CN114480450B - 一种何首乌白藜芦醇合酶基因FmRS2及其编码产物和应用 - Google Patents
一种何首乌白藜芦醇合酶基因FmRS2及其编码产物和应用 Download PDFInfo
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- CN114480450B CN114480450B CN202111630385.6A CN202111630385A CN114480450B CN 114480450 B CN114480450 B CN 114480450B CN 202111630385 A CN202111630385 A CN 202111630385A CN 114480450 B CN114480450 B CN 114480450B
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Abstract
本发明涉及一种何首乌白藜芦醇合酶基因FmRS2及其编码产物和应用,该基因具有SEQ ID NO.1所示的核苷酸序列,该基因编码产物氨基酸序列如SEQ ID NO.2所示。同时还涉及该基因和该基因编码产物在制备二苯乙烯类化合物中的应用。本发明成功从何首乌块根中克隆出了一种何首乌白藜芦醇合酶基因FmRS2的编码基因,该酶可以应用于以对香豆酰辅酶A和丙二酰辅酶A为底物制备白藜芦醇的合成。
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FmRS2及其编码产物和应用。
背景技术
白藜芦醇是一种从植物中提取的天然的非黄酮类多酚化合物,属于二苯乙烯类化合物。化学名为3,4,5-三羟基二苯乙烯,分子式为C14H12O3,分子量为228.25kDa,为无色针状晶体,难溶于水,易溶于乙醚、氯仿和醇类溶剂等。白藜芦醇现今已广泛应用于医药、保健品、化妆品等行业。现有的研究表明,白藜芦醇具有抗氧化、抗炎、抗癌、雌激素、神经保护、心脏保护、抗动脉粥样硬化、抗衰老、抗糖尿病和抗骨质疏松等作用,具有广泛的生理病理效应。
白藜芦醇的生物合成途径是苯丙类素生物合成途径,在白藜芦醇合成全过程中,白藜芦醇合酶(Resveratrol synthase,RS)是合成途径中最后一个起作用的关键酶,也是合成途径中唯一必需的合成酶,它催化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。白藜芦醇合成的底物在植物体内广泛存在,但是大多数植物不含白藜芦醇合酶或含量很低,从而使得植物体中不含白藜芦醇或含少量的白藜芦醇。
何首乌Falopia multiflora(Thunb.)Harald.系蓼科何首乌属多年生草本植物。二苯乙烯类、蒽醌类、酚类等成分是何首乌中主要化学成分,其中二苯乙烯类化合物就包含了白藜芦醇,所以对何首乌白藜芦醇合酶FmRS2基因进行研究,能够为通过基因工程技术提高何首乌中白藜芦醇成分的含量提供重要技术支持,以及为白藜芦醇合成提供新的方向。
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FmRS2,该基因具有SEQ ID NO.1所示的核苷酸序列。
一种如上述所述的何首乌白藜芦醇合酶基因FmRS2所编码的产物,所述产物氨基酸序列如SEQ ID NO.2所示。
含有如上述所述的何首乌白藜芦醇合酶基因FmRS2的重组表达载体。
进一步地,所述重组表达载体为质粒pET-28a。
含有如上述所述的何首乌白藜芦醇合酶基因FmRS2或含有上述所述何首乌白藜芦醇合酶基因FmRS2的重组表达载体的宿主细胞。
进一步地,所述宿主细胞为组培获得的何首乌愈伤组织、何首乌植株的细胞和BL21(DE3)细胞中的任意一种。
如上述所述的何首乌白藜芦醇合酶基因FmRS2或上述所述首乌白藜芦醇合酶基因FmRS2所编码的产物在制备二苯乙烯类化合物中的应用。
进一步地,所述二苯乙烯类化合物为白藜芦醇。
本发明的有益效果如下:
本发明成功从何首乌块根中克隆出了一种何首乌白藜芦醇合酶基因FmRS2的编码基因,该酶可以应用于以对香豆酰辅酶A和丙二酰辅酶A为底物制备白藜芦醇的合成。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二苯乙烯类成分的含量,该技术可以用于后续通过在菌体系产生大量的二苯乙烯类化合物,有助于优质何首乌的基因工程育种。
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FmRS2的琼脂糖凝胶电泳图;
图2为何首乌白藜芦醇合酶基因FmRS2结构功能域预测分析;
图3为何首乌白藜芦醇合酶FmRS2蛋白的二级结构预测分析;
图4为何首乌白藜芦醇合酶FmRS2蛋白序列的跨膜结构域预测分析;
图5为何首乌白藜芦醇合酶FmRS2蛋白的三级结构预测分析;
图6为FmRS2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FmRS2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Takara Bio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 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-FidelityPCR 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;其他试剂均为进口或国产分析纯试剂。
一、何首乌白藜芦醇合酶基因FmRS2的克隆
FmRS2的克隆利用正向引物:上游引物:FmRS2-F:5’-ATGGAGGCTTCAATTGAGGAGATTA-3’;下游引物:FmRS2-R:5’-TCATCTTACCATGATGAAAAACACG-3’。以何首乌白藜芦醇合酶基因FmRS2编码基因全长序列为模板进行PCR扩增。扩增体系如下:2×Phusion Master Mix 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。至此获得了何首乌白藜芦醇合酶FmRS2基因克隆。何首乌白藜芦醇合酶基因FmRS2的琼脂糖凝胶电泳如图1所示,图1中M表示Marker,泳道1为目的基因。目的基因FmRS2片段大小约为1100bp,符合预期。
