CN114480452B - 一种何首乌白藜芦醇合酶基因FmRS1及其编码产物和应用 - Google Patents
一种何首乌白藜芦醇合酶基因FmRS1及其编码产物和应用 Download PDFInfo
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- CN114480452B CN114480452B CN202111681337.XA CN202111681337A CN114480452B CN 114480452 B CN114480452 B CN 114480452B CN 202111681337 A CN202111681337 A CN 202111681337A CN 114480452 B CN114480452 B CN 114480452B
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- resveratrol
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- polygonum multiflorum
- resveratrol synthase
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Abstract
本发明涉及一种何首乌白藜芦醇合酶基因FmRS1及其编码产物与应用,从何首乌块根中克隆得到白藜芦醇合酶FmRS1基因,通过体外验证实验表明FmRS1基因具有催化1分子对香豆酰辅酶A和3分子丙二酰辅酶A反应合成白藜芦醇的活性。利用本发明可以通过基因工程技术来提高何首乌等植物中白藜芦醇等成分的含量,或者通过生物合成技术制备白藜芦醇等成分。
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白藜芦醇合酶基因FmRS1及其编码产物与应用。
背景技术
何首乌作为药物,始载于宋《开宝本草》,此后历代本草均有记载,是一味临床常用中药材。2020年版《中国药典》收载了何首乌和制首乌,前者来源于蓼科植物何首乌Falopiamultiflora(Thunb.)Harald.的干燥块根,有润肠、解毒、截虐的功效;后者为生首乌的炮制加工品,具有补肝肾、益精血、壮筋骨、乌须发之效。何首乌中主要活性成分为二苯乙烯类、蒽醌类、黄酮类、鞣质和磷脂等,其中,二苯乙烯苷、大黄素和大黄素甲醚为药典规定的指标性成。现代药理学研究表明,何首乌具有抗氧化、抗衰老、抑制缺血脑损伤、降血脂、抗动脉粥样硬化、抗炎、抗癌、抗肿瘤和保肝等药理作用。
白藜芦醇,别名茋三酚,属于二苯乙烯类化合物。根据有效的数据研究表明,白藜芦醇具有抗癌、抗菌、抗炎症、保护心血管等多种生理功效,已经被广泛用于膳食、医药、化妆品和保健品等行业。白藜芦醇合酶(Resveratrol synthase,RS)具有催化1分子对香豆酰辅酶A和3分子丙二酰辅酶A反应合成白藜芦醇的作用。因此研究何首乌白藜芦醇合酶基因的功能以及序列特征,为初步揭示何首乌白藜芦醇合酶的生物学功能,为进一步探索白藜芦醇的生物合成机理奠定基础。
发明内容
针对上述问题,本发明采用以下技术方案:
一种何首乌白藜芦醇合酶基因FmRS1,所述何首乌白藜芦醇合酶基因FmRS1如(a)或(b)或(c):
(a)其核苷酸序列为SEQ ID No.1所示的DNA分子;
(b)其核苷酸序列为SEQ ID No.1第1-1110位所示的DNA分子;
(c)在严格条件下与(a)或(b)限定的DNA序列杂交且编码具有何首乌白藜芦醇合酶活性的蛋白质的DNA分子。
本发明还提供一种上述所述的何首乌白藜芦醇合酶基因FmRS1编码的产物,该产物氨基酸序列如SEQ ID NO.2所示。
另外,本发明还提供含有上述所述基因的重组载体、转基因细胞系或重组菌。
进一步地,重组表达载体为SEQ ID No.1所示的FmRS1基因插入pET-28a载体的BamHI酶切位点间得到的载体。
本发明还提供了上述所述的基因或上述所述重组载体、转基因细胞系或重组菌在制备何首乌白藜芦醇合酶中的应用,该应用为重组载体、转基因细胞系或重组菌在催化对香豆酰辅酶A和丙二酰辅酶A反应合成白藜芦醇中的应用。
本发明还提供了一种制备白藜芦醇合酶的方法,包括如下步骤:将上述所述的重组表达载体接种于LB培养基中振荡培养,诱导培养4小时后,收集培养物,得到白藜芦醇合酶。
本发明的有益效果如下:
本发明首次从何首乌块根中克隆得到白藜芦醇合酶FmRS1基因,通过体外验证实验表明FmRS1基因具有催化1分子对香豆酰辅酶A和3分子丙二酰辅酶A反应合成白藜芦醇的活性。利用本发明可以通过基因工程技术来提高何首乌等植物中白藜芦醇等成分的含量,或者通过生物合成技术制备白藜芦醇等成分。
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为何首乌白藜芦醇基因FmRS1的琼脂糖凝胶电泳图;
图2为何首乌白藜芦醇合酶基因FmRS1结构功能域预测分析;
图3为何首乌白藜芦醇合酶基因FmRS1二级结构预测分析;
图4为何首乌白藜芦醇合酶基因FmRS1跨膜结构域预测分析;
图5为何首乌白藜芦醇合酶基因FmRS1三级结构预测分析;
图6为FmRS1蛋白质的SDS-聚丙烯酰胺凝胶电泳结果;
图7为白藜芦醇对照品的MRM色谱图;
图8为FmRS1蛋白质催化对香豆酰辅酶A和丙二酰辅酶A产物的MRM色谱图;
