CN114807183B - 一种青稞矢车菊素氧甲基转移酶基因的新用途 - Google Patents
一种青稞矢车菊素氧甲基转移酶基因的新用途 Download PDFInfo
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- CN114807183B CN114807183B CN202111480017.8A CN202111480017A CN114807183B CN 114807183 B CN114807183 B CN 114807183B CN 202111480017 A CN202111480017 A CN 202111480017A CN 114807183 B CN114807183 B CN 114807183B
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- paeoniflorin
- cyanidin
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- highland barley
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Abstract
本发明属于基因工程技术领域，具体涉及一种青稞矢车菊素氧甲基转移酶基因的新用途。本发明提供了青稞来源的矢车菊素氧甲基转移酶基因HOVUSG2104500、该基因的重组载体、其重组菌及矢车菊素氧甲基转移酶的新用途。本发明还提供了生产芍药素3‑O丙二酰葡萄糖苷的转基因植物的制备方法。本发明的矢车菊素氧甲基转移酶能够以硫腺苷甲硫氨酸作为甲基供体，以矢车菊素3‑O丙二酰葡萄糖苷作为甲基受体，合成芍药素3‑O丙二酰葡萄糖苷，应用前景优良。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种青稞矢车菊素氧甲基转移酶基因的新用途。
背景技术
重要活性成分花青素不仅具有抗氧化、清除自由基、抑制炎症和抗癌等作用，同时也能预防慢性等疾病。目前花青素在植物体内多以糖基化修饰的形式存在，但目前对负责花青素的修饰的基因还鲜有报道。
青藏高原是青稞的驯化地，青稞种质资源极其丰富，黑色、紫色和蓝色等深色青稞(下文中称有色青稞) 又是其中最为珍贵的种质资源。有色青稞是一类珍贵的青稞种质资源，主要包括黑青稞、紫青稞、蓝青稞等。有色青稞中主要富集大量的花青素，对优良品种进行种子色泽进行改良培育，将会成为青稞种色育种的亮点，且具有重要的市场应用价值。
花青素骨架物质如花翠素、芍药素在植物中不稳定，必须经过甲基、丙二酰、糖基化等修饰化后才能以稳定的结果存在于植物体内（Wang et al. Molecular plant, 12(7), 899-919）。有报道研究发现糖基化修饰的花青素必须通过酰基化修饰后才能被花青素转运蛋白识别进而转运到液泡中储存 （Zhao et al. Trends Plant Sci. 20:576–585）。其中，芍药素3-O丙二酰葡萄糖苷即是芍药素经过丙二酰基、糖基化修饰后产生的一种花青素衍生物。
目前制备花青素及其衍生物的方法主要是利用酶法提取从葡萄、桑葚、紫薯等组织中进行提取。现有技术仍然无法做到用基因工程等手段实现花青素的大量合成和修饰。
发明内容
针对现有技术的缺陷，本发明提供一种青稞矢车菊素氧甲基转移酶基因的新用途，能够有效将矢车菊素3-O-丙二酰葡萄糖苷和硫腺苷甲硫氨酸转化为芍药素3-O-丙二酰葡萄糖苷。
核苷酸序列如SEQ ID NO.1所示的基因片段在制备芍药素3-O丙二酰葡萄糖苷中的用途。
本发明还提供含有核苷酸序列如SEQ ID NO.1所示的基因片段的重组载体在制备芍药素3-O丙二酰葡萄糖苷中的用途。
优选的，所述重组载体是重组pGEX-6P-1载体。
本发明还提供含有SEQ ID NO.1所示的基因片段的重组菌在制备芍药素3-O丙二酰葡萄糖苷中的用途。
优选的，所述重组菌为重组大肠杆菌。
优选的，所述重组菌为Transeta(DE3)。
本发明还提供氨基酸序列如SEQ ID NO.2所示的蛋白在制备芍药素3-O丙二酰葡萄糖苷中的用途。
本发明还提供一种生产芍药素3-O丙二酰葡萄糖苷的转基因植物的构建方法，取核苷酸序列如SEQ ID NO.1所示的基因片段，转入植物中，获得表达氨基酸序列如SEQ IDNO.2所示的蛋白的植株，即可。
优选的，所述转入植物的方法是农杆菌法、基因枪法、电转法、PEG介导法、脂质体法和磷酸钙-DNA共沉淀法中的一种。
优选的，所述植物为烟草。