二、FmRS2基因的生物信息学分析
本发明所获得的何首乌白藜芦醇合酶基因全长cDNA,FmRS2基因的开放阅读框(ORF)的长度为1041bp,其序列如序列表中SEQ ID NO.1所示。将FmRS2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基因有很高的相似度,均属于Ⅲ型聚酮合酶家族,如图2所示。图3示出了何首乌白藜芦醇合酶基因FmRS2二级结构,何首乌白藜芦醇合酶FmRS2蛋白二级结构由α螺旋,延伸链和无规则卷曲构成。FmRS2无跨膜结构,为膜外蛋白,如图4所示。利用Swiss Model预测FmRS2蛋白的三级结构,如图5所示,FmRS2蛋白模型3a5q.1.A,蛋白序列的相似性为83.04%。
三、FmRS2基因原核表达载体的构建
以FmRS2cDNA为模板,设计特异性上游引物和下游引物(如表1所示),进行PCR扩增反应,引物中划线部分为酶切位点。
表1特异性上游引物和下游引物碱基序列
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-28a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-28a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。
四、工程菌的诱导表达
用目标质粒pET-28a-FmRS2转化BL21(DE3)感受态细胞,培养筛选含有pET-28a-FmRS2质粒的阳性菌株BL21(DE3)-FmRS2。取转化表达菌液按照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0.8mMIPTG(异丙基-β-D-硫代半乳糖苷)于16℃低温诱导4h,以同样条件处理pET-28a空载作为空白对照。菌液离心去上清以获得菌体,加5mL Buffer A(20mM Na3PO4·12H2O、500mM NaCl、20mM咪唑),重悬,转移至15mL离心管置于超声破碎仪中超声破碎有效时长为15min(其中超声功率比25%,超声破碎过程超声开5s,关5s,间断超声)。离心管插入装有冰的烧杯中,于冰上操作。超声破碎裂解液于4℃离心15min后获得FmRS2基因的上清,进行12%SDS-PAGE电泳分析。SDS-聚丙烯酰胺凝胶电泳结果如图4。结果表明与pET-28a空载相比,含有FmRS2基因重组蛋白超声破碎后的上清在39.18kDa处出现明显的目的蛋白条带,符合预期FmRS2蛋白分子量大小,其中泳道M为Protein Marker,泳道1为含有pET-28a空载体的BL21(DE3)全菌;泳道2为未诱导的FmRS2全菌;泳道3为诱导的FmRS2上清。
五、体外酶功能验证
FmRS2酶功能验证: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,分析色谱柱为ACQUITYBEH C181.7μm 2.1×100mm柱对产物和标准品白藜芦醇进行分析。
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40℃;流速0.4mL/min;进样量5μL。
质谱分析条件: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FmRS2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:FmRS2催化样品在保留时间为2.18min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-FmRS2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-FmRS2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资源中心
<120> 一种何首乌白藜芦醇合酶基因FmRS2及其编码产物和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1041
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcatcttacc atgatgaaaa acacggtagc acttgacatg tttccatagt cgttcaacac 60
ttgtctagtt gccttaagtt tctccttttt gagaccggca gtggcttcaa catggtccag 120
gatagcaggg ccaccagggt gcgtgatcca gaatagggag ttccaattgc taatgttccg 180
aggggtgaaa gcctctgaaa ggcaagtttc gatgtaatta gaaattagta cgggaagagt 240
cttggacaaa tggcaactaa gtccagattc aagcaaatgg ccctcaactg caccatcaga 300
ttcgggtaca atagtttggg ctgtccaaac caactcaaaa atcggcctct caacagttag 360
gtccggattt gcgccaacta tgactgctgc agccccgtca cctaataccg atgtccctat 420
catggaggat atgtgggttt cagatggccc acggaaacaa attgccgtca tctctgagca 480
aacgatgaga acacgagctc ccttattgtt ctcagttatg tcctttgcaa ggcgaaggac 540
agtcccacca gcgtaacatc cgaggtggta aaacataaag cgtttaactg aagggtgaag 600
gtcaagaagt ttagtgagtt gataatctgt gccgggcatg tcaacaccgg ctaagcaaca 660
cacgatgaga tgtgtgatct tagactttgg ttggccccat tctgtgatag ccttgagggc 720
