CN110872594B - 黑莓糖基转移酶基因及其应用 - Google Patents
黑莓糖基转移酶基因及其应用 Download PDFInfo
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- CN110872594B CN110872594B CN201911254551.XA CN201911254551A CN110872594B CN 110872594 B CN110872594 B CN 110872594B CN 201911254551 A CN201911254551 A CN 201911254551A CN 110872594 B CN110872594 B CN 110872594B
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Abstract
本发明公开了黑莓糖基转移酶基因及其应用，UGT78H2基因编码的氨基酸序列如SEQ ID No.1所示，突变基因mUGT78H2基因编码的氨基酸序列如SEQ ID No.2所示。本发明提供的UGT78H2基因和以及通过UGT78H2基因进行点位突变得到的mUGT78H2基因具有重要的应用价值，将上述基因通过表达载体，转化至宿主菌进行表达，可快速获得重组UGT78H2蛋白和mUGT78H2蛋白，实现体外重组蛋白的应用，提高蛋白生产效率；同时上述基因赋予植物对UDP‑活化糖和槲皮素的特异性糖基化反应，因此利用重组UGT78H2蛋白和mUGT78H2蛋白可以实现槲皮素糖苷的定向生产应用，适用于蔷薇科悬钩子属的野生黑树莓插田泡与栽培黑莓无刺品种。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及黑莓糖基转移酶基因及其应用。
背景技术
糖基转移酶(Glycosyltransferases，GTs，EC 2.4.X.Y)是广泛分布于所有生物的功能高度分化的一类蛋白质(秦晶晶等,2018；Li et al.,2018)。它可催化糖基自活化的供体小分子转移至对应的受体小分子。糖基转移酶主要通过改变受体小分子的水溶性、稳定性、在细胞内和植株中的运输特性、与受体的识别特性，从而参与植物激素平衡的调节、次生代谢产物的修饰、内外源毒性物质的解毒以及防御反应等生物学进程(Lim and Bowles,2004；Xiao et al.,2014)。因此，对糖基转移酶进行结构与功能的分析与研究，是了解植物生长发育规律的基础，在次生代谢产物定向分子设计、药物开发生产以及育种实践上均具有重要的理论价值和实际意义。
在GTs的106个超家族(http://www.cazy.org/GlycosylTransferases.html)中，GT1的成员最多，包含了来源于古细菌、细菌、病毒以及真核生物的序列。GT1家族的成员通常以UDP活化糖(如UDP-葡萄糖、UDP-阿拉伯糖、UDP-木糖、UDP-鼠李糖以及UDP-半乳糖)为供体，因此该家族的成员又被称为尿苷二磷酸糖基转移酶(UDP-glycosyltransferases，UGTs)(陈清等,2015)。
UGTs命名标准为：最前面的阿拉伯数字表示家族，阿拉伯数字后的字母表示亚家族，字母后再用阿拉伯数字表示某个特定的糖基转移酶。其中，UGT71-UGT100家族为植物源，若现有的编号全部用尽，则新发现UGT的编号将以10倍扩大(Campbell et al.,1997；Mackenzie et al.,1997)。同一家族成员之间的氨基酸序列一致性不小于45％，同一亚家族成员之间的氨基酸序列一致性不小于60％。如果UGT的编号后带有标记“P”，即表示该基因为假基因。
与其他超家族成员不同，植物UGT大都含有折叠形成的GT-B空间结构：包含由链接(linker)相连的两个独立的Rossmann折叠结构域，两个结构域之间的裂沟构成了底物结合“口袋”，当前的研究也大都集中在底物结合“口袋”中氨基酸残基与底物识别位点的互作上(Elejalde-Palmett et al.,2019)。