本发明提供了青稞来源的矢车菊素氧甲基转移酶基因HOVUSG2104500（SEQ IDNO.1）、该基因的重组载体、其重组菌及矢车菊素氧甲基转移酶的新用途。本发明还提供了生产芍药素3-O丙二酰葡萄糖苷的转基因植物的制备方法。本发明的矢车菊素氧甲基转移酶能够以硫腺苷甲硫氨酸作为甲基供体，以矢车菊素3-O丙二酰葡萄糖苷作为甲基受体，合成芍药素3-O丙二酰葡萄糖苷，应用前景优良。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为HOVUSG2104500蛋白的SDS-PAGE电泳图，其中，Marker：100,70,55,40,35,25KDa。
图2为体外催化反应的LC-MS图： Cyanidin O-malonylhexoside，矢车菊素3-O丙二酰葡萄糖苷；Peonidin O-malonylhexoside 芍药素3-O丙二酰葡萄糖苷。
图3为转基因植物提取物中芍药素3-O丙二酰葡萄糖苷的质谱图。
具体实施方式
以下实施例和实验例中所用的试剂和材料，无特别说明的均为市售品。
实施例1 HOVUSG2104500基因（SEQ ID NO.1）的分离以及原核表达
本实施例提供HOVUSG2104500基因的获得、载体构建及原核表达的方法。
(1)基因片段和载体的构建
称取2克青稞新鲜叶片，提取青稞RNA，用Thermo Fisher公司的M-MLV ReverseTranscriptase合成cDNA，设计引物，如下：
F:ATGCTAGGTGGCCTACCTCCTCT（SEQ ID NO.3）
R:TCAAGGGTAGACTTCAATTATGGATCGAAATCCTA（SEQ ID NO.4）
进行PCR扩增，得到目的条带大小的片段(结果如图1所示)。PCR产物用胶回收试剂盒(Gel Extraction Kit D2500-02，OMEGA)纯化。
扩增得到的目的片段HOVUSG2104500基因的核苷酸序列(SEQ ID NO.1)为：
ATGCTAGGTGGCCTACCTCCTCTCCTCCCTAGTGACCAGTTGAGAAGAAAACATATGGTGGTGCTGGTGCATGTCCAAAAAGAAACTGGAAACGATGTGATCATCAGCACGGAGGGGTTACTCGAAGCTCAGCTTGAGCTCTACCATAACGCCATGGCATACGTCAAGTCCGCGGTGGTGAGGGCTGCCTTGGACCTACGCATCCCCGACGCCATCCACCGTCGCGGCGGTGCCGCCACCTTGTCCGATATCGCCACCGAGGCCGGGGTCCAGCCGACAAAGGTTTCCCACCTCCGTCGGCTCATGCGCGCCCTCACCATCTTTGATGTCTTCTCAGTCCACCGGGGCACTCACCATGATGATGCCATCGACGTGCACTATAAGCTCACCCCCCTCTCGCGCCTCCTCGTCGGGGACAGCTCGTGCACCCAGTCCCCCATCATGCGCGTGCTCGTGGACCCGCTGTCCTTGACCGCCCTCTGCAGTATAGGTGAGTGGTTCACGGACGAGAGGGCGTCGGCTCTGACACTCTTCGAAGTGGCGCATGGGTGCAAACGGGATGAGATGACAATGAAGAAGGGCACGCGTAGCATGTTCAACGCTGGCATGGTCTCCGATAGTCGCCTTCTTATGGAGACCGTCATCAAAGATCACTGCAACATCTTTGAGGGCGTTAGCTCTCTTGTTGACGCCGGCGGTGCTCATGGTGCCACGGCGGAAGCCGTCACTAAGGCATTCCCACACATCAGGTGCACCGTGTTGGACCTCCCACATGCGATCGACGGGGCACCTGCCATCGGTAATGTCGAGTTTGTTGCTGGTGATTTGTTTGAGTATGTGCCACCAGCAGACGTTGTTCTACTCAAGTGGGTTTTGTGCTTGTGGCAAGATGAAGATGCTGTCAAGGTACTACGACGGTGCAAAGAAGCAATAACAAGTAGAGGTTCCAAAGGGAAGGTGATAATCATTGATGTCGTGATAGACTCCGGGATGTCACAGGATGATCTTCTTCTTAGGGAGACGCAAGTTCTATTCGATGTCCAAATGATGCGTGTTGATGGGGGCGAGCGAGACGAGAAGGAGTGGAGGAAGATTTTCATTGAAGCCGGATTCAAGGATTATAATATCACTCCAATGCTAGGATTTCGATCCATAATTGAAGTCTACCCTTGA。
