CN114807184B - 一种青稞矢车菊素5-氧糖基转移酶的用途 - Google Patents
一种青稞矢车菊素5-氧糖基转移酶的用途 Download PDFInfo
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- CN114807184B CN114807184B CN202111536444.3A CN202111536444A CN114807184B CN 114807184 B CN114807184 B CN 114807184B CN 202111536444 A CN202111536444 A CN 202111536444A CN 114807184 B CN114807184 B CN 114807184B
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Abstract
本发明提供了一种青稞矢车菊素5‑氧糖基转移酶的用途，涉及基因工程技术领域。该青稞矢车菊素5‑氧糖基转移酶是如SEQ ID NO.2所示的氨基酸序列。表达该青稞矢车菊素5‑氧糖基转移酶的基因片段如SEQ ID NO.1所示。本发明矢车菊素5‑氧糖基转移酶基因可以用UDP‑葡萄糖作为糖基供体、矢车菊素3‑O‑葡萄糖苷作为糖基受体，合成矢车菊素3,5‑O‑双葡萄糖苷。将该基因片段，以及含有该基因片段的重组载体、重组菌，以及该基因片段表达的蛋白植入植物中，可以提高植物的应用价值，具有优良的应用前景。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种青稞矢车菊素5-氧糖基转移酶的用途。
背景技术
花青素，又称花色素，是自然界一类广泛存在于植物中的天然色素，是花色苷水解而得的有颜色的苷元。花青素具有优异的生物活性，其不仅具有抗氧化、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。
花青素骨架物质，如矢车菊素、芍药素等在植物中不稳定，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。有报道研究发现糖基化修饰后花青素不仅可以增加花青素的稳定性还可以增加花青素在溶液中的溶解度，更有利于人们对花青素的吸收利用。但是，目前对负责花青素的修饰的基因还鲜有报道。如何通过基因修饰提高植物中花青素稳定性，增加花青素含量还没有相关研究。
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。
目前国内外对青稞种子花青素的合成和修饰的机理了解甚少。如何对青稞进行基因修饰，对青稞进行定向改良，提高其花青素含量。对提高青稞价值具有重要意义。
发明内容
本发明提供了一种青稞矢车菊素5-氧糖基转移酶的用途。本发明通过对青稞的研究，发现了一种青稞矢车菊素糖基转移酶基因，其可以有效将矢车菊素3-O葡萄糖苷和葡萄糖转化为矢车菊素3,5-O-双葡萄糖苷。
本发明提供了核苷酸序列如SEQ ID NO.1所示的基因片段在制备矢车菊素3,5-O-双葡萄糖苷中的用途。
本发明还提供了含有核苷酸序列如SEQ ID NO.1所示的基因片段的重组载体在制备矢车菊素3,5-O-双葡萄糖苷中的用途；
优选地，所述重组载体是重组pGEX-6P-1(Novagen)。
本发明还提供了含有核苷酸序列如SEQ ID NO.1所示的基因片段的重组菌在制备矢车菊素3,5-O-双葡萄糖苷中的用途；
优选地，所述重组菌为Transetta(DE3)。
本发明还提供了含有携带核苷酸序列如SEQ ID NO.1所示的基因片段的重组质粒的重组菌在制备矢车菊素3,5-O-双葡萄糖苷中的用途；
优选地，所述重组菌为Transetta(DE3)。
进一步地，所述重组菌为重组农杆菌；优选为重组农杆菌EHA105。
本发明还提供了氨基酸序列如SEQ ID NO.2所示的蛋白在制备矢车菊素3,5-O-双葡萄糖苷中的用途。
本发明还提供了一种制备矢车菊素3,5-O-双葡萄糖苷的方法，它是采用前述的基因片段、重组载体、重组菌、蛋白，以葡萄糖作为糖基供体，矢车菊素3-O-葡萄糖苷作为糖基受体，制备矢车菊素3,5-O-双葡萄糖苷。
本发明还提供了一种生产矢车菊素3,5-O-双葡萄糖苷的转基因植物的构建方法，取前述的基因片段、重组载体、重组菌、蛋白，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。
进一步地，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；
和/或，所述转基因植物为转基因烟草。
本发明还提供了前述的基因片段、重组载体、重组菌、蛋白在制备高产矢车菊素3,5-O-双葡萄糖苷的青稞品种的用途。
3,5-O-双葡萄糖苷的积累可以增加花青素在植物中的稳定性，同时可以增加花青素在溶液中的溶解度，利于人们对花青素的吸收利用。而本发明发现青稞中的一种基因：青稞矢车菊素5-氧糖基转移酶基因，该基因表达的蛋白——青稞矢车菊素5-氧糖基转移酶，可以将矢车菊素3-O葡萄糖苷转化为矢车菊素3,5-O-双葡萄糖苷，增加青稞中矢车菊素3,5-O-双葡萄糖苷的积累，提高青稞的保健价值。本发明还以该基因片段进行体外表达，获得了青稞矢车菊素5-氧糖基转移酶，在体外反应中，成功地将葡萄糖作为糖基供体，矢车菊素3-O葡萄糖苷作为受体，制备得到了矢车菊素3,5-O-双葡萄糖苷；本发明还将该基因转移到烟草中，使得烟草植株中也表达矢车菊素5-氧糖基转移酶，进一步产生矢车菊素3,5-O-双葡萄糖苷，提高其价值。本发明提供的新的基因，及其重组载体、重组菌、转基因植物均具有良好的应用前景。
