CN111100849B - 茶树类胡萝卜素裂解双加氧酶CsCCD4及其在催化合成β-紫罗酮上的应用 - Google Patents
茶树类胡萝卜素裂解双加氧酶CsCCD4及其在催化合成β-紫罗酮上的应用 Download PDFInfo
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Abstract
本发明公开了茶树类胡萝卜素裂解双加氧酶CsCCD4及其在催化合成β‑紫罗酮上的应用，茶树类胡萝卜素裂解双加氧酶CsCCD4，其氨基酸序列如SEQ ID No.2所示。茶树类胡萝卜素裂解双加氧酶基因CsCCD4，其核苷酸序列为a、b或c中的任一种：a、如SEQ ID No.1所示，b、与SEQ ID No.1所示核苷酸序列互补的核苷酸序列，c、编码SEQ ID No.2所示氨基酸序列的核苷酸序列。本发明茶树类胡萝卜素裂解双加氧酶CsCCD4或茶树类胡萝卜素裂解双加氧酶基因CsCCD4可应用于催化β‑胡萝卜素生成β‑紫罗酮中。
Description
技术领域
本发明属于基因工程领域，具体涉及一种茶树类胡萝卜素裂解双加氧酶CsCCD4及其在催化合成β-紫罗酮上的应用。
背景技术
花香可以影响高等植物与传粉者相互作用，也可以起到阻吓食草动物和吸引天敌的作用。作为最传统的无酒精饮料之一，茶(Camellia sinensis(L.)O.Kuntze)以其温和的口感、宜人的香气及其营养价值在世界享有盛誉。挥发性香气是评价茶叶品质的重要指标，在茶叶生产过程中已发现600多种挥发性化合物，包括脂肪酸衍生物(FADVs)、氨基酸衍生物(AADVs)、挥发性萜烯(VTs)、类胡萝卜素衍生物(CDVs)。在茶叶众多的香气物质中，由类胡萝卜素降解产生的香气物质占成品茶香气总量的4.3-46.5％，是茶叶香气的重要组成部分。这些香气物质大多源自次生代谢途径，例如在类胡萝卜素的生物合成中，以类胡萝卜素提供前体生成的α，β-紫罗酮，β-大马酮等，凭借其极低的气味阈值在茶叶风味中起着关键作用。
在类胡萝卜素的酶促氧化途径中，类胡萝卜素裂解双加氧酶(carotenoidcleavage dioxygenases，CCDs)是类胡萝卜素降解的关键酶，在类胡萝卜素的酶促氧化中起着至关重要的作用。
1997年研究人员从玉米中提取得到VP14，并被鉴定为脱落酸(Abscisic Acid,ABA)生物合成途径中的关键酶,Chernys等在拟南芥中鉴定出可以根据不同底物，在9,10和/或9',10' 双键对称地裂解各种类胡萝卜素，形成二醛和C13产物的酶。经过一段时间的研究与鉴定后，研究者们将类胡萝卜素裂解酶基因分为两个亚家族，分别是类胡萝卜素裂解双加氧酶基因 (Carotenoid Cleavage Dioxygenases,CCD)和9-顺式-环氧类胡萝卜素裂解双加氧酶基因 (nine-cis-epoxy-carotenoid dioxygenases,NCEDs)。CCDs是一类非血红素铁氧合酶，在亚铁离子的协助下，可将类胡萝卜素裂解为脱辅基类胡萝卜素或香气化合物。CCDs在自然界中分布广泛，在原核细胞、动物、真菌和绿藻中具有重要的代谢和激素调节功能，此外，在高等植物中，类胡萝卜素裂解产物也可作为信号参与调控花的香味、果实的颜色和香气，吸引传粉者和传播媒介。其中，NCED是ABA合成途径的关键基因。根据降解底物双键的位置，CCD又被分为CCD1、CCD4、CCD7、CCD8，CCD7和CCD8是合成独脚金内酯的关键基因，CCD1和CCD4可裂解多种胡萝卜素和脱辅基胡萝卜素生成具有花果香气的香气物质。其中，已有文献报道β-紫罗酮是典型的类胡萝卜氧化降解产生的香气物质，它可在茶叶加工过程中通过酶促氧化降解类胡萝卜素得到，是绿茶和红茶重要的香气物质。
β-紫罗酮是一种重要的香料，室温下具有木质香和愉悦诱人的紫罗兰香，在食品、化妆品、烟草和日化用品中有着广泛的应用。GB 2760－96规定其为暂时允许使用的食用香料，主要用以配制树莓、樱桃、葡萄、草莓、黑莓、菠萝等型香精,用于日化、食品香精，香水中，还可大量用于生产维生素A、E和胡萝卜素。
尽管β-紫罗酮已被证实是绿茶和红茶的重要风味物质之一,但茶树中CCDs降解类胡萝卜素生成β-紫罗酮的机理尚不明确。
发明内容
本发明所要解决的技术问题为：如何提供一种茶树类胡萝卜素裂解双加氧酶CsCCD4及其编码基因，为人工合成β-紫罗酮提供一种新的途径。
本发明的技术方案为：茶树类胡萝卜素裂解双加氧酶CsCCD4，其氨基酸序列如SEQID No.2所示。
茶树类胡萝卜素裂解双加氧酶基因CsCCD4，其核苷酸序列为a、b或c中的任一种：
a、如SEQ ID No.1所示，
b、与SEQ ID No.1所示核苷酸序列互补的核苷酸序列，
