CN116855465A - 一种异鼠李素生物合成相关的黄酮醇3’-o-氧甲基转移酶及其应用 - Google Patents
一种异鼠李素生物合成相关的黄酮醇3’-o-氧甲基转移酶及其应用 Download PDFInfo
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- CN116855465A CN116855465A CN202210340160.5A CN202210340160A CN116855465A CN 116855465 A CN116855465 A CN 116855465A CN 202210340160 A CN202210340160 A CN 202210340160A CN 116855465 A CN116855465 A CN 116855465A
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- flavonol
- oxymethyl
- transferase
- expression vector
- isorhamnetin
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Classifications
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Abstract
本发明提供了一种黄酮醇3’‑O‑氧甲基转移酶、表达载体和以及含有该表达载体的转基因细胞系和宿主菌。本发明首次克隆并验证了桃异鼠李素生物合成相关的PpFOMT1基因的功能，实现了PpFOMT1基因的异源活性表达，重组蛋白可以利用底物SAM和槲皮素，高效转化成异鼠李素。本发明还提供了PpFOMT1基因及其编码蛋白、含有该PpFOMT1核苷酸序列的表达载体以及含有该表达载体的转基因细胞系和宿主菌在异鼠李素生物合成中的用途。本发明进一步提供了表达载体及含有表达载体的转基因细胞系或宿主菌在异鼠李素生物合成的用途。
Description
技术领域
本发明属于分子生物学领域，涉及重组蛋白和基因工程，具体涉及一种异鼠李素生物合成相关的黄酮醇3’-O-氧甲基转移酶及其应用。
背景技术
黄酮醇是植物体内一种重要的次生代谢物，且研究表明其化合物具有广泛的生物及药理活性。常见的黄酮醇苷元主要包括：槲皮素、山奈酚、杨梅素以及异鼠李素，同时黄酮醇化合物常存在不同类型及程度的修饰，包括糖基化、甲基化和酰基化等。其中，槲皮素在3'位羟基处可发生甲基化从而生成异鼠李素。相关研究表明异鼠李素具有良好抗癌和心血脑血管保护活性。
担任黄酮醇化合物修饰的重要角色之一的氧甲基转移酶(OMT)具有催化羟基氧甲基化功能，根据分子量和二价金属离子依赖性可将其分为两个主要亚家族：咖啡酸O-甲基转移酶(COMT)和咖啡酰辅酶A O-甲基转移酶(CCoAOMT)，参与类黄酮甲基化的类黄酮OMT(FOMT)通常属于COMT亚家族，尽管越来越多的FOMT被鉴定，一些FOMT也聚类在CCoAOMT亚家族中，但是一些常见且营养价值丰富的水果的OMT基因及特征功能并未被表征或解析。
桃(Prunus persica)是蔷薇科桃属果树，原产中国，世界各地均有栽培，且栽培面积逐年递增。桃因果实风味浓郁，富含黄酮醇等具有生物活性的天然产物，而受到人们的喜爱。异鼠李素是桃果实中重要的黄酮醇组分，其中以异鼠李素芸香糖苷最为丰富，因此鉴别参与异鼠李素生物合成的FOMT，对于阐明异鼠李素生物合成机制具有重要意义，可应用于基于基因工程的植物基因组改造，定向改良黄酮醇组分及含量，增加食物的保健功能，具有重要的应用价值。
发明内容