本发明的基因片段和重组载体，可以用来提高青稞对矢车菊素3,5-O-双葡萄糖苷的合成，实现青稞的定向改良。本发明的转基因烟草构建方法，为青稞的定向改良提供了重要的参考。
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HOVUSG3539700蛋白的SDS-PAGE电泳图，Marker：100,70,55,40,35,25KDa。
图2为体外催化反应的LC-MS图：Cyanidin 3-O-galactoside，矢车菊素3-O葡萄糖苷；Cyanidin 3,5-O-diglucoside，矢车菊素3,5-O-双葡萄糖苷。
图3为转基因植物提取物中矢车菊素3,5-O-双葡萄糖苷的质谱图。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品，通过购买市售产品获得。
实施例1、HOVUSG3539700基因的分离以及原核表达
本实施例主要是HOVUSG3539700基因获得、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。
进行PCR扩增后，提纯蛋白，得到目的条带大小的片段。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HOVUSG3539700基因的核苷酸序列(SEQ ID NO.1)为：
ATGGCCACCAACGACAAGCCGCACGCCGTCTTCGTGCCGTTCCCGGCG
CAGGGGCACGTCACGCCGATGATGAAGCTAGCCAAGGTCCTCCACCGC
AAGGGCTTCCATGTCACCTTTGTCAACACCGAGTACAACCAGCGCCGC
CTTGTCCGCTCCCGCGGCCCCGACGCCGTGGCCGGCCTCCCGGACTTC
CGCTTCGCCACCATCCCAGACGGCCTGCCCACGTCCAAAGCAGACGCC
GACGCTGACGCCACGCAGGACCCGCCGTCCCTTTGCTACTACACCATG
ACCACCTGCCTCCCCCATTTGAAGAACCTGCTCCGCGACCTCAACGCC
GCCGTTGGGGCGCCGTCGGTCAGCTGCGTCGTGGGTGACGGCGTCATG
AGCTTCTGCGTGGACGCGGCCGCGGAGCTCGGCGTGCCGTGCGCGCTG
TTCTGGACTGCCAGCGCCTGCGGCTTCATGGGCTACCGCAACTTCCGGT
TCCTCCTAGACGAGGGCCTCACCCCTCTCAAAGACGAAGAGCAAGTGA
AGAACGGGTACCTGGACACGCCGGTGACGCAGGCACGTGGGATGAGC
AAGCACATGCGCCTCCGAGACTTCTCCTCCTTCGTCCGCACCACGGAC
CGCAGCGACATCCTCTTCAACTTCCTGCTGCACGAGGTCGAGCAGTCG
GATCGCGCGACCGCCATCGTCATCAACACCATTGACGAGCTCGAGCAG
ACGGCGCTCGACGCCATGCGCGCCATCCTCCCCGTGCCCGTCTACACCA
TCGGCCCGCTTAACTTCCTCACCCAGCAGCTGGTCTCAGAAGGCGATG
GCGGCGGAAGCGAGCTCGCGGCGATGCGCTCCAGCCTCTGGAGAGAA
GATCAGTCATGTCTCGAGTGGCTCCAGGGCAGGGAGCCGCGGTCCGTG
GTGTACGTCAACTACGGGAGCGTGACCACCATGTCGAAGCAGGAGCTG
GTGGAGTTCGCGTGGGGACTGGCCAACTGCGGCTACGACTTCCTCTGG
ATCGTGAGGAACGACCTGGTGAAGGGCGATGCCGCCGTGCTGCCTCCC
GAGTTCATCGAGGCCACCAAGGGCAGATGCCTCCTGGCAAGCTGGTGC
GAGCAGGAGGCGGTCATGCGTCACGAGGCGGTGGGCGCCTTCTTGACG
CACTGCGGGTGGAACTCCATGATGGAGGGGCTCGGCGCCGGCGTGCCG
ATGCTCTGCTGGCCCTTCTTCGCCGAGCAGCAGACAAACAGCCGCTAT
GCGTGTGTGGAGTGGGGCGTTGGGATGGAGGTCGGCGATGATGTGCGT
CGGGTGGTGGTCGAGGCGAGGATAAGGGAGGTGATGGGAGGGGGAGA
AGTAGGAAGGGAGATGCGGAGGAGGGCGGCAGAGTGGAAGGAGGTC
GCTTCTCGCTCGACCGCGCAGCCTGGTGGCCGGTCGTTGGCCAACCTT
GAGAGTCTGCTCAAAGATGTACTGAAGTGA
前述核苷酸序列所述的HOVUSG3539700基因可以通过采用上述方法获得，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
①PCR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2μL 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。
