CN114480451B - 一种何首乌查尔酮合酶基因FmCHS2及其编码产物和应用 - Google Patents
一种何首乌查尔酮合酶基因FmCHS2及其编码产物和应用 Download PDFInfo
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- CN114480451B CN114480451B CN202111630425.7A CN202111630425A CN114480451B CN 114480451 B CN114480451 B CN 114480451B CN 202111630425 A CN202111630425 A CN 202111630425A CN 114480451 B CN114480451 B CN 114480451B
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Abstract
本发明涉及一种何首乌查尔酮合酶基因FmCHS2及其编码产物和应用,所述基因FmCHS2的核苷酸序列如SEQ ID NO.1所示;所述编码产物氨基酸序列如SEQ ID No.2所示。何首乌查尔酮合酶基因FmCHS2是何首乌黄酮类化合物合成途径的关键调控基因,可用于调节何首乌的柚皮素含量,该酶可以应用于以对香豆酰辅酶A和的丙二酰辅酶A制备柚皮素的通路。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种何首乌查尔酮合酶基因FmCHS2及其编码产物和应用。
背景技术
何首乌Fallopia multiflora(Thunb.)Harald.系蓼科何首乌属多年生草本植物,分布于中国陕西南部、甘肃南部、华东、华中、华南、四川、云南及贵州等地。何首乌通常长在山谷灌丛、山坡林下、沟边石隙,海拔200-3000米等地方。2020年版《中国药典》记载何首乌的干燥块根做何首乌入药,具有解毒,消痈,截疟,润肠通便的功效。用于疮痈,瘵病,风疹瘙痒,久疟体虚,肠燥便秘等的治疗,为中医常用药材之一。其有效成分有二苯乙烯苷类、蒽醌类、黄酮类与酚类等。
黄酮类化合物的生物合成途径一般可分为三个阶段,第一阶段是由苯丙氨酸到香豆酰辅酶A,这是许多次生代谢共有的;第二阶段由香豆酰辅酶A到查尔酮,这是黄酮代谢的关键反应,它合成芦丁和其他黄酮类物质,所有黄酮类化合物合成的前体物都是对香豆酰辅酶A和丙二酰辅酶A在查尔酮合成酶(Chalcone synthase,CHS)的作用下产生查尔酮开始的;第三阶段是各种黄酮类化合物的合成。
目前,在很多植物中发现了CHS多基因家族,如葡萄、矮牵牛、郁金香、甘薯、拟南芥等。但对于何首乌FmCHS基因的克隆、表达模式及CHS蛋白编码序列目前尚不清楚。未有任何与何首乌CHS蛋白及其编码基因相关的文献报道。
发明内容
针对上述问题,本发明采用以下技术方案:
一种何首乌查尔酮合酶基因FmCHS2,该基因的核苷酸序列如SEQ ID NO.1所示。
一种如上述所述的何首乌查尔酮合酶基因FmCHS2编码的产物,其氨基酸序列如SEQ ID No.2所示。
一种含有如上述所述的何首乌查尔酮合酶基因FmCHS2的重组表达载体。
进一步地,所述重组表达载体以基因FmCHS2的cDNA为模板,如SEQ ID NO:3-4所示的引物,进行PCR扩增;将PCR产物和FmCHS2表达载体进行单酶切,回收纯化,连接转化大肠杆菌获得重组表达载体pET-32a-FmCHS2。
一种如上述所述的基因FmCHS2或上述所述的重组表达载体在制备黄酮类化合物中的应用。
进一步地,所述应用包括在细胞中转入所述何首乌查尔酮合酶基因FmCHS2或利用何首乌查尔酮合酶促进黄酮类化合物的合成。
进一步地,所述黄酮类化合物为柚皮素。
本发明的有益效果如下:
本发明所提供的何首乌查尔酮合酶基因FmCHS2是首次从何首乌植物中克隆制备所得。何首乌查尔酮合酶基因FmCHS2是何首乌黄酮类化合物合成途径的关键调控基因,可用于调节何首乌的柚皮素含量,该酶可以应用于以对香豆酰辅酶A和的丙二酰辅酶A制备柚皮素的通路。利用本发明所提供的基因可以通过基因工程技术提高何首乌药效成分黄酮类化合物的含量,可用于其大量生成,为制备何首乌的黄酮类化合物提供了研究方向。
本发明的其它特征和优点将在随后的说明书中阐述,并且,部分地从说明书中变得显而易见,或者通过实施本发明而了解。本发明的目的和其他优点可通过在说明书、权利要求书以及附图中所指出的结构来实现和获得。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例。
图1为何首乌查尔酮合酶基因FmCHS2的琼脂糖凝胶电泳图;