c、编码SEQ ID No.2所示氨基酸序列的核苷酸序列。
上述所述的茶树类胡萝卜素裂解双加氧酶CsCCD4或类胡萝卜素裂解双加氧酶基因 CsCCD4在催化β-胡萝卜素生成β-紫罗酮上的应用。
本发明的茶树类胡萝卜素裂解双加氧酶CsCCD4的制备方法，包括以下步骤：
将茶树类胡萝卜素裂解双加氧酶基因CsCCD4连接到pGEX-4T1载体中，转入大肠杆菌中培养，添加IPTG后，16-37℃诱导20-24小时，用GST-树脂进行纯化，纯化后的蛋白用SDS-Page进行检测。
与现有技术相比，本发明具有以下有益效果：
本发明通过筛选得到类胡萝卜素裂解双加氧酶CsCCD4及其编码基因，通过原核表达其编码的蛋白能够特异性催化β-胡萝卜素生成大量β-紫罗酮，该发明不仅对茶树β-紫罗酮的积累及分子育种提供分子标记，也为其在茶叶加工过程中的调控提供理论依据。
附图说明
图1CsCCD4重组蛋白条带，M，Marker；
图2HPLC检测底物变化情况。与对照相比，转入CsCCD4的工程菌中，β-胡萝卜素菌块颜色由黄变浅，β-胡萝卜素的含量明显降低；
图3GC-MS检测反应产物，虚线代表对照，实线代表转入目的基因。“1”为检测到的产物：β-紫罗酮；离子谱图代表β-紫罗酮；
图4茶树各部位中β-紫罗酮的相对含量，采用单因素方差分析(ANOVA)和Duncan(s)进行统计学分析。不同字母代表各组之间的差异有统计学意义(P<0.05)；
图5茶树各部位中CsCCD4的表达量，采用单因素方差分析(ANOVA)和Duncan(s)进行统计学分析。不同字母代表各组之间的差异有统计学意义(P<0.05)；
图6不同应激处理对CsCCD4表达量的影响，对茶苗分别进行低温、失水、低温+机械损伤(采摘)、失水+机械损伤(采摘)处理后CsCCD4表达量的变化。“-”，完整茶苗；“+”，机械损伤(采摘)。采用单因素方差分析(ANOVA)和Duncan(s)进行统计学分析。不同字母代表各组之间的差异有统计学意义(P<0.05)。
具体实施方式
1、基因CsCCD4的克隆
CsCCD4基因序列由茶树基因转录组数据筛选得到,并对其ORF序列进行特异性引物设计。引物序列如下：
5'-GGATCTGGTTCCGCGTGGATCCATGGATGCCTTCTCATCCTCAT
3'-GCTCGAGTCGACCCGGGTTAGAGCTTGTTGAGGTCGC
使用Taq mix酶进行PCR反应，反应条件为：94℃预变性3min，94℃变性30s，55℃退火30s，72℃延伸3min，共进行35个循环，最后72℃延伸10min，4℃保存。PCR产物用1.2％琼脂糖凝胶电泳(AGE)检测.反应体系如下：
2、类胡萝卜素裂解双加氧酶CsCCD4的制备
把上述基因连接到pGEX-4T1载体中，构建得到重组表达质粒pGEX-4T1-CsCCD4；转入BL21或Rosetta(DE3)大肠杆菌中37℃培养至OD600＝0.6-0.8，添加1mM IPTG后，16-18℃诱导20-24小时(也可以室温或37℃诱导)，用GST-树脂进行纯化，纯化后的蛋白用SDS-Page进行检测(如图1)。
3、双质粒共转化感受态JM109
将能编码合成类胡萝卜素的基因质粒DNA，其编号如下：#100(合成β-胡萝卜素)、#102(合成玉米黄质)、#834(合成番茄红素)、#835(合成Zeta-胡萝卜素)，pGEX-4T1载体质粒，以及成功构建的重组表达质粒pGEX-4T1-CsCCD4各1ul,分别共转化感受态JM109，转化体系如下：
表1
转化方法如下：
(1)100μL感受态细胞JM109(×4)悬液冰上溶解，分别各取30μL于4个1.5mL 离心管中，按表1分别分组加入两种质粒DNA各1μL。
(2)用移液器枪头轻轻将体系搅拌均匀，置于冰上30分钟后，于42℃恒温热水浴中加热90s，然后迅速在冰上冷却3～5min。
(3)加入500μl LB液体培养基，37℃200rpm振荡培养l h，使菌体恢复正常生长状态，并扩大培养。
(4)取第一组、第二组500μL菌液均匀涂在同时含有两种抗性的LB平板上 (Amp+,100mg/L；Cl-25mg/L),待LB培养基完全吸收菌液后，将涂布好的培养皿倒放，在 37℃培养箱中过夜培养。
(5)挑取单菌落，通过菌落PCR验证转化结果。
4、双质粒重组蛋白共表达
将两组双质粒共转化成功的重组质粒：#100+pGEX-4TI，#100+CCD4，#102+pGEX-4TI，#102+CCD4，#834+pGEX-4TI，#834+CCD4，#835+pGEX-4TI，#835+CCD4进行体外表达，加入0.2mM IPTG诱导后纯化，离心收集菌液沉淀，观察到β-胡萝卜素颜色变化明显。对沉淀进行萃取后，利用HPLC检测底物变化，发现与对照相比，转入CsCCD4的工程菌中，β-胡萝卜素的含量大幅度下降。(如图2)