⑤50mL Lysis buffer重悬菌体，涡旋混匀，转入50mL离心管中，分别加入50μLPMSF，10μLβ-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20μl作为总蛋白样品。再取1mL样品4℃，13000rpm离心10min，取20μL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。
⑧SDS-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。
⑨GST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80kDa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54kDa，与氨基酸计算的分子量相同，说明本发明制备得到了带有GST标签的目的蛋白。
目的蛋白的氨基酸序列(SEQ ID NO.2)如下：
MATNDKPHAVFVPFPAQGHVTPMMKLAKVLHRKGFHVTFVNTEYNQRR
LVRSRGPDAVAGLPDFRFATIPDGLPTSKADADADATQDPPSLCYYTMTTC
LPHLKNLLRDLNAAVGAPSVSCVVGDGVMSFCVDAAAELGVPCALFWT
ASACGFMGYRNFRFLLDEGLTPLKDEEQVKNGYLDTPVTQARGMSKHM
RLRDFSSFVRTTDRSDILFNFLLHEVEQSDRATAIVINTIDELEQTALDAMR
AILPVPVYTIGPLNFLTQQLVSEGDGGGSELAAMRSSLWREDQSCLEWLQ
GREPRSVVYVNYGSVTTMSKQELVEFAWGLANCGYDFLWIVRNDLVKGD
AAVLPPEFIEATKGRCLLASWCEQEAVMRHEAVGAFLTHCGWNSMMEGL
GAGVPMLCWPFFAEQQTNSRYACVEWGVGMEVGDDVRRVVVEARIREV
MGGGEVGREMRRRAAEWKEVASRSTAQPGGRSLANLESLLKDVLK
实施例2、转基因烟草的构建
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500μl含有相应抗生素(kn)的LB中，培养20-24小时；
③转接200μl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 MgCl2，100μM UDP-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、HOVUSG3539700蛋白的酶活检测
1.方法
1.1HOVUSG3539700蛋白的获取
实施例1方法制备得到的分子量80kDa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-葡萄糖苷作为糖基受体、100μM UDP-葡萄糖为糖基供体和500ng纯化蛋白的总体积100μl进行体外糖基转移酶测定。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，显示生成的物质是矢车菊素3,5-O-双葡萄糖苷(图2)。说明本发明HOVUSG3539700蛋白具有催化矢车菊素3-O-葡萄糖苷糖基化转换为矢车菊素3,5-O-双葡萄糖苷的能力，市场应用前景良好。
试验例2、转基因烟草生产矢车菊素3,5-O-双葡萄糖苷
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2ml EP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
矢车菊素3,5-O-双葡萄糖苷的峰度值为2.5E+06(图3)，表明HOVUSG3539700蛋白的活性高。
实验结果说明，本发明将基因HOVUSG3539700转移到烟草中，使得烟草植株可以表达矢车菊素5-氧糖基转移酶基因，并诱导烟草积累矢车菊素3,5-O-双葡萄糖苷，提高烟草的应用价值，同时为高产矢车菊素3,5-O-双葡萄糖苷的青稞的品种制备提供依据。
综上，本发明提供了一种基因片段在制备矢车菊素3,5-O-双葡萄糖苷的用途，其核苷酸序列如SEQ ID NO.1所示。本发明还提供了包含前述基因片段的重组载体、重组菌在制备矢车菊素3,5-O-双葡萄糖苷的用途。以及该基因片段表达的SEQ ID NO.2所示的蛋白制备矢车菊素3,5-O-双葡萄糖苷的用途。本发明通过在植物中转入该基因片段，不仅可以增加花青素的稳定性还可以增加花青素在溶液中的溶解度，更有利于人们对花青素的吸收利用。
SEQUENCE LISTING
<110> 西藏自治区农牧科学院农业研究所
西藏自治区农牧科学院农业资源与环境研究所
<120> 一种青稞矢车菊素5-氧糖基转移酶的用途
<130> GY462-2021P0114335CC
<140> 2021115364443
<141> 2021-12-06
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1467
<212> DNA
<213> 人工序列
<400> 1
atggccacca acgacaagcc gcacgccgtc ttcgtgccgt tcccggcgca ggggcacgtc 60
acgccgatga tgaagctagc caaggtcctc caccgcaagg gcttccatgt cacctttgtc 120
aacaccgagt acaaccagcg ccgccttgtc cgctcccgcg gccccgacgc cgtggccggc 180
