CN115074375A - 一种丹参2-酮戊二酸依赖性双加氧酶基因及其应用 - Google Patents
一种丹参2-酮戊二酸依赖性双加氧酶基因及其应用 Download PDFInfo
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- CN115074375A CN115074375A CN202210226322.2A CN202210226322A CN115074375A CN 115074375 A CN115074375 A CN 115074375A CN 202210226322 A CN202210226322 A CN 202210226322A CN 115074375 A CN115074375 A CN 115074375A
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Abstract
本发明公开了一种丹参2‑酮戊二酸依赖性双加氧酶基因及其应用，属于基因工程及分子生物学技术领域。丹参2‑酮戊二酸依赖性双加氧酶基因核苷酸序列如SEQ ID NO.1所示。该基因编码的蛋白质氨基酸序列如SEQ ID No.2所示。本发明丹参2‑酮戊二酸依赖性双加氧酶基因为丹参酮类化合物生物合成途径中的关键基因之一，其所编码的蛋白能够催化隐丹参酮合成丹参酮IIA，并且可催化二氢丹参酮I转化为丹参酮I。该基因为今后在丹参育种以及丹参酮类成分工业生产的应用方面提供了重要的理论依据，具有广阔的应用前景和极大的经济价值。
Description
技术领域
本发明属于基因工程及分子生物学技术领域，具体涉及一种丹参2-酮戊二酸依赖性双加氧酶基因及其应用。
背景技术
丹参始载于《神农本草经》，列为上品，味苦微寒。丹参酮类化合物是丹参中主要的脂溶性成分，主要包括隐丹参酮、丹参酮IIA、丹参酮I、二氢丹参酮I等。现有研究表明，丹参酮类化合物具有治疗心血管疾病、抗菌抗炎、抗肿瘤、肝保护、神经保护的作用。
目前已知二萜合酶SmCPS1、SmCPS2、SmKSL1以及多个细胞色素P450酶CYP76AH1、CYP76AH3、CYP76AK1、CYP71D373、CYP71D375、CYP71D411参与丹参酮的生物合成。然而丹参酮类化合物生物合成途径仍未被完全解析。
因此，如何提供进一步揭示丹参酮类化合物生物合成途径，寻找丹参酮类化合物生物合成途径中的关键基因是本领域亟待解决的问题。
发明内容
本发明公开了一种丹参2-酮戊二酸依赖性双加氧酶基因Sm2OGD3，其能够催化丹参酮类化合物D环的15，16位脱氢形成双键，参与丹参酮IIA、丹参酮I的合成。
为了实现上述目的，本发明采用如下技术方案：
一种丹参2-酮戊二酸依赖性双加氧酶基因Sm2OGD3，核苷酸序列如SEQ ID NO.1所示：ATGGACTCATCCAGCTTGAAAAATTGCTCGCAAGAAAACGAGGCCGATCGCGTGCACGAGCTGAACGCTTTCGAGGCCACAAAAGCCGGCGTGAAGGGGCTCACCGACTCCGGCGTCCAGAAGGTTCCGAGAATGTTCATCAGGCCAGCCGACGAGCTCGTCGAGGAGCGCAACCGGAGCCGCTCCCCGCTGCAAGCTCCGGTGATAGACCTCGGCCGGATCGGGGAGGGCGAGGGGCGGGAGAAGGTGGTGAGCGAGGTGAGATGGGCGTCGAAGGAGCTCGGGATCTTCCAGATCGTGAACCACGGGGTGGCCGTGGAGGTCATGGACGCGATGATCGACGGCGTGAGGAAGTTTCACGAGCAAGATGCGGAGGCGAAGAAGCAGTTCCACACGCGCGACGCCATGCGCAAGGTGATGTACGCGAGCAACGTCGATCTGTACAAGTCGCGCGCGGCGAATTGGAGGGACACGTTCTCGGTCGCGCTCATGGGTTCCGACAGCGTTGAGCCGGAAGAGTTGCCGGAGATTTGCAGAGACTCAACAATCAAGTATCTTGATGAAGTCACGAATCTCGCACACACTCTATTTGAGCTGCTCTCGGAAGCTCTTGGGCTCGAACAAGGTTGCCTAGGAGCCTTGAAATGTGGCCGAGGACGCACGTTCGTCGGCCAGTACTACCCCGCGTGCCCCGAGCCGGAGCTCACGATGGGCATGACCAACCACACCGATCCTTGTTTCCTAACTATTCTTCTCCAAGATCATATCGGAGGCCTCCAAGCTCTGCACAATAGTCAGTACATAAATGTGGAGCCTCTGCCTGCTAGCTTCGTCGTCAACATTGGAGATATGTTGCAGATTGTGTCAAATGATGAGTTCATAAGCCCAATTCATAGAGTTCATGCAAATCGGGCCGGGCCAAGAATCTCGATTGCGGGCTTTTTCACCGGTGATGCTATTTCAGGGACAATATACGGCCCGATCAAAGAGTTGGTATCAAAGAACAATGGGGCTCGATACAAAGAGTTCACAGTGGGAGAGTACATGTCCAAGTTTTTAGAGCGGCCAATTGATAAATCTGGTCTTGATGAATGCAGATTGCAAGATGAAGACAATGTCTAA。
上述基因编码的蛋白，氨基酸序列如SEQ ID NO.2所示：MDSSSLKNCSQENEADRVHELNAFEATKAGVKGLTDSGVQKVPRMFIRPADELVEERNRSRSPLQAPVIDLGRIGEGEGREKVVSEVRWASKELGIFQIVNHGVAVEVMDAMIDGVRKFHEQDAEAKKQFHTRDAMRKVMYASNVDLYKSRAANWRDTFSVALMGSDSVEPEELPEICRDSTIKYLDEVTNLAHTLFELLSEALGLEQGCLGALKCGRGRTFVGQYYPACPEPELTMGMTNHTDPCFLTILLQDHIGGLQALHNSQYINVEPLPASFVVNIGDMLQIVSNDEFISPIHRVHANRAGPRISIAGFFTGDAISGTIYGPIKELVSKNNGARYKEFTVGEYMSKFLERPIDKSGLDECRLQDEDNV。
克隆上述基因全长的引物对，
上游引物Sm2OGD3-F(5'to 3')：
GGATCCATGGACTCATCCAGCTTGAAAAAT，SEQ ID No.3：
下游引物Sm2OGD3-R(5'to 3')：
GCGGCCGCTTAGACATTGTCTTCATCTTGC，SEQ ID No.4。
含有上述基因的重组表达载体、表达盒、转基因细胞系或宿主菌。
含有上述基因的重组表达载体，以pET-30a为出发载体，基因的插入位点为NotI和BamHI。
上述基因或上述蛋白在制备丹参酮类化合物中的应用。
上述基因编码的蛋白催化隐丹参酮合成丹参酮IIA，催化二氢丹参酮I转化为丹参酮I。
综上所述，本发明2-酮戊二酸依赖性双加氧酶基因Sm2OGD3是丹参主要活性成分丹参酮类物质生物合成的关键酶基因，也是丹参中第一个揭示催化反应的2-酮戊二酸依赖性双加氧酶。利用本发明提供的基因可以通过基因工程技术提高丹参药效成分丹参酮IIA和丹参酮I的含量，可用于其工业生产大量合成。同时，为进一步解析丹参酮生物合成通路提供了研究方向。
附图说明
图1所示为丹参Sm2OGD3基因克隆鉴定电泳结果；
其中，泳道1：DNAmarker DL2000；泳道2和3：Sm2OGD3；
图2所示为Sm2OGD3基因工程菌SDS-聚丙烯酰胺凝胶电泳结果；
其中，泳道1：蛋白Marker(10-180KDa)；泳道2：携带pET-30a空载质粒的BL21(DE3)；泳道3、4、5：携带pET-30a-Sm2OGD3重组质粒的BL21(DE3)；表达目的蛋白条带图中用星号标示。
图3所示为Sm2OGD3原核表达体系中催化反应UPLC检测的产物结果；
其中，A为Sm2OGD3原核表达体系中催化隐丹参酮的UPLC检测的产物结果；B为Sm2OGD3原核表达体系中催化二氢丹参酮I的UPLC检测的产物结果；
图中，Control为空白对照，Standard为标准品。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅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。