本发明首次表征了桃PpFOMT1基因在参与异鼠李素生物合成基因中的独特作用。从桃组织中成功克隆PpFOMT1基因，即PpFOMT1的cDNA序列(参见SEQ ID NO.1所示序列)，通过PCR扩增此DNA片段，然后将次片段连接至T-easy载体上(参见实施例1和实施列2)，验证成功后构建表达载体pET-32a(+)在原核细胞中首次表达了PpFOMT1多肽或蛋白质，即黄酮醇3’-O-氧甲基转移酶(参见SEQ ID NO.2所示序列和实施例2)。进一步，本发明所述的黄酮醇3’-O-氧甲基转移酶是由所述的PpFOMT1基因经原核细胞内的复制、转录和翻译过程而来。所述的复制指的是细胞以所述PpFOMT1基因的核苷酸为模板产生多个相同的基因的过程。所述的转录是指细胞以所述多个相同的PpFOMT1基因的核苷酸为模板，根据核苷酸互补配对原则，以核糖核苷酸为原料，合成对应的PpFOMT1基因mRNA的过程。所述的翻译是指，细胞进一步以所述的PpFOMT1基因mRNA为模板，以各种氨基酸为原料，合成出PpFOMT1基因对应得多肽或蛋白质的过程。所述的PpFOMT1基因的CDS序列如SEQ:NO.1所示，编码序列全长为705个核苷酸，可编码一个含234个氨基酸的蛋白。其氨基酸序列如SEQ:NO.2所示，在甲氧基转移酶大家族中属于CCOAOMT亚家族。
本发明提供了一种黄酮醇3’-O-氧甲基转移酶，其特征在于，至少含有下列1)～4)特征之一：
1)所述黄酮醇3’-O-氧甲基转移酶编码基因的核苷酸序列为SEQ:NO.1所示；
2)所述黄酮醇3’-O-氧甲基转移酶的氨基酸序列为SEQ:NO.2所示；
3)与SEQ ID NO.1所示的DNA序列杂交的核苷酸序列；
4)与SEQ ID NO.1编码相同功能蛋白质的核苷酸序列。
优选的，本发明所述黄酮醇3’-O-氧甲基转移酶编码基因由桃中分离得到；进一步，所述黄酮醇3’-O-氧甲基转移酶编码基因由桃的叶、花、果中得到。只要是编码具有PpFOMT1黄酮醇3’-O-氧甲基化反应功能蛋白质的核苷酸序列，就应包含在本发明涉及的黄酮醇3’-O-氧甲基转移酶基因中。
本发明还提供了一种基因表达载体，其特征在于：所述的载体含有如前所述黄酮醇3’-O-氧甲基转移酶基的核苷酸序列或氨基酸序列；进一步，所述基因的核苷酸序列如SEQ:NO.1所示，该基因编码蛋白的氨基酸序列如SEQ:NO.2所示。具体地，所述的基因表达载体含有如前所述的PpFOMT1基因的核苷酸序列或含有如前所述的PpFOMT1基因的氨基酸序列。本发明所述的基因可插入到现有的真核或原核表达载体中，适宜的载体包括细菌质粒、慢病毒、腺病毒、腺相关病毒、逆转录病毒等。所述的载体是一种环状DNA分子，能够在细胞内自主复制和转录表达，是基因工程中最常用的工具之一。
本发明还提供了一种转基因细胞系或宿主菌，其特征在于：所述的转基因细胞系或宿主菌含有如前所述的黄酮醇3’-O-氧甲基转移酶或基因表达载体。具体地，所述的转基因细胞系或宿主菌含有如前所述的具有PpFOMT1核苷酸序列或PpFOMT1氨基酸序列的基因表达载体。本发明所述的含有PpFOMT1基因的载体可以用来转化到适当细胞系或宿主菌，所述的细胞系可以来自动物或植物细胞，例如昆虫细胞、哺乳动物细胞，所述的宿主菌可以是经过修饰的基因工程菌，例如酵母菌、大肠杆菌等。
本发明还提供了如前所述的黄酮醇3’-O-氧甲基转移酶、基因表达载体、转基因细胞系或宿主菌在异鼠李素苷制备中的应用；进一步，所述的应用包括生产基因工程产品、培育植物新品种、制备食品等。所述的基因工程产品包括利用基因工程技术获取的药品、食品、化妆品、保养保健品等。
本发明还提供了一种异鼠李素的制备方法，其特征在于，所述的制备方法选自以下的任意一种：1)向如前所述的糖基转移酶直接提供底物-腺苷甲硫氨酸(SAM)和槲皮素；2)通向细胞系或宿主菌中导入如前所述的含有黄酮醇3’-O-氧甲基转移酶核苷酸序列的基因表达载体，诱导转基因细胞系或宿主菌表达重组蛋白；向重组蛋白提供SAM和槲皮素，从而合成异鼠李素；3)向细胞系或宿主菌中导入如前所述的含有黄酮醇3’-O-氧甲基转移酶核苷酸序列的基因表达载体，并向宿主菌提供原料SAM和槲皮素，从而合成异鼠李素。
以大肠杆菌宿主菌为例，诱导其翻译表达PpFOMT1蛋白，然后提供原料SAM和槲皮素，从而合成类异鼠李素。如图4和实施例3所示，以大肠杆菌宿主菌为例，提供原料SAM和槲皮素，则生成了异鼠李素。
本发明对研究更多物种具有独特催化活力和特性的黄酮醇3’-O-氧甲基转移酶具有指导性意义，为开发工程微生物菌或基于基因工程技术的植物异鼠李素组分改良奠定基础。
附图说明