前述HOVUSG2104500基因可以通过上述方法获得，也可以直接进行合成。
得到的基因片段转入载体pGEX-6P-1中，后重组载体转入Transetta(DE3)菌株中，得到含有目的片段的重组菌株。
(2)基因的表达
包括如下步骤：
1.PCR检测阳性克隆，抽提质粒测序。
2.将测序正确的质粒载体热击转化大肠杆菌transeta(DE3)，抗性CN。
3.早上9点随机挑取2个正常大小克隆于5mL含有Amp的LB培养基中，37℃摇至下午4点。选其中一个，将4mL活化菌液(浓度为1×106～107cfu/ml)按照1:50比例转接到200mL大瓶LB培养基中，大摇床37℃培养，转速200rpm。3～4小时后，200mL培养基中加入2uL 1MIPTG诱导剂。20℃，160rpm条件下诱导过夜。剩余1mL菌液用来保菌。
4.第二天早上8点收集菌体。500mL离心瓶，4000rpm离心10min即可。
5 .50mL Lysis buffer重悬菌体，涡旋混匀，转入50mL离心管中，分别加入50uLPMSF,10uLβ-巯基乙醇，混匀放置冰上。
6.采用高压破碎仪进行大肠杆菌细胞破碎实验。
7 .破碎完成后样品，取20ul作为总蛋白样品。再取1mL样品4℃，13000rpm离心10min，取20uL上清作为上清液样品。加入等体积2*Loading buffer，煮沸5min,SDS-PAGE电泳检测蛋白表达情况。剩余上清可暂冻存于-20℃冰箱。剩余未离心样品可冻存于-80℃冰箱。
8.SDS-PAGE电泳完毕，加入考马斯亮蓝染色液，微波炉煮沸1min后染色半小时，加入脱色液脱色。每隔1h换一次脱色液，直至蛋白条带清晰，转至清水中。
9.GST标签融合蛋白纯化。将破碎未离心的所有样品离心，上清与1mL树脂在4℃混匀仪上混合3h。混匀结束后，将混合液穿过层析柱，穿流2次效果更佳。先用预冷的Lysisbuffer冲洗树脂(Glutathione SepharoseTM 4B，GE公司)，同时流出液用Bradford Assay检测，直至不变蓝色表示杂蛋白冲洗干净。然后，用15mmol/L还原型谷胱甘肽溶液(0.09g溶解于20mL lysis buffer)洗脱目的蛋白，每次加入1mL,层析柱底部用1.5mL的离心管收集，每管约1mL，分别记为E1、E2、E3、E4、E5、E6，直至Bradford Assay检测洗脱液中不含蛋白。未用完的还原型谷胱甘肽溶液继续全部洗脱树脂，然后分别用Lysis buffer，ddH2O 20％乙醇冲洗，保存于20％乙醇中。
10 .收集的蛋白用SDS-PAGE检测，得到69kDa的条带(图1)，GST标签的分子量为26kDa，剩余目的蛋白的分子量为43kDa，与氨基酸计算的分子量相同，说明本实施例制备得到了带有GST标签的目的蛋白。
目的蛋白的氨基酸序列(SEQ ID NO.2)如下：
MLGGLPPLLPSDQLRRKHMVVLVHVQKETGNDVIISTEGLLEAQLELYHNAMAYVKSAVVRAALDLRIPDAIHRRGGAATLSDIATEAGVQPTKVSHLRRLMRALTIFDVFSVHRGTHHDDAIDVHYKLTPLSRLLVGDSSCTQSPIMRVLVDPLSLTALCSIGEWFTDERASALTLFEVAHGCKRDEMTMKKGTRSMFNAGMVSDSRLLMETVIKDHCNIFEGVSSLVDAGGAHGATAEAVTKAFPHIRCTVLDLPHAIDGAPAIGNVEFVAGDLFEYVPPADVVLLKWVLCLWQDEDAVKVLRRCKEAITSRGSKGKVIIIDVVIDSGMSQDDLLLRETQVLFDVQMMRVDGGERDEKEWRKIFIEAGFKDYNITPMLGFRSIIEVYP。
实施例2转基因烟草的构建
本实施例构建含有目标基因（SEQ ID NO.1）的转基因烟草。具体包括如下步骤：
①将含有目标基因的瞬时表达载体(瞬时表达载体pEAQ ，来自John InnesCentre)转化农杆菌(EHA105)；
②挑取阳性农杆菌克隆于500ul含有相应抗生素(kn)的LB中，培养20-24小时；
③转接200ul于5ml含有相应抗生素(kn)的LB中，28℃摇床220rpm至OD＝2.0左右。
④10000rpm常温离心2min收集菌体，用提前配制转化缓冲液进行菌体的重悬，摇床震荡3h；缓冲液工作液成分以及浓度如下：10mM MES(pH5.7)，10mM MgCl2，100μUDP-葡萄糖。