图2为何首乌查尔酮合酶基因FmCHS2结构功能域预测分析;
图3为何首乌查尔酮合酶基因FmCHS2二级结构预测分析;
图4为何首乌查尔酮合酶基因FmCHS2跨膜结构域预测分析;
图5为何首乌查尔酮合酶基因FmCHS2三级结构预测分析;
图6为何首乌查尔酮合酶基因FmCHS2系统进化树;
图7为何首乌查尔酮合酶FmCHS2蛋白质的SDS-聚丙烯酰胺凝胶电泳结果;
图8为柚皮素对照品的MRM色谱图;
图9为何首乌查尔酮合酶FmCHS2催化对香豆酰辅酶A和丙二酰辅酶A产物的MRM色谱图;
图10为pET-32a空载体催化对香豆酰辅酶A和丙二酰辅酶A产物的MRM色谱图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地说明,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实验中使用的试剂盒如反转录试剂盒PrimeScriptTMII 1st Strand cDNASynthesis Kit购自Takara Bio公司;切胶回收试剂盒EasyPure Quick Gel ExtractionKit、T载体pEASY-Blunt Zero Cloning Kit、原核表达感受态细胞BL21(DE3)购自北京全式金生物技术有限公司;引物由上海生工生物工程股份有限公司合成;高保真酶High-Fidelity PCR Master Mix with HF Buffer、BamH I限制性内切酶等购自纽英伦(NEB)生物技术北京有限公司;超微量紫外分光光度计购自美国Denovix公司,型号为DS-11+,S-03512);其他试剂均为进口或国产分析纯试剂。
一、何首乌总RNA的提取及cDNA合成
按照RNA prep Pure Plant Kit试剂盒操作说明提取何首乌块根中总RNA,用1%琼脂糖凝胶电泳检测总RNA完整性。利用超微量紫外分光光度计测定何首乌总RNA的A260nm和A280nm(A260nm为核酸最高吸收峰的吸收波长,A280nm为蛋白和酚类物质最高吸收峰的吸收波长),选择A260nm/A280nm为1.8-2.0的总RNA反转录成cDNA。以RNA为模板,采用TaKaRa反转录试剂盒(PrimeScriptTMII 1st Strand cDNA Synthesis Kit)进行反转录,得到何首乌的cDNA,将合成产物保存于-20℃冰箱中。
二、何首乌FmCHS2的克隆
依据何首乌转录组中FmCHS2序列设计引物,以何首乌cDNA为模板进行PCR扩增。扩增体系(50μL)如下:2×Phusion Master Mix为25μL,引物primer-F和引物primer-R各2.5μL,模板cDNA 1μL,其余用无菌双蒸水补足。反应条件:98℃预变性2min、98℃变性10s、60℃退火30s、72℃延伸2min、40个循环后72℃延伸5min、4℃保存。获得何首乌FmCHS2基因克隆,何首乌查尔酮合酶基因FmCHS2的琼脂糖凝胶电泳如图1所示,图1中,M表示Marker,泳道1表示目的基因。目的基因FmCHS2片段大小约为1200bp,符合预期。
三、何首乌FmCHS2基因的生物信息学分析
本发明实施例中所获得的何首乌查尔酮合酶基因全长cDNA,FmCHS2基因的开放阅读框(ORF)的长度为1182bp,其序列如序列表中SEQ ID NO.1所示。根据FmCHS2全长cDNA编码393个氨基酸,其序列如序列表中SEQ ID NO.2所示。将FmCHS2基因序列用NCBI数据库中的BLAST程序在Non-redundant GenBank+EMBL+DDBJ+PDB和Non-redundant GenBankCDStranslation+PDB+Swissprot+Superdate+PIR数据库中进行核苷酸同源性检索,该基因在氨基酸水平上与其他物种中的CHS有较高的同源性,如图2所示。FmCHS2蛋白二级结构由α螺旋,延伸链和无规则卷曲,如图3所示。FmCHS2无跨膜结构,为膜外蛋白,如图4所示。利用Swiss Model预测蛋白质的三级结构,如图5所示。以1jwx.1.A蛋白模型,FmCHS2蛋白序列的相似性为80.36%,得分为0.56;用MEGA6软件构建Neighbor-joining系统进化树,如图6所示,何首乌FmCHS2氨基酸序列与蓼科植物虎杖(Polygonum cuspidatum Siebold etZucc.)CHS2氨基酸基因序列位于同一分支点,其亲缘关系最高。
四、何首乌FmCHS2基因的原核表达载体构建与诱导表达