图9为pET-28a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TakaraBio公司;无毒型4S Green Plus核酸染料购于上海生工生物工程股份有限公司;切胶回收试剂盒EasyPure Quick Gel Extraction Kit、T载体pEASY-Blunt Zero CloningKit、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 PCRMaster 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;其他试剂均为进口或国产分析纯试剂。
1、白藜芦醇合酶基因FmRS1的克隆与生物信息学分析
按照RNA prep Pure Plant Kit试剂盒操作说明提取何首乌块根中总RNA,以何首乌RNA为模板,采用TaKaRa反转录试剂盒(PrimeScriptTMII 1st Strand cDNA SynthesisKit)进行反转录,得到何首乌的cDNA模板,利用正向引物FmRS1-F:5’-ATGGCGGCTTCAATTGAAGAGATTA-3’,反向引物FmRS1-R:5’-TCAAAACAAAGCACCCCACTCCAGT-3’进行PCR扩增,获得了何首乌白藜芦醇合酶FmRS1基因克隆。何首乌白藜芦醇合酶基因FmRS1的琼脂糖凝胶电泳如图1所示,其中M表示Marker,泳道1表示目的基因。目的基因FmRS1片段大小约为1100bp,符合预期。
上述扩增体系如下:2×Phusion Master Mix 25μL、10μM primer-F和10μMprimer-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。产物经1%琼脂糖凝胶电泳检测、回收,使用EasyPure Quick Gel Extraction Kit试剂盒切胶回收目的条带。将扩增产物连接至克隆载体pEASY-Blunt Zero,转化至大肠杆菌Trans1-T1感受态细胞中,挑菌并进行菌液PCR验证,将阳性克隆菌液送至苏州金唯智公司完成测序。
经过测序,PCR产物具有序列表中SEQ ID No.1所示的核苷酸,编码369个氨基酸,该蛋白的氨基酸序列为序列表中SEQ ID No.2,核苷酸所示的基因命名为FmRS1,编码的蛋白命名为FmRS1。
本发明实施例中所获得的何首乌白藜芦醇合酶FmRS1基因全长cDNA,FmRS1基因的开放阅读框(ORF)的长度为1100bp,其序列如序列表中SEQ ID NO.1所示。根据FmRS1全长cDNA编码369个氨基酸,其序列如序列表中SEQ ID NO.2所示。将FmRS1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,RS基因和CHS基因有较高的相似性,均属于Ⅲ型聚酮合酶家族,如图2所示。FmRS1蛋白二级结构由α螺旋,延伸链和无规则卷曲,如图3所示。FmRS1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3a5q.1.A为蛋白模型,FmRS1蛋白序列的相似性为85.12%。
2、白藜芦醇合酶基因FmRS1的功能研究
2.1重组载体的制备
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-FmRS1-F:
5’-TCCAGGGGCCCGAATTCGGAATGGCGGCTTCAATTGAAGAGATTA-3’;下游引物:BamH I-FmRS1-R:
5’-CGACGGAGCTCGAATTCGGATCAAAACAAAGCACCCCACTCCAGT-3’。以重组质粒为模板,进行PCR扩增。得到的酶切产物与经过相同酶切的pET-28a连接,得到重组载体。
该重组载体为将序列表中SEQ ID No.1所示的FmRS1基因插入pET-28a载体的BamHI酶切位点间得到的载体,命名为pET-28a-FmRS1。
2.2 FmRS1蛋白的表达
将目标质粒pET-28a-FmRS1导入BL21(DE3)感受态细胞,得到重组菌BL21(DE3)-pET-28a-FmRS1。
将空载质粒pET-28a-FmRS1导入BL21(DE3)感受态细胞,得到对照菌BL21(DE3)-pET-28a。
将重组菌BL21(DE3)-pET-28a-FmRS1接种于含50ug/ml卡那霉素抗生素的LB培养基中,37℃下振荡培养,OD600=0.6,以1:100比例接种于新鲜的含卡那霉素抗生素培养基中,37℃下振荡培养3~5h进行扩大培养,然后加入异丙基硫代-β-D-半乳糖苷(IPTG,终浓度为0.8mM),在16℃振荡诱导培养,以BL21(DE3)-pET-28a为阴性对照。
诱导培养4小时后,收集培养物,离心收集上清液。
将上清液进行SDS-聚丙烯酰胺凝胶电泳,结果如图6所示,图6中,M表示ProteinMarker,1为pET-28a空载体,2为未诱导的FmRS1,3为FmRS1全菌,4为FmRS1上清。结果表明与pET-28a空载相比,含有FmRS1基因重组蛋白的全菌和超声破碎后的上清在40.93kDa处出现明显的目的蛋白条带,符合预期FmRS1蛋白分子量大小,未加IPTG诱导的FmRS1基因重组蛋白全菌液在40.93kDa处同样出现目的蛋白条带,表现为蛋白本底表达。
2.3 FmRS1蛋白的功能验证
1)FmRS1蛋白粗提取物
将重组菌BL21(DE3)-pET-28a-FmRS1按照1:100的比例加到含卡那霉素抗生素的LB培养液中,37℃,200rpm振荡培养至A600=0.4~0.6,加入终浓度为0.8mM IPTG于16℃低温诱导4h,离心去上清以获得菌体,加入3mL His BufferA重悬菌体,冰浴下超声破碎10min(超声破碎过程超声开5s,关5s,间断超声),4℃离心后获得FmRS1基因的蛋白粗提取物。