⑤拿准备好的1ml的注射器，去掉针头，选取出口光滑的注射器吸入菌液，取1月龄的本氏烟草(Nicotiana benthamiana)，用手按住叶片，从叶片反面注射，使农杆菌渗透进去。给每株打过的烟草做好标记，可在叶片上圈出农杆菌渗透的区域，并选取转化缓冲液打烟草做对照。
⑥注射了农杆菌的烟草黑暗培养24h，然后移至烟草培养箱中光照培养24-48小时即可取样(注意打过的烟草不可直接在叶片上喷洒水)。
以下通过试验例的方式来说明本发明的有益效果：
试验例1 HOVUSG2104500蛋白的酶活检测
1.方法
1.1 HOVUSG2104500蛋白的获取
实施例1方法制备得到的分子量69kDa的、带有GST标签的目的蛋白。
1.2酶活检测
在Tris-HCl缓冲液(100mM，pH 7.4)中，以含有200μM矢车菊素3-O丙二酰葡萄糖苷作为甲基受体、100μM 硫腺苷甲硫氨酸为甲基供体和500ng纯化蛋白的总体积100μl进行体外甲基转移酶测定。孵育10min后，加入300μL冰冷甲醇，停止反应。然后将反应混合物通过0.2μm的过滤器(微孔)过滤，然后用于LC-MS分析。
2.结果
经过上述催化反应后，将产物通过LC-MS/MS，显示生成的物质是芍药素3-O丙二酰葡萄糖苷(图2)。说明本发明的HOVUSG2104500蛋白具有催化矢车菊素氧甲基基化转换为芍药素3-O丙二酰葡萄糖苷的能力，市场应用前景良好。
试验例2转基因烟草生产芍药素3-O丙二酰葡萄糖苷
1.方法
1.1转基因烟草的构建
按照实施例2构建。
1.2产物收集和纯化
剪取农杆菌渗透区域的叶片，放在已称重的装有钢珠的EP管中，做好标记，迅速放置液氮中，进行冻干。冻干后的样本，利用研磨仪(MM 400,Retsch)在30Hz条件下研磨60s，将研磨好的样本粉末装入2mlEP管内。用电子天平称取每个EP管的重量并记录；将已研磨好的样本取适量(范围30-60mg)于EP管中，称量并记录，算出所有EP管中样本的净重。已知每份样本的净重，按体积V＝样本净重(mg)*12μL/mg在4℃冰上操作加入70％MeOH溶液。混匀，涡旋15s，每隔半小时涡旋一次，共涡旋4次，放4℃冰箱内提取12h以上。后离心。先将离心机开机预冷到4℃，设置时间10min和转速12000rpm，将样本涡旋后放入离心，使用离心机注意对称平衡，离心后吸取上清液。将上清液用微孔滤膜(0.22μm pore size)过滤，装入上样瓶中，准备LC-MS检测。
1.3目的产物检测
将装有样本提取液的进样瓶放入自动进样器内的样品盘，并记录每个进样瓶编号所对应的进样孔位置。同时打开软件Analyst Software，双击Hardware Configuration，选择LCMS-V(有切换阀模式) ，点击Activate Profile，并选择Acquire Mode模式，点击Acquire，点击图上方的Equilibrate键，一般设定时间为3min，此操作目的是预热仪器，使高压输液泵、色谱柱、柱温箱、离子源温度等达到方法中设置的条件。待各仪器部件状态Ready后，功能区Start Sample键成为可点击状态，此时表明仪器正常，分析条件正常，然后点击Start Sample开始跑样，首次跑样前先提交4针空白样。
2.结果
芍药素3-O丙二酰葡萄糖苷的峰度值为0.8E+06(图3)，表明HOVUSG2104500蛋白的活性高。
实验结果说明，本发明将基因HOVUSG2104500转移到烟草中，使得烟草植株可以表达矢车菊素氧甲基转移酶，并诱导烟草积累芍药素3-O丙二酰葡萄糖苷，提高烟草的应用价值，同时为高产芍药素3-O丙二酰葡萄糖苷的青稞的品种制备提供依据。
SEQUENCE LISTING
<110> 西藏自治区农牧科学院农业研究所
<120> 一种青稞矢车菊素氧甲基转移酶基因的新用途
<130> GY462-2021P0114338CC
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1173
<212> DNA
<213> 青稞
<400> 1
atgctaggtg gcctacctcc tctcctccct agtgaccagt tgagaagaaa acatatggtg 60
gtgctggtgc atgtccaaaa agaaactgga aacgatgtga tcatcagcac ggaggggtta 120
ctcgaagctc agcttgagct ctaccataac gccatggcat acgtcaagtc cgcggtggtg 180