对克隆成功的序列进行分析,设计带有酶切位点的引物。以BamH I作为上下游引物的酶切位点,设计引物,上游引物:BamH I-FmCHS2-F:5'-AGGCCATGGCTGATATCGGAATGGCTCCGGCGGTTGCGGATATCA-3';BamH I-FmCHS2-R:5'-CGACGGAGCTCGAATTCGGATTAGTTAGCCACCGGCACACTGTGG-3'。以重组质粒为模板,进行PCR扩增。将扩增后的产物进行1%的琼脂糖凝胶电泳检测,并对其进行切胶回收。对切胶回收后的产物与表达载体pET-32a质粒分别进行BamH I酶切处理,并进行切胶回收。将切胶回收后的目的片段与表达载体pET-32a用无缝拼接试剂盒在50℃连接30min,将连接产物转化至大肠杆菌Trans1-T1感受态细胞中,挑取单克隆进行菌液PCR阳性检验、测序及提取质粒,将原核表达载体质粒转化到BL21(DE3)表达感受态中,构建转化表达宿主菌。取转化表达菌液按照1:100的比例加到含100mg/LAmp抗性的LB培养液中,37℃下以200rpm的搅拌速度振荡培养至A600=0.4~0.6,加入终浓度为1mMIPTG(异丙基-β-D-硫代半乳糖苷)于20℃条件下低温诱导12h,以同样条件处理pET-32a空载作为空白对照。取1mL菌液离心获取沉淀作为全菌,剩余菌液在4℃条件下5000×g条件下离心10min,弃去上清,加入5mL His BufferA(20mM Na3PO4·12H2O、500mM NaCl、20mM咪唑)重悬,5000×g条件下离心10min,再次弃去上清,取出立即置于冰上冷却。加3mL Buffer A重悬,于冰上超声破碎。超声破碎后,4℃条件下以10000×g的离心力离心15min,分别取50μL上清进行12%SDS-PAGE(聚丙烯酰胺凝胶电泳,其中丙烯酰胺浓度为12%)电泳分析。用80V电压电泳20min,再用120V电压电泳150min,电泳结束后,用考马斯亮蓝R250染色1h,再用脱色液脱色2h至条带清晰,观察结果并扫描保存胶图,如图7所示,图7中M表示ProteinMarker,1为pET-32a空载体,2为未诱导的FmCHS2,3为FmCHS2全菌,4为FmCHS2上清。与pET-32a空载相比,含有FmCHS2基因重组蛋白的全菌和超声破碎后的上清在53.26kDa处出现明显的目的蛋白条带,符合预期FmCHS2蛋白分子量大小。
五、体外酶功能验证
在1.5mL的离心管中中配制500μL反应体系,反应体系包括280μM丙二酰辅酶A、150μM对香豆酰辅酶A、100μL粗酶(蛋白上清)和0.1M磷酸钾缓冲液,总体系500μL酶促反应条件:30℃水浴反应60min过后,用250μL乙酸乙酯萃取,12000rpm离心10min后取上清(乙酸乙酯重复萃取3次)。氮吹仪吹干后,用100μL质谱甲醇溶解。采用分析平台为AB Sciex QTRAP5500三重四极杆-线性离子肼串联质谱仪(美国AB Sciex),分析色谱柱为BEH C18 1.7μm 2.1×100mm柱对产物和标准品柚皮素进行分析。
色谱分析条件为:流动相:0.1%甲酸水-A相,乙腈-B相;在0~1min时间段内,采用5%B相洗脱;在1~2min时间段内,采用5%~50%B相洗脱;在2~3min时间段内,采用50%~75%B相洗脱;在3~4min时间段内,采用90%~95%B相洗脱;在4~5min时间段内,采用5%B相洗脱。柱温40℃;流速0.4mL/min;进样量2μL。
质谱条件:负离子化模式下,采取多反应监测(MRM)检测喷雾电压4.5kV,离子化温度500℃,喷雾气(Gas1)为45psi,加热辅助气(Gas2)为45psi,气帘气为40psi,柚皮素检测离子对质荷比(m/z)为271/151,去簇电压(DP)为-138V和碰撞电压(CE)为-24.5V。
图8为柚皮素对照品的MRM色谱图,从图8的分析结果可以看出:柚皮素的保留时间为2.34min,图9为FmCHS2催化对香豆酰辅酶A和丙二酰辅酶A产物的MRM色谱图,从图9的分析结果可以看出:FmCHS2催化样品在保留时间为2.35min存在与柚皮素保留时间一致的特征峰;图10为pET-32a空载体催化对香豆酰辅酶A和丙二酰辅酶A产物的MRM色谱图,从图10的分析结果可以看出:pET-32a空载体催化样品在保留时间为2.34min没有存在与柚皮素保留时间一致的特征峰。
LC-MS(液相色谱质谱联用)结果如图9所示,pET32a-FmCHS2可以将一分子的对香豆酰辅酶A和三分子的丙二酰辅酶A转化为柚皮素(质荷比m/z为271/151),因此可以认定pET32a-FmCHS2具有催化一分子的对香豆酰辅酶A和三分子的丙二酰辅酶A合成柚皮素的活性。
尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