5、产物检测：
在上述体系共表达过程中，利用GC-MS对反应产物进行检测。结果显示，CsCCD4能够特异性降解β-胡萝卜素生成β-紫罗酮。值得注意的是，检测到的β-紫罗酮含量极高。 (如图3)
6、应用举例
(1)结合其能降解β-胡萝卜素生成大量β-紫罗酮，通过实时定量PCR对CsCCD4的在茶树中的表达量进行分析，反应体系为12.5μL of SYBR1 Premix ExTaqTMII,2μL cDNA,，上下游引物(10μM)各1μL，总体积25μL。反应条件为95℃ for 3min；40cycles of 95℃ for10s,62℃ for 30s。同时分析茶树各个部位的β-紫罗酮含量，如图4和图5所示，CsCCD4在茶树一二叶中表达量最高，芽次之；在CsCCD4调控β-紫罗酮合成的研究基础上，发现β-紫罗酮在茶树一二叶中含量最高，芽次之，与CsCCD4表达量相对应。为茶树β-紫罗酮的积累及分子育种提供分子标记。
(2)结合茶叶加工过程中存在的环境胁迫，对茶苗分别进行低温、失水、低温+机械损伤(采摘)、失水+机械损伤(采摘)处理，处理后的茶苗收取顶芽至三、四叶，液氮速冻，磨碎样品提取RNA,反转录后进行实时荧光定量，反应体系为12.5μL of SYBR1 PremixExTaqTMII,2μL cDNA,，上下游引物(10μM)各1μL，总体积25μL。反应条件为 95℃ for 3min；40cycles of 95℃ for 10s,62℃ for 30s。对CsCCD4的表达量进行分析，结果如图6所示。发现CsCCD4基因在低温及机械损伤的胁迫下表达量增加，此时可能调控β-紫罗酮含量增加，为其在茶叶加工过程中的调控提供理论依据。
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<110> 安徽农业大学
<120> 茶树类胡萝卜素裂解双加氧酶CsCCD4及其在催化合成β-紫罗酮上的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1842
<212> DNA
<213> Camellia sinensis
<400> 1
atggatgcct tctcatcctc attcctctct acattttcat tctcgaatgt ctctctaaca 60
ccaccaccac caccaccaca accacaacca tcaaatcctc ctctcctcca catctccgct 120
gtccgaatag aagaaaaacc accaacttca acttcctcct cctcctccac caccaccacc 180
accacccccc ctacaagaac acctcaaatt tcaaaaccaa tcccacaaaa atccacatca 240
aaaaaaccaa ccaccacacc accaccacca actgcgagac gagcagagcc gagcttttcc 300
accaccatct tcaacacatt cgacgaaatc atcaacaact tcattgaccc acccctccgc 360
cagtccgtcg acccacgcta cgtcctctcc gacaacttcg cccccgtcga cgaactccct 420
ccgaccaact gcacggtggt ggaaggctcc ctcccgcagt gcctccacgg cggagcgtac 480
atccgcaacg gccccaatcc ccaattcctc ccccgcggcc cctaccacct cttcgacggc 540
gacggcatgc tccacgccat caaaatctcc ggccccaccg ccgccaccct ctgcagccgc 600
tacgtgaaga cctacaagta cacaatcgag tgcaaaaccg gttctccggt catccccaat 660
gtcttctccg gcttcaatgg cctcaccgcc tcagcggcgc gtggggcagt caccgccgct 720
cgagctctcg ccggacaatt caaccccacc aatgggattg ggcttgcgaa cacgagcttg 780
gctctcatcg gatccaaact ctacgctctc ggcgagtctg atcttcctta cgccgtcgag 840
atctcgccgg acggcgatat aatcactctg ggtcgccgtg atttcgccgg aaaattgttc 900