agggctgcct tggacctacg catccccgac gccatccacc gtcgcggcgg tgccgccacc 240
ttgtccgata tcgccaccga ggccggggtc cagccgacaa aggtttccca cctccgtcgg 300
ctcatgcgcg ccctcaccat ctttgatgtc ttctcagtcc accggggcac tcaccatgat 360
gatgccatcg acgtgcacta taagctcacc cccctctcgc gcctcctcgt cggggacagc 420
tcgtgcaccc agtcccccat catgcgcgtg ctcgtggacc cgctgtcctt gaccgccctc 480
tgcagtatag gtgagtggtt cacggacgag agggcgtcgg ctctgacact cttcgaagtg 540
gcgcatgggt gcaaacggga tgagatgaca atgaagaagg gcacgcgtag catgttcaac 600
gctggcatgg tctccgatag tcgccttctt atggagaccg tcatcaaaga tcactgcaac 660
atctttgagg gcgttagctc tcttgttgac gccggcggtg ctcatggtgc cacggcggaa 720
gccgtcacta aggcattccc acacatcagg tgcaccgtgt tggacctccc acatgcgatc 780
gacggggcac ctgccatcgg taatgtcgag tttgttgctg gtgatttgtt tgagtatgtg 840
ccaccagcag acgttgttct actcaagtgg gttttgtgct tgtggcaaga tgaagatgct 900
gtcaaggtac tacgacggtg caaagaagca ataacaagta gaggttccaa agggaaggtg 960
ataatcattg atgtcgtgat agactccggg atgtcacagg atgatcttct tcttagggag 1020
acgcaagttc tattcgatgt ccaaatgatg cgtgttgatg ggggcgagcg agacgagaag 1080
gagtggagga agattttcat tgaagccgga ttcaaggatt ataatatcac tccaatgcta 1140
ggatttcgat ccataattga agtctaccct tga 1173
<210> 2
<211> 390
<212> PRT
<213> 青稞
<400> 2
Met Leu Gly Gly Leu Pro Pro Leu Leu Pro Ser Asp Gln Leu Arg Arg
1 5 10 15
Lys His Met Val Val Leu Val His Val Gln Lys Glu Thr Gly Asn Asp
20 25 30
Val Ile Ile Ser Thr Glu Gly Leu Leu Glu Ala Gln Leu Glu Leu Tyr
35 40 45
His Asn Ala Met Ala Tyr Val Lys Ser Ala Val Val Arg Ala Ala Leu
50 55 60
Asp Leu Arg Ile Pro Asp Ala Ile His Arg Arg Gly Gly Ala Ala Thr
65 70 75 80
Leu Ser Asp Ile Ala Thr Glu Ala Gly Val Gln Pro Thr Lys Val Ser
85 90 95
His Leu Arg Arg Leu Met Arg Ala Leu Thr Ile Phe Asp Val Phe Ser
100 105 110
Val His Arg Gly Thr His His Asp Asp Ala Ile Asp Val His Tyr Lys
115 120 125
Leu Thr Pro Leu Ser Arg Leu Leu Val Gly Asp Ser Ser Cys Thr Gln
130 135 140
Ser Pro Ile Met Arg Val Leu Val Asp Pro Leu Ser Leu Thr Ala Leu
145 150 155 160