序列表
<110> 安徽中医药大学
中国中医科学院中药资源中心
<120> 一种何首乌查尔酮合酶基因FmCHS2及其编码产物和应用
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ctcaccatcg gcaccgccac tcctcccaac tgtgtctacc agaaggacta ccctgactac 120
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gtaagcgaag ttccaaggct tggcaaagaa gccgcccaaa aggcgatcaa agagtggggt 360
caacccaagt caaagatcac ccacgtcatc atgtgcacga cctccggcgt cgacatgccc 420
ggcgccgact accagctcac gaagctcctc ggcctccgcc cctccgtcaa gcgcttcatg 480
atgtaccagc aaggctgctt cgccggcggc accgtcctcc gcctcgccaa ggacctcgcc 540
gagaacaacc gaggcgcgcg cgttctcgtg gtttgctccg aaataaccgc tatctgcttc 600
cgcggcccta cggacacaca tttggactcc atggtgggcc aggctttgtt cggtgacgga 660
tccggggccg tcattattgg ggctgacccg gatttgtcaa ttgaaaagcc catatttgag 720
ctcgtgtgga cggcccaaac aatcctcccg gactccgaag gtgcaatcga cggtcacttg 780
agggaagtgg ggctcacttt ccatctcctc aaggacgtgc ccgggctaat ttccaagaac 840
attgagaaga gtcttacgga ggcgttttcg cctcttaatg tgagcgactg gaactcgctt 900
ttctggatcg cgcaccctgg ggggcccgcg atccttgatc aggttgaaag taagttgggg 960
ctcaaggaag agaagcttaa ggcgactaga caagtgttga atgactatgg aaacatgtcg 1020
agcgcgtgtg ttctgtttat catggatgag atgaggaaga agtcggttga gaatggacat 1080
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Pro Ala Thr Val Leu Thr Ile Gly Thr Ala Thr Pro Pro Asn Cys Val
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Tyr Gln Lys Asp Tyr Pro Asp Tyr Tyr Phe Arg Val Thr Asn Ser Asp
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His Met Thr Asp Leu Lys Glu Lys Phe Arg Arg Met Cys Asp Lys Ser
50 55 60
Asn Ile Glu Lys Arg Tyr Met Tyr Leu Thr Glu Glu Ile Leu Lys Glu
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Asn Pro Asn Met Cys Ala Tyr Met Gln Thr Ser Ser Leu Asp Thr Arg
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Gln Asp Met Val Val Ser Glu Val Pro Arg Leu Gly Lys Glu Ala Ala
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Gln Lys Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His