实施例1：丹参Sm2OGD3基因分离克隆与原核表达载体构建
以丹参成熟红根为材料，使用EASYspin Plus多糖多酚复杂植物RNA快速提取试剂盒(艾德莱，中国)提取丹参成熟红根的总RNA，方法参照说明书。使用
IIIFirst-Strand Synthesis System for RT-PCR(invitrogen，美国)反转录试剂盒进行反转录得到丹参成熟红根的cDNA，方法参照说明书。
原核表达载体采用酶切连接的方法构建。根据pET-30a载体的多克隆位点和Sm2OGD3基因的编码区序列上的酶切位点，选择Not I和BamH I作为内切酶。设计引物时在引物的5’端各自加上了酶切位点，
上游引物Sm2OGD3-F(5'to 3')：
GGATCCATGGACTCATCCAGCTTGAAAAAT，SEQ ID No.3：
下游引物Sm2OGD3-R(5'to 3')：
GCGGCCGCTTAGACATTGTCTTCATCTTGC，SEQ ID No.4。
以cDNA为模板，使用上述引物进行扩增反应，获得Sm2OGD3基因的全长序列(其核苷酸序列如SEQ ID NO.1所示，编码的蛋白氨基酸序列如SEQ ID NO.2所示)。
PCR反应体系：cDNA 1μL，上游引物1μL，下游引物1μL，2×A9LongHiFiPCRMasterMix 10μL，ddH2O 7μL。高保真酶2×A9 LongHiFi PCR MasterMix购自北京艾德莱生物科技有限公司。
PCR的设定程序：1)95℃，3min；2)95℃，10s；3)55℃，15s；4)72℃，30s；5)72℃，3min。循环从2)到4)，循环数25个。PCR产物经1％的琼脂糖凝胶电泳检测后(图1)，使用AxyPrep DNA Gel Extraction Kit(Axygen，美国)回收目的DNA片段，方法参照说明书。
使用Nanodrop测定回收的目的DNA片段浓度，按照说明书中的推荐用量将目的DNA连接至pTOPO-Blunt-Vector载体(购自北京艾德莱生物科技有限公司)。取连接产物10μL转化大肠杆菌感受态DH5α，在含有100mg/L卡那霉素的LB固体平板中筛选出阳性克隆，抽提质粒进行PCR鉴定，重组质粒样品T-Sm2OGD3送生物公司测序。
将测序正确的T-Sm2OGD3质粒和pET30a质粒分别进行双酶切反应。反应体系50μL，质粒20μL，10×CutSmart缓冲液5μL，BamH I 1μL，Not I 1μL(购自NEB公司)；37℃温育4h，酶切产物使用胶回收试剂盒回收，测定回收产物的浓度，之后进行连接反应。
连接反应：反应为10μL体系。3μL酶切后的T-Sm2OGD3质粒DNA和5μL酶切后的pET30a质粒DNA，加入1μL 10×T4 DNALigase buffer，加入1μL连接酶，16℃连接过夜。
将上述连接产物转化大肠杆菌BL21(DE3)感受态(购自北京全式金生物技术有限公司)，将菌液涂布在含有100mg/L的卡那霉素的LB固体培养基上，37℃培养箱中过夜培养。次日挑取单克隆进行菌落PCR验证以及酶切验证，得到重组表达质粒pET30a-Sm2OGD3。
实施例2：工程菌的诱导表达
将重组表达质粒pET30a-Sm2OGD3转化大肠杆菌BL21(DE3)感受态，得到含有目的基因的大肠杆菌菌株，同时转化pET30a空载质粒作为阴性对照，在含有100mg/L的卡那霉素的LB固体培养基上37℃生长过夜。
挑选阳性克隆的单菌落接种于含有100mg/L的卡那霉素的LB液体培养基中37℃下生长过夜。次日，按照1：100的比例进行扩大培养，于37℃培养至菌液的OD600达到0.5时，加入终浓度为0.5mM IPTG，于20℃低温诱导20h，以同样条件处理pET-30a空载作为空白对照。
取40μL菌液煮沸作为全菌，进行12％SDS-PAGE电泳分析。SDS-聚丙烯酰胺凝胶电泳结果表明(图2)，在分子量55kDa和43kDa之间，出现一条明显的特异蛋白质表达条带，与理论值约47kDa一致。
实施例3：对工程菌饲喂隐丹参酮并检测产物