Cys Ser Ile Gly Glu Trp Phe Thr Asp Glu Arg Ala Ser Ala Leu Thr
165 170 175
Leu Phe Glu Val Ala His Gly Cys Lys Arg Asp Glu Met Thr Met Lys
180 185 190
Lys Gly Thr Arg Ser Met Phe Asn Ala Gly Met Val Ser Asp Ser Arg
195 200 205
Leu Leu Met Glu Thr Val Ile Lys Asp His Cys Asn Ile Phe Glu Gly
210 215 220
Val Ser Ser Leu Val Asp Ala Gly Gly Ala His Gly Ala Thr Ala Glu
225 230 235 240
Ala Val Thr Lys Ala Phe Pro His Ile Arg Cys Thr Val Leu Asp Leu
245 250 255
Pro His Ala Ile Asp Gly Ala Pro Ala Ile Gly Asn Val Glu Phe Val
260 265 270
Ala Gly Asp Leu Phe Glu Tyr Val Pro Pro Ala Asp Val Val Leu Leu
275 280 285
Lys Trp Val Leu Cys Leu Trp Gln Asp Glu Asp Ala Val Lys Val Leu
290 295 300
Arg Arg Cys Lys Glu Ala Ile Thr Ser Arg Gly Ser Lys Gly Lys Val
305 310 315 320
Ile Ile Ile Asp Val Val Ile Asp Ser Gly Met Ser Gln Asp Asp Leu
325 330 335
Leu Leu Arg Glu Thr Gln Val Leu Phe Asp Val Gln Met Met Arg Val
340 345 350
Asp Gly Gly Glu Arg Asp Glu Lys Glu Trp Arg Lys Ile Phe Ile Glu
355 360 365
Ala Gly Phe Lys Asp Tyr Asn Ile Thr Pro Met Leu Gly Phe Arg Ser
370 375 380
Ile Ile Glu Val Tyr Pro
385 390
<210> 3
<211> 23
<212> DNA
<213> 人工合成
<400> 3
atgctaggtg gcctacctcc tct 23
<210> 4
<211> 35
<212> DNA
<213> 人工合成
<400> 4
tcaagggtag acttcaatta tggatcgaaa tccta 35
Claims (9)
1.核苷酸序列如SEQ ID NO.1所示的基因片段在制备芍药素3-O丙二酰葡萄糖苷中的用途。
2.含有核苷酸序列如SEQ ID NO.1所示的基因片段的重组载体在制备芍药素3-O丙二酰葡萄糖苷中的用途。
3.根据权利要求2所述的用途，其特征在于：所述重组载体是重组pGEX-6P-1载体。
4.含有SEQ ID NO.1所示的基因片段的重组菌在制备芍药素3-O丙二酰葡萄糖苷中的用途。
5.根据权利要求4所述的用途，其特征在于：所述重组菌为重组大肠杆菌。
6.根据权利要求5所述的用途，其特征在于：所述重组菌为Transeta(DE3)。
7.氨基酸序列如SEQ ID NO.2所示的蛋白在制备芍药素3-O丙二酰葡萄糖苷中的用途。
8.一种生产芍药素3-O丙二酰葡萄糖苷的转基因植物的构建方法，其特征在于：取核苷酸序列如SEQ ID NO.1所示的基因片段，转入植物中，获得表达氨基酸序列如SEQ ID NO.2所示的蛋白的植株；所述植物为烟草。
9.根据权利要求8所述的构建方法，其特征在于：所述转入植物的方法是农杆菌法、基因枪法、电转法、PEG介导法、脂质体法和磷酸钙-DNA共沉淀法中的一种。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06292574A (ja) * | 1992-03-27 | 1994-10-21 | Toyobo Co Ltd | α−グルコシダーゼ活性を有する蛋白質及びその遺伝情報を有するＤＮＡ並びにα−グルコシダーゼの製造法 |