ctcccggact tccgcttcgc caccatccca gacggcctgc ccacgtccaa agcagacgcc 240
gacgctgacg ccacgcagga cccgccgtcc ctttgctact acaccatgac cacctgcctc 300
ccccatttga agaacctgct ccgcgacctc aacgccgccg ttggggcgcc gtcggtcagc 360
tgcgtcgtgg gtgacggcgt catgagcttc tgcgtggacg cggccgcgga gctcggcgtg 420
ccgtgcgcgc tgttctggac tgccagcgcc tgcggcttca tgggctaccg caacttccgg 480
ttcctcctag acgagggcct cacccctctc aaagacgaag agcaagtgaa gaacgggtac 540
ctggacacgc cggtgacgca ggcacgtggg atgagcaagc acatgcgcct ccgagacttc 600
tcctccttcg tccgcaccac ggaccgcagc gacatcctct tcaacttcct gctgcacgag 660
gtcgagcagt cggatcgcgc gaccgccatc gtcatcaaca ccattgacga gctcgagcag 720
acggcgctcg acgccatgcg cgccatcctc cccgtgcccg tctacaccat cggcccgctt 780
aacttcctca cccagcagct ggtctcagaa ggcgatggcg gcggaagcga gctcgcggcg 840
atgcgctcca gcctctggag agaagatcag tcatgtctcg agtggctcca gggcagggag 900
ccgcggtccg tggtgtacgt caactacggg agcgtgacca ccatgtcgaa gcaggagctg 960
gtggagttcg cgtggggact ggccaactgc ggctacgact tcctctggat cgtgaggaac 1020
gacctggtga agggcgatgc cgccgtgctg cctcccgagt tcatcgaggc caccaagggc 1080
agatgcctcc tggcaagctg gtgcgagcag gaggcggtca tgcgtcacga ggcggtgggc 1140
gccttcttga cgcactgcgg gtggaactcc atgatggagg ggctcggcgc cggcgtgccg 1200
atgctctgct ggcccttctt cgccgagcag cagacaaaca gccgctatgc gtgtgtggag 1260
tggggcgttg ggatggaggt cggcgatgat gtgcgtcggg tggtggtcga ggcgaggata 1320
agggaggtga tgggaggggg agaagtagga agggagatgc ggaggagggc ggcagagtgg 1380
aaggaggtcg cttctcgctc gaccgcgcag cctggtggcc ggtcgttggc caaccttgag 1440
agtctgctca aagatgtact gaagtga 1467
<210> 2
<211> 488
<212> PRT
<213> 人工序列
<400> 2
Met Ala Thr Asn Asp Lys Pro His Ala Val Phe Val Pro Phe Pro Ala
1 5 10 15
Gln Gly His Val Thr Pro Met Met Lys Leu Ala Lys Val Leu His Arg
20 25 30
Lys Gly Phe His Val Thr Phe Val Asn Thr Glu Tyr Asn Gln Arg Arg
35 40 45
Leu Val Arg Ser Arg Gly Pro Asp Ala Val Ala Gly Leu Pro Asp Phe
50 55 60
Arg Phe Ala Thr Ile Pro Asp Gly Leu Pro Thr Ser Lys Ala Asp Ala
65 70 75 80
Asp Ala Asp Ala Thr Gln Asp Pro Pro Ser Leu Cys Tyr Tyr Thr Met
85 90 95
Thr Thr Cys Leu Pro His Leu Lys Asn Leu Leu Arg Asp Leu Asn Ala
100 105 110
Ala Val Gly Ala Pro Ser Val Ser Cys Val Val Gly Asp Gly Val Met
115 120 125
Ser Phe Cys Val Asp Ala Ala Ala Glu Leu Gly Val Pro Cys Ala Leu
130 135 140
Phe Trp Thr Ala Ser Ala Cys Gly Phe Met Gly Tyr Arg Asn Phe Arg
145 150 155 160
Phe Leu Leu Asp Glu Gly Leu Thr Pro Leu Lys Asp Glu Glu Gln Val
165 170 175