挑选阳性克隆的单菌落接种于含有100mg/L的卡那霉素的LB液体培养基中37℃下生长过夜。次日，按照1：100的比例扩大培养至10mL，于37℃培养至菌液的OD600达到0.5时，加入终浓度为0.5mM IPTG，于20℃低温诱导5h后再加入100μM的隐丹参酮作为底物，于20℃继续反应20h。随后，3000rpm，离心3min收集菌体，并用2mL甲醇重悬。超声30min裂解大肠杆菌释放代谢物。8000rpm离心5min，上清再经过0.22μm的滤膜过滤，即得样品，使用UPLC进行检测。
以同样条件处理pET-30a空载作为空白对照。
使用Waters公司的UPLC仪器以及C18色谱柱(1.7μm,2.1×100mm)进行分析。流动相：(A)：0.1％甲酸水，(B)：乙腈。梯度洗脱如下：0min–5min,40％B；5min–20min,40％–60％B。流速0.4ml/min，进样量2μL，柱温20℃，检测波长270nm。
获得如下结果(如图3A)：相对于转化pET-30a空载的空白对照菌株，含pET30a-Sm2OGD3的菌株在19.169min出现产物丹参酮IIA。说明Sm2OGD3能够催化隐丹参酮生成丹参酮IIA。
实施例4：对工程菌饲喂二氢丹参酮I并检测产物
具体实施方法基本与实施例3相同，不同之处在于，加入终浓度为0.5mM IPTG，于20℃低温诱导5h后再加入100μM的二氢丹参酮I作为底物。
获得如下结果(如图3B)：相对于转化pET-30a空载的空白对照菌株，含pET30a-Sm2OGD3的菌株在13.938min出现产物丹参酮I。说明Sm2OGD3能够催化二氢丹参酮I生成丹参酮I。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对上述实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 中国医学科学院药用植物研究所
<120> 一种丹参2-酮戊二酸依赖性双加氧酶基因及其应用
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atggactcat ccagcttgaa aaattgctcg caagaaaacg aggccgatcg cgtgcacgag 60
ctgaacgctt tcgaggccac aaaagccggc gtgaaggggc tcaccgactc cggcgtccag 120
aaggttccga gaatgttcat caggccagcc gacgagctcg tcgaggagcg caaccggagc 180
cgctccccgc tgcaagctcc ggtgatagac ctcggccgga tcggggaggg cgaggggcgg 240
gagaaggtgg tgagcgaggt gagatgggcg tcgaaggagc tcgggatctt ccagatcgtg 300
aaccacgggg tggccgtgga ggtcatggac gcgatgatcg acggcgtgag gaagtttcac 360
gagcaagatg cggaggcgaa gaagcagttc cacacgcgcg acgccatgcg caaggtgatg 420
tacgcgagca acgtcgatct gtacaagtcg cgcgcggcga attggaggga cacgttctcg 480
gtcgcgctca tgggttccga cagcgttgag ccggaagagt tgccggagat ttgcagagac 540
tcaacaatca agtatcttga tgaagtcacg aatctcgcac acactctatt tgagctgctc 600
tcggaagctc ttgggctcga acaaggttgc ctaggagcct tgaaatgtgg ccgaggacgc 660
acgttcgtcg gccagtacta ccccgcgtgc cccgagccgg agctcacgat gggcatgacc 720
aaccacaccg atccttgttt cctaactatt cttctccaag atcatatcgg aggcctccaa 780