| JP2003289884A (ja) * | 2002-01-31 | 2003-10-14 | Suntory Ltd | 新規糖転移活性を有する蛋白質をコードする遺伝子 |
| CN103987848A (zh) * | 2011-10-21 | 2014-08-13 | 巴斯夫植物科学有限公司 | 具有增强的产量相关性状的植物及其制备方法 |
| WO2015066609A1 (en) * | 2013-11-01 | 2015-05-07 | Conagen Inc. | Methods of using o-methyltransferase for biosynthetic production of pterostilbene |
| CN108624572A (zh) * | 2017-08-11 | 2018-10-09 | 中国农业科学院生物技术研究所 | 糖苷类化合物的甲基化转移酶基因 |
| CN114790460A (zh) * | 2021-12-09 | 2022-07-26 | 西藏自治区农牧科学院农业研究所 | 一种青稞矢车菊素丙二酰基转移酶基因及其用途 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7214786B2 (en) * | 2000-12-14 | 2007-05-08 | Kovalic David K | Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
| AUPS017402A0 (en) * | 2002-01-25 | 2002-02-14 | International Flower Developments Pty Ltd | Genetic sequences and uses therefor |
| CN101217967B (zh) * | 2005-05-04 | 2014-09-10 | 诺松制药股份公司 | 镜像异构体的新用途 |
-
2021
- 2021-12-06 CN CN202111480017.8A patent/CN114807183B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06292574A (ja) * | 1992-03-27 | 1994-10-21 | Toyobo Co Ltd | α−グルコシダーゼ活性を有する蛋白質及びその遺伝情報を有するＤＮＡ並びにα−グルコシダーゼの製造法 |
| JP2003289884A (ja) * | 2002-01-31 | 2003-10-14 | Suntory Ltd | 新規糖転移活性を有する蛋白質をコードする遺伝子 |
| CN103987848A (zh) * | 2011-10-21 | 2014-08-13 | 巴斯夫植物科学有限公司 | 具有增强的产量相关性状的植物及其制备方法 |
| WO2015066609A1 (en) * | 2013-11-01 | 2015-05-07 | Conagen Inc. | Methods of using o-methyltransferase for biosynthetic production of pterostilbene |
| CN108624572A (zh) * | 2017-08-11 | 2018-10-09 | 中国农业科学院生物技术研究所 | 糖苷类化合物的甲基化转移酶基因 |
| CN114790460A (zh) * | 2021-12-09 | 2022-07-26 | 西藏自治区农牧科学院农业研究所 | 一种青稞矢车菊素丙二酰基转移酶基因及其用途 |
Non-Patent Citations (3)
| Title |
|---|
| methyltransferase ZRP4 [Hordeum vulgare];Nyima,T.等;GenBank Database;Accession No.KAE8803999.1 * |
| Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin O-methyltransferase of Nemophila menziesii Hook;Arn.，Okitsu, N等;PLANT BIOTECHNOLOGY;第35卷(第1期);第9-16页 * |
| 海拔高度对青钱柳代谢产物的影响及生态位预测研究;余斌斌;中国硕士学位论文全文数据库;摘要 * |
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