图1是桃PpFOMT1蛋白与其他植物OMT系统发育树分析；PpAOMT1(XP_007221607)，PpAOMT2(XP_020412429),VvAOMT1(Z54233)，AtOMT1(AT5G54160)，McPFOMT(AY145521)，CrOMT1(Ciclev10026344m)，GmSOMT-9(NP_001236240)，OsROMT-15(XM_483167)，OsROMT-17(XM_507282)，PtCCoAOMT(ACE95173)，AtCCoAOMT7(At4g26220)，PtCCoAOMT2(KX219802)，VvCCoAOMT(CAA90969)，BpCCoAOMT(AAT37172)，NtCCoAOMT(AAC49913)，EgCCoAOMT(CAA72911)，PaCCoAOMT1(ANG84009)，MsCCoAOMT(AAC28973)，VvCOMT(CAQ76879)，CaCOMT2(AAA86982)，AtCOMT(AY081565)，PdCOMT(Q43609)，PtCOMT(AAF63200)，MsCOMT(AAB46623)，ShCOMT(2119166A)，SbCOMT(AAL57301)，ZmCOMT(Q06509)，NtCOMT(AAL91506)，TaOMT2(AY226581)，CrOMT2(HM641694)，CrOMT6(AY343490)，GeHIOMT(Q84KK6)，MsIOMT(AAC49928)，LjHIOMT(Q84KK4)，MpOMT1A(AY337457)，MpOMT1B(AY337458)，MpOMT2(AY337459)，MpOMT3(AY337460)，MpOMT4(AY337461)，MtIOMT1(XP_003621488)，MtIOMT2(XP_039683998)，MtIOMT7(XP_013455259)。
图2是桃PpFOMT1重组蛋白的SDS-PAGE分析图。
图3是重组蛋白PpFOMT1对槲皮素体外酶活性分析HPLC图谱。
图4是重组蛋白PpFOMT1体外酶活性产物LC-MS图谱。-ESI EIC(负离子模式产物分子量)；异鼠李素负离子模式分子量：315。
具体实施方式
下面对本发明的实施例和附图作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：桃RNA提取及PpFOMT1基因克隆
一、实验方法
1、材料：以‘湖景蜜露’桃果实为材料，设置四个生物学重复，每个重复6个果实，取果皮组织，迅速用液氮冻透，放至-80℃冰箱中保存。
2、利用CTAB法提取桃果肉的RNA，按照PrimeScriptTMRT reagent Kit with gDNAEraser(Takara)试剂说明书操作合成cDNA。以反转录产物cDNA为模板，用SEQ:NO.3和SEQ:NO.4所示引物进行PCR扩增，PCR反应体系为50μL，成分分别为：2×Phanta Max Buffer 25μL，dNTP Mix(10mM each)1μL，DNA polymerse(1U/μL)1μL，上下游引物(10μM)各2μL，cDNA 1μL，H2O 18μL。PCR程序为：95℃预变性3min，35个循环的95℃15s，58℃15s和72℃1min 20s，72℃5min，4℃hold。得到扩增产物。
3、将PCR扩增产物连接到T-easy载体，转化大肠杆菌DH5α，挑取单菌落进行菌落PCR验证，获得阳性菌落进行测序。
二、实验结果
1、测序结果返还之后经过比对分析得到与基因组数据库相匹配的PpFOMT1基因序列SEQ:NO.1，含有705个核苷酸，编码234个氨基酸的蛋白质，如SEQ:NO.2所示。将SEQ:NO.2与已报道其他植物OMT进行系统发育树分析，得到图1所示结果。
实施例2：PpFOMT1基因的原核表达
一、实验方法
1、设计带有表达载体pET-32a(+)载体的多克隆酶切位点的特异引物，其引物序列如SEQ:NO.5和SEQ:NO.6所示。
2、以测序正确T-easy载体为模板，用SEQ:NO.5和SEQ:NO.6所示引物进行PCR扩增，PCR反应体系为50μL，成分分别为：2×Phanta Max Buffer 25μL，dNTP Mix(10mM each)1μL，DNA polymerse(1U/μL)1μL，上下游引物(10μM)各2μL，cDNA 1μL，H2O 18μL。PCR程序为：95℃预变性3min，35个循环的95℃15s，58℃15s和72℃1min 20s，72℃5min，4℃hold。