植物UGT家族成员众多，在蛋白质一级结构上差异较大。在不同物种中，同一类群的植物UGT数量差异极大，功能的冗余或者新功能的获得难以预料(Yonekura-Sakakibaraet al.,2011)。例如，对于同为Group F的植物UGT，来自拟南芥的UGT78D1以UDP-鼠李糖为供体(Jones et al.,2003)，同一亚家族拟南芥的UGT78D2和UGT78D3主要以UDP-阿拉伯糖为供体(Yonekura-Sakakibara et al.,2008)，而同一家族当归(Angelica sinensis(Oliv.)Diels)的UGT78A2却以UDP-半乳糖为供体(Nagashima et al.,2004)。
植物糖基转移酶对糖基供体的选择性：植物UGT的C末端含有一段由44个氨基酸组成的高度保守的序列，被称为植物次生代谢产物糖基转移酶保守区(PSPG结构域)(Noguchiet al.,2009)。研究表明，PSPG保守结构域的几个特定氨基酸残基在选择糖基供体的种类、糖基位点以及转移效率上起重要作用，因为某些氨基酸残基定点突变后会导致酶活性改变或者完全丧失(Modolo et al.,2009b；Adepoju et al.,2014)，但尚未确定其中某个氨基酸决定某种糖基供体的一一对应关系。除PSPG结构域以外，在活性“口袋”中能和糖基供体发生接触或相互作用的其他氨基酸也会影响植物UGT对糖基供体的特异性识别，这一特性在多功能性植物UGT蛋白中尤为突出(
et al.,2005；He et al.,2006)。另有研究发现，植物UGT蛋白N端序列也可能参与对糖基供体的识别(Kim et al.,2013；King,2016)。除此之外，植物UGT蛋白中双结合氨基酸位点的存在，增加了植物UGT对底物的识别机制的复杂性(Dai et al.,2017)。因此，植物UGT对糖基供体的特异性识别机制仍有待研究。
植物糖基转移酶对糖基受体的选择性：植物UGT蛋白窄而深的“口袋”结构是结合糖基受体的主要区域。相比供体识别区，植物UGT受体识别区的保守特征不明显，空间结构的多样性对应于糖基受体种类的多样性。关于N端氨基酸的突变研究比PSPG结构域的突变研究更少(Noguchi et al.,2007；Ghose et al.,2015)。研究植物UGT对糖基受体的特异性识别机制，还包括对糖基受体的区域选择性(Regioselectivity)的确定，即对于一个特定的糖基受体，当多个受体位点同时存在时，其催化反应如何识别和选择。值得注意的是，目前发现的植物UGT对糖基受体的特异性选择绝大部分都是通过重组蛋白体外催化反应确定的，这些试验结果和植物UGT在植物体内的功能是否一致仍需谨慎对待(Terasaka et al.,2012)。植物UGT对糖基受体区域选择性的识别机制的解析仍需更多研究。
黑莓作为一种特种经济果树，因其结果期早、见效快，经济效益显著，已被很多国家和地区引种栽培，其种植面积和产量逐年增加。对野生资源进行筛选，是进行黑莓和插田泡品种选育的传统方法。传统方法的劣势在于：育种周期长，工作量大。利用现代生物技术手段对现有的黑莓和插田泡品种进行遗传改良以培育新的具有自主知识产权的新品种是重要的解决方法。
在关于黑莓果实发育过程中类黄酮代谢的生理和分子机理研究中，发现了一个糖基转移酶基因RuGT1，命名为UGT78H2。生物信息学分析表明该酶具有植物UGT特有的PSPG保守结构域，但是UGT78H2蛋白在PSPG结构域中保守氨基酸残基第3位天冬酰胺(即整个氨基酸序列中中第340位)和第23位的赖氨酸(即整个氨基酸氨基酸中第360位)与其他同为Group F的蛋白不一致(如图1所示)(胡玥阳，2017)。
关于植物UGT蛋白PSPG结构域内关键氨基酸位点的研究，大都集中于第350位脯氨酸Pro(Noguchi et al.,2009)、第392位谷氨酸Glu(Noguchi et al.,2007)、第391位丝氨酸Ser(
et al.,2005)、第355位色氨酸(Ghose et al.,2015)、第382位谷氨酰胺Gln(Kubo et al.,2004)、第381谷氨酸Glu(Shao et al.,2005)、第377位半胱氨酸Cys(Masada et al.,2007)以及第389位苏氨酸Thr(Yang et al.,2014)。UGT78H2在氨基酸关键位点的差异对其功能的影响是不清楚的，因此对黑莓UGT78H2蛋白的第340位天冬酰胺Asn和第360位赖氨酸Lys进行定点突变，有利于研究黑莓mUGT78H2蛋白对底物的特异性识别机制。
