)的质谱图。
具体实施方式
本发明的第一种典型实施方式,一种艾叶氧甲基转移酶FOMT4,所述艾叶氧甲基转移酶FOMT4的氨基酸序列为以下之一:
(1)SEQ ID NO.1所示的氨基酸序列;
(2)将SEQ ID NO.1所示的氨基酸序列经过若干个氨基酸取代/添加/缺失且功能相同的蛋白质。
本发明的第二种典型实施方式,第一种典型实施方式所述的艾叶氧甲基转移酶FOMT4的编码基因,所述编码基因的核苷酸序列为以下之一:
(1)SEQ ID NO.2所示的核苷酸序列;
(2)与SEQ ID NO.2所示的核苷酸序列同源性大于90%且编码具有相同功能蛋白质的核苷酸序列。
用于扩增上述编码基因的全长引物对,其核苷酸序列分别为SEQ ID NO.3和SEQID NO.4所示。
用于扩增上述编码基因的带酶切接头引物对,带有EcoRI正向引物其核苷酸序列为SEQ ID NO.5所示,带有XbaI反向引物其核苷酸序列为SEQ:NO.6所示。
本发明的第三种典型实施方式,第二种典型实施方式所述的编码基因的真核表达载体,其特征在于,所述表达载体为插入第二种典型实施方式所述的编码基因的表达载体pPICZαA。
本发明的第四种典型实施方式,第一种典型实施方式,所述的艾叶氧甲基转移酶FOMT4在催化类黄酮化合物特定位点的羟基发生甲基化中的应用。
该实施方式的一种或多种实施例中,所述类黄酮化合物包括野黄芩素或异泽兰黄素。
该实施方式的一种或多种实施例中,所述特定位点为4’位、6位、7位中的一个或多个。
该实施方式的一种或多种实施例中,具体为在催化野黄芩素生成高车前素、柳穿鱼黄素、鼠尾草素以及催化棕矢车菊素生成异泽兰黄素中的应用。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例与对比例详细说明本发明的技术方案。
下述实施例中常规的基因操作方法参照《分子克隆实验指南》(第三版)。
实施例1
基因克隆:利用同源比对和保守结构域(PF01596)从艾叶基因组数据库中调取类黄酮生物合成通路上的FOMT4基因序列,其核苷酸序列为SEQ:NO.2。结合引物对的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示,以艾叶cDNA为模板进行PCR扩增,其中PCR体系为:Buffer 5μl,dNTP 1μl,上下游引物(10μM)各0.5μl,酶0.5μl,DEPC处理的水39.5μl,cDNA 3μl;PCR程序为:94℃预变形5min;94℃变性30s,58℃退火30s,72℃延伸1min30s,30个热循环;72℃延伸5min,4℃保存。PCR产物经1%琼脂糖凝胶电泳后回收,连接至pGEM-T Easyvector(Promega),转化到大肠杆菌(DH5α)中,最后通过菌落PCR和后续的测序验证挑选阳性单克隆并保存。
如图1所示,成功从艾叶中克隆得到FOMT4全长序列1077bp,核苷酸序列如SEQ IDNO.2所示,其编码的氨基酸序列如图SEQ ID NO.1所示,并构建了插入FOMT4全长序列的FOMT4-T质粒。
实施例2
目的基因重组载体构建
根据pPICZαA载体的序列,选择EcoRI酶及XbaI酶。设计FOMT4全长序列F/R端添加酶切位点与载体序列的引物对(核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示),以实施例1中得到的FOMT4-T为模板,按照实例1中的扩增体系和程序克隆FOMT4全长序列(不包含终止子)。用EcoRI酶和XbaI酶(NEB)线性化pPICZαA载体,将PCR胶回收产物和双酶切回收产物进行一步重组法(诺唯赞,南京)反应,37℃反应30min。将反应产物用热激方法转化大肠杆菌DH5α,培养12h,挑取单克隆,通过菌落PCR和测序验证挑选阳性单克隆,测序正确的菌液,提取质粒,即完成酵母表达载体构建。
重组载体的线性化
取10μg以上质粒,用SacI进行单酶切反应,37℃酶切2.5h,反应完成后用PCR回收试剂盒进行纯化回收,核酸浓度的测定采用Termer的Nano Drop 2000测定。
酵母感受态制备