3、将PCR扩增产物连接到用限制性内切酶BamHI和XhoI双酶切过的线性pET-32a(+)载体，获得pET-32a(+)-PpFOMT1重组质粒。
4、将pET-32a(+)-PpFOMT1重组质粒转化到大肠杆菌BL21(DE3)PlysS表达宿主菌中，经菌落PCR验证，挑取阳性菌落接种到500mL LB(Amp+)液体培养基，37℃培养，直至OD600为0.6～1.0，获得转基因工程菌。
5、在上述转基因的工程菌中加入IPTG至终浓度为1mM，16℃诱导24h，收集菌体，500mL收集到1管中，加入20mL 1×PBS缓冲液，充分重悬浮菌体，-80℃放置12h以上。将菌体置于30℃水浴锅解冻后，用超声破碎仪破碎5min。在4℃温度下，10000rpm离心30min，收集上清液。用Clontech HisTALON重力纯化试剂盒进一步纯化，获得目的蛋白。
二、实验结果
利用SDS-PAGE方法检测蛋白表达和纯化效果，结果如图2所示。图中可看出，加上重组标签后在45.16kDa左右有明显的重组蛋白条带，重组蛋白条带大小与预测的一致。纯化的蛋白可用于进一步的酶学分析。
实施例3：PpFOMT1重组蛋白的酶学活性检测分析
一、实验方法
1、对于酶活性检测，是在总体积100μl，0.1M pH 7.5的Tris-HCl缓冲液中进行，缓冲液包含10μl 100mM腺苷甲硫氨酸(SAM)制备液作为甲基供体，10μl1mg/ml槲皮素，5μg纯化后的重组蛋白和0.1％的DTT。
2、酶反应体系在37℃反应30min后加入等量甲醇停止反应，反应均以空载蛋白作为对照，获得酶反应产物。
3、酶反应产物经HPLC进行检测鉴定，所述HPLC检测条件如下：Waters2695-2996DAD检测器，ODS C18柱(4.6×250mm)色谱柱。以含0.1％甲酸水溶液(溶液A)和含0.1％甲酸的100％乙腈(溶液B)为流动相，洗脱梯度为：0-7min，10％-50％B；7-10min，50％B；10-15min，50％-100％B；15-15.1min，100％-10％B；15.1-20min，10％B。检测波长为370nm，柱温为25℃，流速为1ml/min，进样体积为10μl。
二、实验结果
结果如图3和图4所示，PpFOMT1重组蛋白以SAM作为甲基供体，可催化槲皮素生成与标准品一致的异鼠李素，说明PpFOMT1重组蛋白具有催化槲皮素生成异鼠李素的3'氧甲基化功能。
序列表
<110> 浙江大学山东（临沂）现代农业研究院
<120> 一种异鼠李素生物合成相关的3’-O-氧甲基转移酶及其应用
<160> 6
<210> 1
<211> 705
<212> DNA
<213> Prunus persica
<400> 1
atgggagaca aggtagagaa gatcatcctc aaaagcccgg cacttttaaa gtacatcttc 60
gaaacaagct gcttcccaag agaacacgag caattgaagc aactaaggga agccactgtc 120
gagaaatacc aattttggag tctcatgaat gtgcctgtag atgaaggcct gcttctctcg 180
atgattctaa agctcatgaa tgcaaacaag acactggaac ttggtgtatt cactggctac 240
tctcttctta caactgctct tgcaatacct catgacggca aaataacggc aatcgatcca 300
gataaagaag cctatgagtt tggattgcca tacattcaga gggctggggt ggatcataag 360
attaatttct gtcactcaga tgccctcact gtcctaaatg atctcattgc caatgggaag 420
gaagaaggga gctttgattt tgcatttgtg gacgcgaaca aggacgcata catcaaatat 480