Lys Asn Gly Tyr Leu Asp Thr Pro Val Thr Gln Ala Arg Gly Met Ser
180 185 190
Lys His Met Arg Leu Arg Asp Phe Ser Ser Phe Val Arg Thr Thr Asp
195 200 205
Arg Ser Asp Ile Leu Phe Asn Phe Leu Leu His Glu Val Glu Gln Ser
210 215 220
Asp Arg Ala Thr Ala Ile Val Ile Asn Thr Ile Asp Glu Leu Glu Gln
225 230 235 240
Thr Ala Leu Asp Ala Met Arg Ala Ile Leu Pro Val Pro Val Tyr Thr
245 250 255
Ile Gly Pro Leu Asn Phe Leu Thr Gln Gln Leu Val Ser Glu Gly Asp
260 265 270
Gly Gly Gly Ser Glu Leu Ala Ala Met Arg Ser Ser Leu Trp Arg Glu
275 280 285
Asp Gln Ser Cys Leu Glu Trp Leu Gln Gly Arg Glu Pro Arg Ser Val
290 295 300
Val Tyr Val Asn Tyr Gly Ser Val Thr Thr Met Ser Lys Gln Glu Leu
305 310 315 320
Val Glu Phe Ala Trp Gly Leu Ala Asn Cys Gly Tyr Asp Phe Leu Trp
325 330 335
Ile Val Arg Asn Asp Leu Val Lys Gly Asp Ala Ala Val Leu Pro Pro
340 345 350
Glu Phe Ile Glu Ala Thr Lys Gly Arg Cys Leu Leu Ala Ser Trp Cys
355 360 365
Glu Gln Glu Ala Val Met Arg His Glu Ala Val Gly Ala Phe Leu Thr
370 375 380
His Cys Gly Trp Asn Ser Met Met Glu Gly Leu Gly Ala Gly Val Pro
385 390 395 400
Met Leu Cys Trp Pro Phe Phe Ala Glu Gln Gln Thr Asn Ser Arg Tyr
405 410 415
Ala Cys Val Glu Trp Gly Val Gly Met Glu Val Gly Asp Asp Val Arg
420 425 430
Arg Val Val Val Glu Ala Arg Ile Arg Glu Val Met Gly Gly Gly Glu
435 440 445
Val Gly Arg Glu Met Arg Arg Arg Ala Ala Glu Trp Lys Glu Val Ala
450 455 460
Ser Arg Ser Thr Ala Gln Pro Gly Gly Arg Ser Leu Ala Asn Leu Glu
465 470 475 480
Ser Leu Leu Lys Asp Val Leu Lys
485
Claims (14)
1.核苷酸序列如SEQ ID NO.1所示的基因片段在制备矢车菊素3,5-O-双葡萄糖苷中的用途。
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矢车菊素3,5-O-双葡萄糖苷中的用途。
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。
4.含有核苷酸序列如SEQ ID NO.1所示的基因片段的重组菌在制备矢车菊素3,5-O-双葡萄糖苷中的用途。
5.根据权利要求4所述的用途，其特征在于：所述重组菌为Transetta(DE3)。
6.含有携带核苷酸序列如SEQ ID NO.1所示的基因片段的重组质粒的重组菌在制备矢车菊素3,5-O-双葡萄糖苷中的用途。
7.根据权利要求6所述的用途，其特征在于：所述重组菌为Transetta(DE3)。
8.根据权利要求6所述的用途，其特征在于：所述重组菌为重组农杆菌。
9.根据权利要求8所述的用途，其特征在于：所述重组农杆菌为重组农杆菌EHA105。
10.氨基酸序列如SEQ ID NO.2所示的蛋白在制备矢车菊素3,5-O-双葡萄糖苷中的用途。
11.一种制备矢车菊素3,5-O-双葡萄糖苷的方法，其特征在于：它是采用氨基酸序列如SEQ ID NO.2所示的蛋白，以UDP-葡萄糖作为糖基供体，矢车菊素3-O-葡萄糖苷作为糖基受体，制备矢车菊素3,5-O-双葡萄糖苷。
12.一种生产矢车菊素3,5-O-双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 IDNO.2所示的蛋白的植株，即可；
所述转基因植物为转基因烟草。
13.根据权利要求12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。
14.核苷酸序列如SEQ ID NO.1所示的基因片段、氨基酸序列如SEQ ID NO.2所示的蛋白在制备高产矢车菊素3,5-O-双葡萄糖苷的青稞品种的用途。
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