发明内容
综上所述，本发明目的在于提供黑莓糖基转移酶基因UGT78H2基因和mUGT78H2基因，通过将基因连接至表达载体，转化至宿主进行表达，可快速获得重组UGT78H2蛋白和mUGT78H2蛋白，实现蛋白的高效生产，再利用重组UGT78H2蛋白和mUGT78H2蛋白可以实现槲皮素糖苷的定向生产应用。
本发明通过下述技术方案实现：
黑莓糖基转移酶基因的突变基因，所述突变基因为mUGT78H2基因，其编码的氨基酸序列如SEQ ID No.2所示，所述mUGT78H2基因为将UGT78H2基因编码蛋白的PSPG结构域中第3位天冬酰胺突变为脯氨酸，第23位赖氨酸突变为天冬酰胺的基因；所述UGT78H2基因编码的氨基酸序列如SEQ ID No.1所示。
基于突变基因的重组蛋白，所述重组蛋白为mUGT78H2蛋白，其氨基酸序列如SEQID NO.2所示
基于UGT78H2基因和mUGT78H2基因的应用，用于体外获取重组蛋白。
基于UGT78H2蛋白和mUGT78H2蛋白的应用，用于生产槲皮素糖苷。
基于UGT78H2基因和mUGT78H2基因应用，包括以下步骤：
步骤A，克隆得到黑莓UGT78H2基因；
步骤B，设计特异引物，采用OE-PCR技术，将UGT78H2基因中用于UGT78H2蛋白PSPG结构域中第3位表达为天冬酰胺的基因片段突变为表达为脯氨酸的基因片段，第23位表达为赖氨酸基因片段突变为天冬酰胺基因片段，从而获得mUGT78H2基因；
步骤C，将UGT78H2基因或者mUGT78H2基因通过表达载体转入宿主进行表达获取重组蛋白。
采用本发明提供的UGT78H2基因和以及通过UGT78H2基因进行点位突变得到的mUGT78H2基因具有重要的应用价值，将上述基因通过表达载体，转化至宿主进行表达，可快速获得重组UGT78H2蛋白和mUGT78H2蛋白，实现体外重组蛋白的应用，提高蛋白生产效率；同时上述基因赋予植物对UDP-活化糖和槲皮素的特异性糖基化反应，因此利用重组UGT78H2蛋白和mUGT78H2蛋白可以实现槲皮素糖苷的定向生产应用，适用于蔷薇科悬钩子属的野生黑树莓插田泡与栽培黑莓无刺品种。
本发明与现有技术相比，具有如下的优点和有益效果：
本发明的UGT78H2基因和mUGT78H2基因，连接至表达载体，转化烟草或者其他生物反应器，可快速获得重组UGT78H2和mUGT78H2蛋白，再利用重组UGT78H2和mUGT78H2蛋白实现槲皮素-4'-O-葡萄糖苷、槲皮素-3-O-半乳糖苷、槲皮素-7-O-葡糖醛苷的定向生产。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解，构成本申请的一部分，并不构成对本发明实施例的限定。在附图中：
图1为Group F进化组部分成员的PSPG结构域特征。
图2为UGT78H2和mUGT78H2蛋白纯化后的免疫印迹检测。
图3为UGT78H2和mUGT78H2催化的糖基化反应结果检测图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例1
黑莓UGT78H2基因与突变mUGT78H2基因的获取：1)设计特异性引物(SEQ ID NO.3和SEQ ID NO.4)，利用PCR技术，以黑莓叶片总DNA为模板，克隆得到黑莓UGT78H2基因的全长序列。UGT78H2基因DNA长度为1377bp；2)以克隆得到的黑莓UGT78H2基因全长序列，设计三对即六个特异DNA引物(如序列表中SEQ ID NO.5-SEQ ID NO.10)，采用OE-PCR技术，将UGT78H2的PSPG结构域中第3位天冬酰胺和第23位赖氨酸同时突变为脯氨酸和天冬酰胺，从而获得突变mUGT78H2基因的全长序列。
实施例2
UGT78H2和mUGT78H2的序列获取：委托生物公司化学合成UGT78H2和mUGT78H2的全基因序列。
实施例3
UGT78H2和mUGT78H2的蛋白质序列：黑莓UGT78H2基因编码的蛋白质序列如序列表中SEQ ID NO.1所示，突变mUGT78H2基因编码的蛋白质序列如序列表中SEQ ID NO.2所示。