cacgagctgc tgctaaagct tgtgaaggtc ggaggaataa tagcttatga caacacattg 540
tggtttggga cagtggtaga agctgaggag aatgtggagg aattcgcaaa gaaaggcaga 600
aagcatttgc tgcaactcaa cagctttctt gccgccgacg atcgcatcga gttagctctt 660
gtttccatcg gagatggact caccctctgc aggcgtctct attag 705
<210> 2
<211> 234
<212> PRT
<213> Prunus persica
<400> 2
MGDKVEKIIL KSPALLKYIF ETSCFPREHE QLKQLREATV EKYQFWSLMN VPVDEGLLLS 60
MILKLMNANK TLELGVFTGY SLLTTALAIP HDGKITAIDP DKEAYEFGLP YIQRAGVDHK 120
INFCHSDALT VLNDLIANGK EEGSFDFAFV DANKDAYIKY HELLLKLVKV GGIIAYDNTL 180
WFGTVVEAEE NVEEFAKKGR KHLLQLNSFL AADDRIELAL VSIGDGLTLC RRLY 234
<210> 3
<211> 21
<212> DNA
<213>人工序列(Unknown)
<400> 3
atgggagaca aggtagagaa g 21
<210> 4
<211> 21
<212> DNA
<213>人工序列(Unknown)
<400> 4
ctaatagaga cgcctgcaga g 21
<210> 5
<211> 42
<212> DNA
<213>人工序列(Unknown)
<400> 5
gccatggctg atatcggatc catgggagac aaggtagaga ag 42
<210> 6
<211> 38
<212> DNA
<213>人工序列(Unknown)
<400> 6
gtggtggtgg tggtgctcga gatagagacg cctgcaga 38
Claims (6)
1.一种黄酮醇3’-O-氧甲基转移酶，其特征在于，至少含有下列1)～4)特征之一：
1)所述黄酮醇3’-O-氧甲基转移酶编码基因的核苷酸序列为SEQ:NO.1所示；
2)所述黄酮醇3’-O-氧甲基转移酶的氨基酸序列为SEQ:NO.2所示；
3)与SEQ ID NO.1所示的DNA序列杂交的核苷酸序列；
4)与SEQ ID NO.1编码相同功能蛋白质的核苷酸序列。
2.根据权利要求1所述的黄酮醇3’-O-氧甲基转移酶，其特征在于，所述黄酮醇3’-O-氧甲基转移酶由桃中分离得到。
3.一种基因表达载体，其特征在于：所述的载体含有编码权利要求1所述黄酮醇3’-O-氧甲基转移酶的核苷酸序列或氨基酸序列；进一步，所述基因的核苷酸序列如SEQ:NO.1所示，该基因编码蛋白的氨基酸序列如SEQ:NO.2所示。
4.一种转基因细胞系或宿主菌，其特征在于：所述的转基因细胞系或宿主菌含有权利要求1所述的黄酮醇3’-O-氧甲基转移酶或权利要求3所述的基因表达载体。
5.权利要求1或2所述的黄酮醇3’-O-氧甲基转移酶、权利要求3所述的基因表达载体、权利要求4所述的转基因细胞系或宿主菌在异鼠李素制备中的应用；进一步，所述的用途选自制备基因工程产品、培育植物新品种、制备食品；进一步，所述的基因工程产品包括利用基因工程技术获取的药品、食品、化妆品、保养保健品等。
6.一种异鼠李素的制备方法，其特征在于，所述的制备方法选自以下的任意一种：
1)向权利要求1或2所述的黄酮醇3’-O-氧甲基转移酶直接提供底物底物SAM和槲皮素。
2)向转基因细胞系或宿主菌中导入权利要求3所述的基因表达载体；向转基因细胞系或宿主菌提供底物SAM和槲皮素。
3)通过权利要求3所述的基因表达载体或权利要求4所述的转基因细胞系或宿主菌获得重组蛋白；向重组蛋白提供底物SAM和槲皮素。
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