gctctgcaca atagtcagta cataaatgtg gagcctctgc ctgctagctt cgtcgtcaac 840
attggagata tgttgcagat tgtgtcaaat gatgagttca taagcccaat tcatagagtt 900
catgcaaatc gggccgggcc aagaatctcg attgcgggct ttttcaccgg tgatgctatt 960
tcagggacaa tatacggccc gatcaaagag ttggtatcaa agaacaatgg ggctcgatac 1020
aaagagttca cagtgggaga gtacatgtcc aagtttttag agcggccaat tgataaatct 1080
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Gln Tyr Tyr Pro Ala Cys Pro Glu Pro Glu Leu Thr Met Gly Met Thr
225 230 235 240
Asn His Thr Asp Pro Cys Phe Leu Thr Ile Leu Leu Gln Asp His Ile
245 250 255
Gly Gly Leu Gln Ala Leu His Asn Ser Gln Tyr Ile Asn Val Glu Pro
260 265 270
Leu Pro Ala Ser Phe Val Val Asn Ile Gly Asp Met Leu Gln Ile Val
275 280 285
Ser Asn Asp Glu Phe Ile Ser Pro Ile His Arg Val His Ala Asn Arg
290 295 300
Ala Gly Pro Arg Ile Ser Ile Ala Gly Phe Phe Thr Gly Asp Ala Ile
305 310 315 320
Ser Gly Thr Ile Tyr Gly Pro Ile Lys Glu Leu Val Ser Lys Asn Asn
325 330 335
Gly Ala Arg Tyr Lys Glu Phe Thr Val Gly Glu Tyr Met Ser Lys Phe
340 345 350
Leu Glu Arg Pro Ile Asp Lys Ser Gly Leu Asp Glu Cys Arg Leu Gln
355 360 365
Asp Glu Asp Asn Val
370
<210> 3
<211> 30
<212> DNA
<213> Artificial
<400> 3
ggatccatgg actcatccag cttgaaaaat 30
<210> 4
<211> 30
<212> DNA
<213> Artificial
<400> 4
gcggccgctt agacattgtc ttcatcttgc 30
Claims (8)
1.一种丹参2-酮戊二酸依赖性双加氧酶基因，其特征在于，
核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述基因编码的蛋白，其特征在于，
氨基酸序列如SEQ ID NO.2所示。
3.克隆权利要求1所述基因全长的引物对，其特征在于，
上游引物如SEQ ID No.3所示，
下游引物如SEQ ID No.4所示。
4.含有权利要求1所述基因的重组表达载体、表达盒、转基因细胞系或宿主菌。
5.含有权利要求1所述基因的重组表达载体，其特征在于，
所述重组表达载体以pET-30a为出发载体，所述基因的插入位点为NotI和BamHI。
6.权利要求1所述基因或权利要求2所述蛋白在制备丹参酮类化合物中的应用。
7.根据权利要求6所述的应用，其特征在于，
所述基因编码的蛋白催化隐丹参酮合成丹参酮IIA。
8.根据权利要求6所述的应用，其特征在于，
所述基因编码的蛋白催化二氢丹参酮I转化为丹参酮I。
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