黑莓UGT78H2基因编码的一个蛋白多肽由459个氨基酸残基组成，分子量为50.6kD，等电点为5.88。利用NCBI的CDD数据库分析表明，UGT78H2蛋白具有植物次生代谢产物糖基转移酶保守区(PSPG结构域)，UGT78H2蛋白属于植物糖基转移酶。mUGT78H2仅将UGT78H2的PSPG保守结构域的第3位天冬酰胺和第23位赖氨酸同时突变为脯氨酸和天冬酰胺，因此，mUGT78H2也属于植物糖基转移酶。
实施例4
体外获取UGT78H2重组蛋白：将黑莓UGT78H2基因克隆到pEAQ-HT等植物转化载体(序列表SEQ ID NO.11和SEQ ID NO.12)，获得pEAQ-UGT重组载体，将重组载体导入GV3101等农杆菌菌株中，用于转化适龄本氏烟草。在转化后第9天，采收烟草叶片，提取叶片总蛋白，经纯化浓缩，以SDS-PAGE电泳与蛋白质免疫印迹检测重组蛋白的表达情况。如图2所示，这说明可以利用常规方法在体外获取黑莓UGT78H2重组蛋白，其中，图中1-4分别为4次重复样本的检测结果。
实施例5
体外获取mUGT78H2重组蛋白：将黑莓突变mUGT78H2基因克隆到pEAQ-HT等植物转化载体(序列表SEQ ID NO.11和SEQ ID NO.12)，获得pEAQ-mUGT重组载体，将重组载体导入GV3101等农杆菌菌株中，用于转化适龄本氏烟草。在转化后第9天，采收烟草叶片，提取叶片总蛋白，经纯化浓缩，以SDS-PAGE电泳与蛋白质免疫印迹检测重组蛋白的表达情况。如图2所示，这说明可以利用常规方法在体外获取黑莓mUGT78H2重组蛋白，其中，图中1-4分别为4次重复样本的检测结果。
实施例6
UGT78H2和mUGT78H2重组蛋白在生产槲皮素糖苷中的应用：将UGT78H2和mUGT78H2重组蛋白，分别与糖基供体(UDP-半乳糖、UDP-葡糖醛酸和UDP-葡萄糖)和糖基受体(槲皮素)进行体外催化反应，反应体系为200μl：5μg重组蛋白/失活的重组蛋白，0.1mM糖基受体，2.0mM糖基供体，100mM Tris-HCl pH 7.5，5mM MgCl2。反应条件为37℃水浴60min。
数据采集仪器系统主要包括超高效液相色谱(Ultra Performance LiquidChromatography,UPLC)(Shim-pack UFLC SHIMADZU CBM30A,http://www.shimadzu.com.cn/)和串联质谱(Tandem mass spectrometry,MS/MS)(AppliedBiosystems 6500QTRAP,http://www.appliedbiosystems.com.cn/0)。
液相条件主要包括：色谱柱：Waters ACQUITY UPLC HSS T3 C18 1.8μm，2.1mm×100mm；流动相：A相为超纯水(0.04％的乙酸)，B相为乙腈(0.04％的乙酸)；洗脱梯度：0min水/乙腈(95:5V/V)，11.0min为5:95V/V，12.0min为5:95V/V，12.1min为95:5V/V，15.0min为95:5V/V；流速0.4mL/min；柱温40℃；进样量2μL。
HPLC检测结果和质谱鉴定结果如图3所示，Ⅰ为空白对照组，Ⅱ为UGT78H2催化槲皮素与UDP半乳糖苷发生反应，Ⅲ为UGT78H2催化槲皮素与UDP葡糖醛酸发生反应，Ⅳ为mUGT78H2催化槲皮素与UDP葡萄糖发生反应，Ⅴ为mUGT78H2催化槲皮素与UDP半乳糖发生反应，Ⅵ为mUGT78H2催化槲皮素与葡糖醛酸发生反应的高效液相色谱检测结果。图中带圈数字①-⑧分别对应的化学物质为表儿茶素，槲皮素，柚皮素，槲皮素-3-O-半乳糖苷，槲皮素-7-O-β-D-葡糖醛苷，槲皮素-4'-O-葡萄糖苷，槲皮素-3-O-半乳糖苷，槲皮素-7-O-β-D-葡糖醛苷；
从图3中的结果可以看出，UGT78H2蛋白能够催化UDP-半乳糖和槲皮素生成槲皮素-3-O-半乳糖苷，也可催化UDP-葡糖醛酸和槲皮素生成槲皮素-7-O-β-D-葡糖醛苷。mUGT78H2蛋白不仅能够催化UDP-葡萄糖和槲皮素生成槲皮素-4'-O-葡萄糖苷；也能催化UDP-半乳糖和槲皮素生成槲皮素-3-O-半乳糖苷，还能催化UDP-葡糖醛酸和槲皮素生成槲皮素-7-O-β-D-葡糖醛苷。这说明可以用黑莓的UGT78H2和mUGT78H2重组蛋白在体外生产槲皮素糖苷。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<120> 黑莓糖基转移酶基因及其应用