atgagcatga cggcgcatcc gaaacttgac ccagataccg gcgaggcatt cgctttccgg 960
tatggtccgg taccgccatt tctaaccttt ttcaggttca acccagatgg ctcgaaacaa 1020
cctgacgtgc cgattttctc attgactact ccttcgtttc ttcatgattt tgcgatcacg 1080
aagaagtacg ccatttttgt tgatattcag atcggaatga accccatgga gatgatcctc 1140
ggagggtcgc cggtgggtac tgattcggcg aaagtgccga gaatcggcgt gattccgagg 1200
tacgcgaagg acgagtcgga gatgaggtgg tttgaagtgc cggggttcaa tataatacat 1260
gcgataaatg cgtgggatga ggacggtggt gatacggtgg tgatggtggc gccgaatata 1320
ttggcggtgg agcatacgtt ggagaggatg gatttgatac atgcgtcggt ggagatggtg 1380
aggattgatc ttaagagtgg ggtggtgtcc aggtttccgg tggcggccag gaatttggat 1440
tttgctgtga ttaatcacaa ctacttggga aagaagaaca ggtacgtgta tgcagcaatt 1500
ggggatccaa tgccgaagat ctccggggtg gtgaaactcg acgtgtcagt atcagaaggc 1560
gaccgccgtg actgcacggt ggctagtcgg ttgttcgggc ggggctgttt tgggggcgag 1620
cccttcttcg tagcaaagga ccctaacaat cccgaggcgg acgaggatga tgggtacgta 1680
gtaacttatg ttcacgatga atacataggc gagtcgaggt ttctagtgat ggacgcaaag 1740
tctccggacc tcgacatcgt ggcagccgtg aaattgcccc ggcgagtacc ttacggcttt 1800
cacgggttgt ttgtgagaga aagcgacctc aacaagctct aa 1842
<210> 2
<211> 613
<212> PRT
<213> Camellia sinensis
<400> 2
Met Asp Ala Phe Ser Ser Ser Phe Leu Ser Thr Phe Ser Phe Ser Asn
1 5 10 15
Val Ser Leu Thr Pro Pro Pro Pro Pro Pro Gln Pro Gln Pro Ser Asn
20 25 30
Pro Pro Leu Leu His Ile Ser Ala Val Arg Ile Glu Glu Lys Pro Pro
35 40 45
Thr Ser Thr Ser Ser Ser Ser Ser Thr Thr Thr Thr Thr Thr Pro Pro
50 55 60
Thr Arg Thr Pro Gln Ile Ser Lys Pro Ile Pro Gln Lys Ser Thr Ser
65 70 75 80
Lys Lys Pro Thr Thr Thr Pro Pro Pro Pro Thr Ala Arg Arg Ala Glu
85 90 95
Pro Ser Phe Ser Thr Thr Ile Phe Asn Thr Phe Asp Glu Ile Ile Asn
100 105 110
Asn Phe Ile Asp Pro Pro Leu Arg Gln Ser Val Asp Pro Arg Tyr Val
115 120 125
Leu Ser Asp Asn Phe Ala Pro Val Asp Glu Leu Pro Pro Thr Asn Cys
130 135 140
Thr Val Val Glu Gly Ser Leu Pro Gln Cys Leu His Gly Gly Ala Tyr
145 150 155 160
Ile Arg Asn Gly Pro Asn Pro Gln Phe Leu Pro Arg Gly Pro Tyr His
165 170 175