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Val Ile Met Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp Tyr
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Gln Leu Thr Lys Leu Leu Gly Leu Arg Pro Ser Val Lys Arg Phe Met
145 150 155 160
Met Tyr Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu Ala
165 170 175
Lys Asp Leu Ala Glu Asn Asn Arg Gly Ala Arg Val Leu Val Val Cys
180 185 190
Ser Glu Ile Thr Ala Ile Cys Phe Arg Gly Pro Thr Asp Thr His Leu
195 200 205
Asp Ser Met Val Gly Gln Ala Leu Phe Gly Asp Gly Ser Gly Ala Val
210 215 220
Ile Ile Gly Ala Asp Pro Asp Leu Ser Ile Glu Lys Pro Ile Phe Glu
225 230 235 240
Leu Val Trp Thr Ala Gln Thr Ile Leu Pro Asp Ser Glu Gly Ala Ile
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Asp Gly His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys Asp
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Val Pro Gly Leu Ile Ser Lys Asn Ile Glu Lys Ser Leu Thr Glu Ala
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Phe Ser Pro Leu Asn Val Ser Asp Trp Asn Ser Leu Phe Trp Ile Ala
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His Pro Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Ser Lys Leu Gly
305 310 315 320
Leu Lys Glu Glu Lys Leu Lys Ala Thr Arg Gln Val Leu Asn Asp Tyr
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Gly Asn Met Ser Ser Ala Cys Val Leu Phe Ile Met Asp Glu Met Arg
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Lys Lys Ser Val Glu Asn Gly His Ala Thr Thr Gly Glu Gly Leu Glu
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Claims (6)
1.一种何首乌查尔酮合酶基因FmCHS2,其特征在于,该基因的核苷酸序列如SEQ IDNO.1所示。
2.一种如权利要求1所述的何首乌查尔酮合酶基因FmCHS2编码的产物,其特征在于,所述产物的氨基酸序列如SEQ ID No.2所示。
3.一种含有如权利要求1所述的何首乌查尔酮合酶基因FmCHS2的重组表达载体。
4.根据权利要求3所述的重组表达载体,其特征在于,所述重组表达载体以基因FmCHS2的cDNA为模板,如SEQ ID NO:3-4所示的引物,进行PCR扩增;将PCR产物和FmCHS2表达载体进行单酶切,回收纯化,连接转化大肠杆菌获得重组表达载体pET-32a-FmCHS2。
5.权利要求1所述的基因FmCHS2或权利要求3-4任一项所述的重组表达载体在制备柚皮素中的应用。
6.如权利要求5所述的一种何首乌查尔酮合酶基因FmCHS2在制备柚皮素中的应用,其特征在于,包括在细胞中转入所述何首乌查尔酮合酶基因FmCHS2或利用何首乌查尔酮合酶促进柚皮素的合成。
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