agcctctttc ccaagctctg caactccttt cacttgaatt ttgtgtcttg cattcaatga 780
cggagcctca taggcaccaa tatttgggtt ttccttgata atctcttcgg tcaattgaaa 840
gtaacgcttc tcgatcattg aattatcaca aatgcgcttg aatttttgct tgaggtgggt 900
gaggtgttca ctcttggtga tgcggaagta ataatcggga aagtcggctt ggtacatgca 960
gttgggaggg ttggcggtgc cgatggccag gacggtagcg ggtgtttgtg ccttcctaat 1020
ctcctcaatt gaagcctcca t 1041
<210> 1
<211> 346
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Glu Ala Ser Ile Glu Glu Ile Arg Lys Ala Gln Thr Pro Ala Thr
1 5 10 15
Val Leu Ala Ile Gly Thr Ala Asn Pro Pro Asn Cys Met Tyr Gln Ala
20 25 30
Asp Phe Pro Asp Tyr Tyr Phe Arg Ile Thr Lys Ser Glu His Leu Thr
35 40 45
His Leu Lys Gln Lys Phe Lys Arg Ile Cys Asp Asn Ser Met Ile Glu
50 55 60
Lys Arg Tyr Phe Gln Leu Thr Glu Glu Ile Ile Lys Glu Asn Pro Asn
65 70 75 80
Ile Gly Ala Tyr Glu Ala Pro Ser Leu Asn Ala 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 Thr 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 Thr
165 170 175
Glu Asn Asn Lys Gly Ala Arg Val Leu Ile Val Cys Ser Glu Met Thr
180 185 190
Ala Ile Cys Phe Arg Gly Pro Ser Glu Thr His Ile Ser 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 Asp Gly Ala Val Glu Gly His Leu
245 250 255
Leu Glu Ser Gly Leu Ser Cys His Leu Ser Lys Thr Leu Pro Val Leu
260 265 270
Ile Ser Asn Tyr Ile Glu Thr Cys Leu Ser Glu Ala Phe Thr Pro Arg
275 280 285
Asn Ile Ser Asn Trp Asn Ser Leu Phe Trp Ile Thr His Pro Gly Gly
290 295 300
Pro Ala Ile Leu Asp His Val Glu Ala Thr Ala Gly Leu Lys Lys Glu
305 310 315 320
Lys Leu Lys Ala Thr Arg Gln Val Leu Asn Asp Tyr Gly Asn Met Ser
325 330 335
Ser Ala Thr Val Phe Phe Ile Met Val Arg
340 345
Claims (6)
1.一种何首乌白藜芦醇合酶基因FmRS2,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所述的何首乌白藜芦醇合酶基因FmRS2所编码的产物,其特征在于,所述产物氨基酸序列如SEQ ID NO.2所示。
3.含有如权利要求1所述的何首乌白藜芦醇合酶基因FmRS2的重组表达载体。
4.根据权利要求3所述的何首乌白藜芦醇合酶基因FmRS2的重组表达载体,其特征在于,所述重组表达载体为质粒pET-28a。
5.含有如权利要求1所述的何首乌白藜芦醇合酶基因FmRS2或含有权利要求3-4任一项所述何首乌白藜芦醇合酶基因FmRS2的重组表达载体的宿主细胞;所述宿主细胞为BL21(DE3)细胞。
6.一种如权利要求1所述的何首乌白藜芦醇合酶基因FmRS2或权利要求2所述的何首乌白藜芦醇合酶基因FmRS2所编码的产物在制备二苯乙烯类化合物中的应用;所述二苯乙烯类化合物为白藜芦醇。
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CN101824404A (zh) * | 2009-03-03 | 2010-09-08 | 中国科学院植物研究所 | 白藜芦醇合酶及其编码基因与应用 |
CN109266663A (zh) * | 2018-10-10 | 2019-01-25 | 江苏科技大学 | 一种桑树白藜芦醇合成酶、其编码基因及重组载体和应用 |
CN113278630A (zh) * | 2021-05-26 | 2021-08-20 | 安徽农业大学 | 一种改良桑树白藜芦醇生物合成的转录因子基因MaMYB14及其应用 |
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CN101824404A (zh) * | 2009-03-03 | 2010-09-08 | 中国科学院植物研究所 | 白藜芦醇合酶及其编码基因与应用 |
CN109266663A (zh) * | 2018-10-10 | 2019-01-25 | 江苏科技大学 | 一种桑树白藜芦醇合成酶、其编码基因及重组载体和应用 |
CN113278630A (zh) * | 2021-05-26 | 2021-08-20 | 安徽农业大学 | 一种改良桑树白藜芦醇生物合成的转录因子基因MaMYB14及其应用 |
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