Leu Phe Asp Gly Asp Gly Met Leu His Ala Ile Lys Ile Ser Gly Pro
180 185 190
Thr Ala Ala Thr Leu Cys Ser Arg Tyr Val Lys Thr Tyr Lys Tyr Thr
195 200 205
Ile Glu Cys Lys Thr Gly Ser Pro Val Ile Pro Asn Val Phe Ser Gly
210 215 220
Phe Asn Gly Leu Thr Ala Ser Ala Ala Arg Gly Ala Val Thr Ala Ala
225 230 235 240
Arg Ala Leu Ala Gly Gln Phe Asn Pro Thr Asn Gly Ile Gly Leu Ala
245 250 255
Asn Thr Ser Leu Ala Leu Ile Gly Ser Lys Leu Tyr Ala Leu Gly Glu
260 265 270
Ser Asp Leu Pro Tyr Ala Val Glu Ile Ser Pro Asp Gly Asp Ile Ile
275 280 285
Thr Leu Gly Arg Arg Asp Phe Ala Gly Lys Leu Phe Met Ser Met Thr
290 295 300
Ala His Pro Lys Leu Asp Pro Asp Thr Gly Glu Ala Phe Ala Phe Arg
305 310 315 320
Tyr Gly Pro Val Pro Pro Phe Leu Thr Phe Phe Arg Phe Asn Pro Asp
325 330 335
Gly Ser Lys Gln Pro Asp Val Pro Ile Phe Ser Leu Thr Thr Pro Ser
340 345 350
Phe Leu His Asp Phe Ala Ile Thr Lys Lys Tyr Ala Ile Phe Val Asp
355 360 365
Ile Gln Ile Gly Met Asn Pro Met Glu Met Ile Leu Gly Gly Ser Pro
370 375 380
Val Gly Thr Asp Ser Ala Lys Val Pro Arg Ile Gly Val Ile Pro Arg
385 390 395 400
Tyr Ala Lys Asp Glu Ser Glu Met Arg Trp Phe Glu Val Pro Gly Phe
405 410 415
Asn Ile Ile His Ala Ile Asn Ala Trp Asp Glu Asp Gly Gly Asp Thr
420 425 430
Val Val Met Val Ala Pro Asn Ile Leu Ala Val Glu His Thr Leu Glu
435 440 445
Arg Met Asp Leu Ile His Ala Ser Val Glu Met Val Arg Ile Asp Leu
450 455 460
Lys Ser Gly Val Val Ser Arg Phe Pro Val Ala Ala Arg Asn Leu Asp
465 470 475 480
Phe Ala Val Ile Asn His Asn Tyr Leu Gly Lys Lys Asn Arg Tyr Val
485 490 495
Tyr Ala Ala Ile Gly Asp Pro Met Pro Lys Ile Ser Gly Val Val Lys
500 505 510
Leu Asp Val Ser Val Ser Glu Gly Asp Arg Arg Asp Cys Thr Val Ala
515 520 525
Ser Arg Leu Phe Gly Arg Gly Cys Phe Gly Gly Glu Pro Phe Phe Val
530 535 540
Ala Lys Asp Pro Asn Asn Pro Glu Ala Asp Glu Asp Asp Gly Tyr Val
545 550 555 560
Val Thr Tyr Val His Asp Glu Tyr Ile Gly Glu Ser Arg Phe Leu Val
565 570 575
Met Asp Ala Lys Ser Pro Asp Leu Asp Ile Val Ala Ala Val Lys Leu
580 585 590
Pro Arg Arg Val Pro Tyr Gly Phe His Gly Leu Phe Val Arg Glu Ser