将BL21(DE3)-pET-28a按照上述方法提取,得到对照蛋白粗提物。
2)FmRS1蛋白粗提取物功能验证
以对香豆酰辅酶A和丙二酰辅酶A为反应底物进行体外酶促实验,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250μL酶促反应条件:35℃水浴反应60min过后,用250μL乙酸乙酯萃取,12000rpm转速下离心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 QTRAP 5500三重四极杆-线性离子肼串联质谱仪(美国ABSciex),分析色谱柱为ACQUITYBEH C181.7μm 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;梯度洗脱: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。柱温为40℃;流速为0.4mL/min;进样量为2μL。
质谱分析条件为:负离子化模式下,采取多反应监测(MRM)检测,喷雾电压为4.5kV,离子化温度为500℃,喷雾气(Gas1)为45psi,加热辅助气(Gas2)为45psi,气帘气为40psi,白藜芦醇检测离子对质荷比(m/z)为227/185,去簇电压(DP)为-75V和碰撞电压(CE)为-30V。
图7为白藜芦醇对照品的MRM色谱图,从图7的分析结果可以看出:白藜芦醇的保留时间为2.17min,图8为FmRS1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8的分析结果可以看出:FmRS1催化样品在保留时间为2.17min存在与白藜芦醇保留时间一致的特征峰;图9为pET-28a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9的分析结果可以看出:pET-28a空载体催化样品在保留时间为2.17min没有存在与白藜芦醇保留时间一致的特征峰。
LC-MS(液相色谱质谱联用)结果如图8所示,pET-28a-FmRS1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转化为白藜芦醇(其质荷比m/z为227/185),因此可以认定pET-28a-FmRS1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合成白藜芦醇的活性。
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研究所
<120> 一种何首乌白藜芦醇合酶基因FmRS1及其编码产物和应用
<160> 2
<170> SIPOSequenceListing 1 .0
<210> 1
<211> 1110
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcaaaacaaa gcaccccact ccagtccttc tccagtggtt gcttgaccat tttcaagcga 60
cttcttcctc atcttatcca tgatgaaaaa cacagtagca cttgacatgt ttccatagtc 120
gttcaacact tgtcttgttg gtttaagttt ctcctttttc agaccaacct tagcctcaac 180
atggtccagg aaagcagggc caccagggtg cgcgatccag aatatggagt tccaattggt 240
aatgtttaga ggggtgaaag cctctaaaag aatagtttcc atgtgattag agattactac 300
gggagttttc tcgtacaaat ggaagctgag tccagattca agcaaatggc cctcaattgc 360
accctcggat tcaggtacaa tagtttgggc agtccaaacc aactcgaaaa tcggcctctc 420
aacagttaag tccggatttg cgccaactat gactgctgca gccccgtcac ctaatacgga 480
tgtccctatc atggagtcta tgtgggtttc agatggccca cggaaacaaa ttgccgtcat 540
ctctgagcaa atgatgagaa cacgagctcc cttattgttc tcagctatgt cctttgcaag 600
gcgaaggaca gtgccaccag cataacatcc taggtggtaa aacataaagc gtttaacaga 660
agggtgaagg tcaagaagtt tagtgagttg gtaatctatg cccggcatgt caacgccggc 720
taagcaacac acgatgagat gtgtgatctt agacttgggt tggccccatt ctgtgatagc 780
cttgagggca gcctctttcc caagctctgc aactcctttc acttgaattt tgtgtcttac 840
attcaatgac ggcgcatcgt aggcagcaat gtttgggttt tccttgagaa tctcttcggt 900
caaatgaaag taacgcttct cgatcattga cttctcacaa atgcgcttga atttctgctt 960
gaggttggtg aggtgatcac tgttggtgac gcggaagtaa taatcgggaa agtcgacttg 1020