取冻存的GS115酿酒酵母在YPDA固体培养基上划线,接种单克隆到3mL 1×YPDA培养基中,30℃250rpm摇床培养8-12h。取5μl摇好的菌液至50mL 1×YPDA培养基中,30℃250rpm摇床培养至OD260达0.15-0.3(约16-20h)。室温下700g离心5min,去上清,使沉淀重新悬浮于100mL新鲜的1×YPDA中,继续培养至OD260达0.4-0.5(约3-5h)。将菌液分为两份,分别50mL,室温下8000rpm离心5min,去上清,使沉淀分别重新悬浮于30mL无菌dd水中。室温下8000rpm离心5min,去上清,使沉淀重新悬浮于1.5mL 1.1×TE/LiAc中。取细胞悬液至两个2mL无菌离心管中,4℃高速离心15s;去上清,使沉淀重新悬浮于600μl 1.1×TE/LiAc中,即可得到酵母感受态,可用于酵母转化。
FOMT4重组载体电击转化酵母
将5-20μg的线性化重组载体溶解在5-10μl TE溶液(或无菌水)中,与100μl的上述步骤所得的感受态菌体混匀,转至0.2cm冰预冷的电转化杯中。电击完毕后,加入1mL 1M的冰预冷的山梨醇溶液将菌体混匀,洗出转至1.5mL的EP管中;500g离心,去上清,加入1mLYPD,27℃,220rpm,摇3h;稍微离心,将菌体悬液涂布于选择平板上,每200~600μl涂布一块平板,将平板置于30℃倒置培养48h。挑取单克隆作阳性检测,将阳性菌液用20%甘油于-80℃下保存。
实施例3
FOMT4重组蛋白表达及检测
小量诱导表达
挑适量实施例2获得的阳性菌液在YPDA液体培养基(100μg/mL Zeocin)中30℃250rpm摇过夜,至菌液浑浊,转接到25mL BMGY。28-30℃250rpm培养24h左右至OD260=2-6。
超净台中,将菌液转移至50mL Corning管,8000rpm离心10min,弃上清,再用100mLBMMH培养基重悬菌体,饥饿1h或者用BMMH洗涤一遍沉淀以除去残留BMGH。
24℃250rpm甲醇诱导表达,每24h补甲醇至终浓度为0.5%。在48h(2d)、72h(3d),取1mL培养基至1-5mL离心管。室温用水平离心机最大转速离心2-3min,去上清保存菌液,进行后续蛋白印迹分析。
蛋白印迹(WB)检测
样品制备。采用生工酵母蛋白提取试剂盒提取目标蛋白。将酵母菌蛋白液,加入一定体积5×SDS-PAGE凝胶上样缓冲液,金属浴100℃煮沸10min,使蛋白充分变性。
SDS-PAGE凝胶电泳。分别吸取5μL未使用甲醇诱导的酵母蛋白液和48h和72h收集的5μL上述蛋白样品用于蛋白质聚丙烯酰氨凝胶电泳(FuturePAGETM蛋白预制胶)跑胶,电泳条件为①80V 30min②120V 60min。
湿转。根据目的蛋白分子量确定所需PVDF膜的面积大小并裁剪好,用甲醇浸泡活化1分钟,在低温冰箱提前预冷的湿转液中,按照黑夹-海绵-滤纸-胶-PVDF膜-滤纸-海绵-白夹的顺序放置“三明治夹”,排除气泡。在湿转槽中注满预冷的新湿转液,并加入冰袋保持低温,整体置于冰浴中,以恒流320mA进行转膜,根据分子量大小决定转膜时间,约1分钟/KD。
封闭。转膜过程结束后,根据Marker位置对比着剪出目标蛋白所在条带,将标记好的膜,置于新配的5%脱脂奶粉溶液(TBST缓冲液稀释),在室温条件下,将孵育盒放在摇床上30-50rpm/min低速缓慢振摇1小时。
一抗孵育。用移液器吸弃封闭液,加入5%的BSA溶液(TBST缓冲液稀释)稀释一抗,稀释比例依据每个抗体的说明书,保证最终一抗溶液(c-myc标签,购于abclonal)能浸没条带,将抗体孵育盒放在摇床上4℃条件下缓慢振摇孵育过夜(12小时以上)。
二抗孵育。用移液器回收一抗,在-25℃低温冰箱中可以保存1周左右,随后小心加入TBST溶液,以150-200rpm/min低速洗涤条带4次,每次4分钟,最后,加入5%BSA溶液(TBST缓冲液稀释)稀释的二抗,稀释比例依据抗体说明书,要保证最终二抗溶液能浸没条带,在室温下放置摇床30-50rpm/min低速缓慢振摇孵育1小时。
化学发光,成像。用移液器回收二抗,在-25℃低温冰箱中可以保存一段时间。随后小心加入TBST溶液,以150-200rpm/min转速洗涤条带4次,每次4分钟,将条带置于凝胶成像系统中,在避光条件加入新鲜混合好的ECL化学发光液100-300μL,最后启动Tanon的凝胶成像系统程序,进行曝光,成像并分析处理实验结果。
如图2所示,诱导所得蛋白样品大小约为40KDa,与FOMT4-pPICZαA重组蛋白理论计算值(39.4KDa)一致,可用于酶活研究。
实施例4
底物共孵育