595 600 605
Asp Leu Asn Lys Leu
610
Claims (1)
1.茶树类胡萝卜素裂解双加氧酶CsCCD4或其编码基因CsCCD4在催化β-胡萝卜素生成β-紫罗酮上的应用；所述茶树类胡萝卜素裂解双加氧酶CsCCD4氨基酸序列如SEQ ID No.2所示，所述编码基因CsCCD4的核苷酸序列为a、或b中的任一种：
a、如SEQ ID No.1所示，
b、编码SEQ ID No.2所示氨基酸序列的核苷酸序列。
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| CN112899293A (zh) * | 2021-03-11 | 2021-06-04 | 广西壮族自治区蚕业技术推广站 | 桑树中类胡萝卜素裂解双加氧酶相关的基因及其原核表达 |
| CN113122547B (zh) * | 2021-04-20 | 2022-03-18 | 安徽农业大学 | CsMYB110基因在调控类胡萝卜素合成中的应用 |
| CN113512556A (zh) * | 2021-06-11 | 2021-10-19 | 南京大学 | 一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用 |
| CN114376257B (zh) * | 2022-01-30 | 2023-05-02 | 广西中烟工业有限责任公司 | 一种菌酶协同处理改善烟叶品质的方法 |
| CN115161298A (zh) * | 2022-06-26 | 2022-10-11 | 上海龙殷生物科技有限公司 | 一种双加氧酶氨基酸序列、基因及其生物生香应用 |
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| CN105368850A (zh) * | 2015-11-25 | 2016-03-02 | 天津大学 | 产生β-紫罗酮香气物质的枸杞类胡萝卜素裂解双加氧酶基因及应用 |
| CN107502614A (zh) * | 2017-03-22 | 2017-12-22 | 中国医学科学院药用植物研究所 | 一种参与栀子西红花苷合成的类胡萝卜素裂解双加氧酶编码基因的筛选及功能验证 |
| CN110546268A (zh) * | 2017-02-24 | 2019-12-06 | 新加坡科技研究局 | 类胡萝卜素和脱辅基类胡萝卜素的生产 |
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| CN1844377A (zh) * | 2006-04-11 | 2006-10-11 | 华南农业大学 | 柱花草9－顺式环氧类胡萝卜素双加氧酶及其编码基因与应用 |
| EP2884280B1 (en) * | 2013-12-15 | 2018-05-09 | Symrise AG | Method for evaluating the scent performance of perfumes and perfume mixtures |
| CN107075522A (zh) * | 2014-07-23 | 2017-08-18 | 国家新技术、能源和可持续经济发展局(Enea) | 类胡萝卜素双加氧酶和在微生物和植物中以生物技术制备衍生自藏红花的化合物的方法 |
| US10364434B2 (en) * | 2015-03-23 | 2019-07-30 | Arch Innotek, Llc | Compositions and methods of biosynthesizing carotenoids and their derivatives |
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| CN105368850A (zh) * | 2015-11-25 | 2016-03-02 | 天津大学 | 产生β-紫罗酮香气物质的枸杞类胡萝卜素裂解双加氧酶基因及应用 |
| CN110546268A (zh) * | 2017-02-24 | 2019-12-06 | 新加坡科技研究局 | 类胡萝卜素和脱辅基类胡萝卜素的生产 |
| CN107502614A (zh) * | 2017-03-22 | 2017-12-22 | 中国医学科学院药用植物研究所 | 一种参与栀子西红花苷合成的类胡萝卜素裂解双加氧酶编码基因的筛选及功能验证 |
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