gtacatgcaa ttgggagggt tggcggtgcc gatggccagg acggtagcgg gtgtttgtgt 1080
cttcctaatc tcttcaattg aagccgccat 1110
<210> 2
<211> 369
<212> PRT
<213> Artificial Sequence
<400> 2
Met Ala Ala Ser Ile Glu Glu Ile Arg Lys Thr Gln Thr Pro Ala Thr
5 10 15
Val Leu Ala Ile Gly Thr Ala Asn Pro Pro Asn Cys Met Tyr Gln Val
20 25 30
Asp Phe Pro Asp Tyr Tyr Phe Arg Val Thr Asn Ser Asp His Leu Thr
35 40 45
Asn Leu Lys Gln Lys Phe Lys Arg Ile Cys Glu Lys Ser Met Ile Glu
50 55 60
Lys Arg Tyr Phe His Leu Thr Glu Glu Ile Leu Lys Glu Asn Pro Asn
65 70 75 80
Ile Ala Ala Tyr Asp Ala Pro Ser Leu Asn Val Arg His Lys Ile Gln
85 90 95
Val Lys Gly Val Ala Glu Leu Gly Lys Glu Ala Ala Leu Lys Ala Ile
100 105 110
Thr Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His Leu Ile Val Cys
115 120 125
Cys Leu Ala Gly Val Asp Met Pro Gly Ile Asp Tyr Gln Leu Thr Lys
130 135 140
Leu Leu Asp Leu His Pro Ser Val Lys Arg Phe Met Phe Tyr His Leu
145 150 155 160
Gly Cys Tyr Ala Gly Gly Thr Val Leu Arg Leu Ala Lys Asp Ile Ala
165 170 175
Glu Asn Asn Lys ly Ala Arg Val Leu Ile Ile Cys Ser Glu Met Thr
180 185 190
Ala Ile Cys Phe Arg Gly Pro Ser Glu Thr His Ile Asp Ser Met Ile
195 200 205
Gly Thr Ser Val Leu Gly Asp Gly Ala Ala Ala Val Ile Val Gly Ala
210 215 220
Asn Pro Asp Leu Thr Val Glu Arg Pro Ile Phe Glu Leu Val Trp Thr
225 230 235 240
Ala Gln Thr Ile Val Pro Glu Ser Glu Gly Ala Ile Glu Gly His Leu
245 250 255
Leu Glu Ser Gly Leu Ser Phe His Leu Tyr Glu Lys Thr Pro Val Val
260 265 270
Ile Ser Asn His Met Glu Thr Ile Leu Leu Glu Ala Phe Thr Pro Leu
275 280 285
Asn Ile Thr Asn Trp Asn Ser Ile Phe Trp Ile Ala His Pro Gly Gly
290 295 300
Pro Ala Phe Leu Asp His Val Glu Ala Lys Val Gly Leu Lys Lys Glu
305 310 315 320
Lys Leu Lys Pro Thr Arg Gln Val Leu Asn Asp Tyr Gly Asn Met Ser
325 330 335
Ser Ala Thr Val Phe Phe Ile Met Asp Lys Met Arg Lys Lys Ser Leu
340 345 350
Glu Asn Gly Gln Ala Thr Thr Gly Glu Gly Leu Glu Trp Gly Ala Leu
355 360 365
Phe
Claims (7)
1.一种何首乌白藜芦醇合酶基因FmRS1,其特征在于,所述何首乌白藜芦醇合酶基因FmRS1如(a):
(a)其核苷酸序列为SEQ ID NO.1所示的DNA分子。
2.一种由权利要求1所述的何首乌白藜芦醇合酶基因FmRS1编码的产物,其特征在于,该产物氨基酸序列如SEQ ID NO.2所示。
3.含有权利要求1所述基因的重组载体或重组菌。
4.根据权利要求3所述的重组载体,其特征在于,所述重组载体为SEQ ID NO.1所示的FmRS1基因插入pET-28a载体的BamHI酶切位点间得到的载体。
5.如权利要求1所述的基因或权利要求3所述重组载体或重组菌在制备何首乌白藜芦醇合酶中的应用。
6.根据权利要求5所述的应用,其特征在于,所述应用为重组载体或重组菌在催化对香豆酰辅酶A和丙二酰辅酶A反应合成白藜芦醇中的应用。
7.一种制备白藜芦醇合酶的方法,包括如下步骤:将如权利要求4所述的重组表达载体接种于LB培养基中振荡培养,诱导培养4小时后,收集培养物,得到白藜芦醇合酶。
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