挑转化成功酵母的单克隆菌体在YPD液体培养基(100μg/mL Zeocin)30℃250rpm摇过夜,至菌液浑浊,转接到25mL BMGH挑取单克隆,接种至25mL BMGY中,28-30℃,250-300rpm摇至OD600=2-6。
超净台中,将菌液转移至50mL Corning管,8000rpm离心10min,弃上清,再用20mLBMMH培养基重悬菌体,饥饿1h或者用BMMH洗涤一遍沉淀以除去残留BMGH。
在主培养接种24h时,所有底物以每个培养容器200μl等分的10mM DMSO溶液的形式提供,最终底物浓度为100μM。
主要培养物在29℃和210转/分钟下生长。每24h在培养基中加入100μL(0.5%)的甲醇。取样时间为72h,收集菌体样本。
LC-MS检测
液相条件:安捷伦1260液相系统;Agilent ECuPSE PLuS C18柱子(2.1×5mm,1.8μm);流动相A:0.8%甲酸纯净水,流动相B:乙腈;流速:0.3mL/min;洗脱梯度:0-0.3min:2%-5%B,0.3min-1.0min:5%B,1.0-10.0min:5%B-28%B,10.0-16.0min:28%B-45%B,16.0-19.0min:45%B-70%B,19.0-20.5min:70%B-98%B,20.5-24.0min:98%B,24.0-24.2min:98%B-2%B,24.2-27.0min:2%B;进样量:0.1μl。
高分辨率质谱分析:三重四极杆QQQQ系统;均使用MRM模式对反应进行检测,所有黄酮物质在正离子模式下进行质谱分析。优化野黄芩素、高车前素、柳穿鱼黄素、鼠尾草素、棕矢车菊素、异泽兰黄素在MRM模式下的母离子、Fragmenotr电压,子离子、碰撞能量(CE)。具体如下:野黄芩素:母离子为287.2,Fragmentor 163v;碎片离子1为123.1,CE 38v;碎片离子2为94.9,CE 50v。高车前素:母离子为301.2,Fragmentor 124v;碎片离子1为286.2,CE26v;碎片离子2为168.1,CE 38v。柳穿鱼黄素:母离子315.3,Fragmentor129v;碎片离子1为300.1,CE24v;碎片离子2为168,CE36v。鼠尾草素:母离子为329.3,Fragmentor 139v;碎片离子1为268.1,CE32v;碎片离子2为296.2CE24v。棕矢车菊素:母离子为331.2,Fragmentor149v;碎片离子1为316.2,CE 26v;碎片离子2为168.1,CE 38v。异泽兰黄素:母离子为345.2,Fragmentor 140v;碎片离子1为330.2,CE 30v;碎片离子2为169.1,CE 40v。
通过对FOMT4与野黄芩素、棕矢车菊素底物共孵育的样品进行LC-MS检测,如图3所示FOMT4可催化野黄芩素生成高车前素、柳穿鱼黄素、鼠尾草素,如图4所示FOMT4可催化棕矢车菊素生成异泽兰黄素。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种艾叶氧甲基转移酶FOMT4,其特征在于,所述艾叶氧甲基转移酶FOMT4的氨基酸序列为以下之一:
(1)SEQ ID NO.1所示的氨基酸序列;
(2)将SEQ ID NO.1所示的氨基酸序列经过若干个氨基酸取代/添加/缺失且功能相同的蛋白质。
2.权利要求1所述的艾叶氧甲基转移酶FOMT4的编码基因,其特征在于,所述编码基因的核苷酸序列为以下之一:
(1)SEQ ID NO.2所示的核苷酸序列;
(2)与SEQ ID NO.2所示的核苷酸序列同源性大于90%且编码具有相同功能蛋白质的核苷酸序列。
3.权利要求2所述的编码基因的真核表达载体,其特征在于,所述表达载体为插入权利要求2所述的编码基因的表达载体pPICZαA。
4.权利要求1所述的艾叶氧甲基转移酶FOMT4在催化类黄酮化合物特定位点的羟基发生甲基化中的应用。
5.如权利要求4所述的应用,其特征在于,所述类黄酮化合物包括野黄芩素或异泽兰黄素。
6.如权利要求4所述的应用,其特征在于,所述特定位点为4’位、6位、7位中的一个或多个。
7.如权利要求4所述的应用,其特征在于,具体为在催化野黄芩素生成高车前素、柳穿鱼黄素、鼠尾草素以及催化棕矢车菊素生成异泽兰黄素中的应用。
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