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Gly Val Pro Met Ile Gly Arg Pro His Phe Ala Asp Gln Asn Leu Asn
370 375 380
Met Arg Ser Val Glu Val Val Trp Lys Ile Gly Met Arg Ile Glu Gly
385 390 395 400
Gly Val Phe Thr Lys Thr Gly Ala Ile Lys Ala Leu Glu Gln Ala Leu
405 410 415
Ser Leu Glu Gln Gly Lys Glu Met Arg Arg Arg Val Gly Val Leu Lys
420 425 430
Gln Leu Ala Gln Glu Ala Val Gly Pro Asn Gly Arg Ser Thr Gln Asp
435 440 445
Leu Lys Ala Leu Val Gln Ile Ile Lys Ser
450 455
<210> 2
<211> 458
<212> PRT
<213> 2
<400> 2
Met Ser Pro Lys Leu Ala Ala Thr Ile Pro Gln Asn Val Ala Val Leu
1 5 10 15
Asn Phe Pro Phe Ala Ser His Pro Ser Ser Leu Leu Arg Phe Ile Arg
20 25 30
Thr Ile Ser Gly Leu Ala Pro Asp Ile Lys Phe Thr Phe Phe Ser Leu
35 40 45
Ser Arg Thr Asn Ser Ser Leu Phe Ser Gly Ser Asn Asn Lys Gly Leu
50 55 60
Asp Asn Ile Lys Pro Tyr Asp Val Trp Asp Gly Leu Pro Glu Gly Tyr
65 70 75 80
Val Pro Pro Ser Gly His Pro Leu Glu Gln Ile Gly Leu Phe Leu Asp
85 90 95
Lys Ala Pro Cys Thr Phe Glu Arg Ala Ile Lys Glu Val Glu Ala Glu
100 105 110
Met Gly His Lys Phe Gly Cys Leu Ile Ser Asp Ala Phe Leu Trp Phe
115 120 125
Ser Gly Asp Ile Ala Glu Lys Met Gln Val Pro Trp Val Thr Val Trp
130 135 140
Ser Gly Pro Arg Pro Leu Leu Val His Leu Glu Thr Asp Ile Ile Arg
145 150 155 160
Glu Lys Val Gly Ala Pro Gly Gln Glu Asp Lys Thr Leu Asp Phe Leu
165 170 175
Pro Gly Phe Ser Asp Glu Phe Arg Ala Ser Asp Leu Pro Lys Glu Val
180 185 190
Val Phe Gly Asp Ile Glu Ser Pro Leu Ala Arg Met Leu His Ser Met
195 200 205
Gly Gln Lys Leu Pro Gln Ala Thr Val Val Ala Val Asn Ser Phe Glu
210 215 220
Ala Met Asp Phe Lys Val Thr Glu Glu Leu Lys Lys Arg Leu Gln Lys
225 230 235 240
Leu Leu Leu Val Gly Pro Leu His Leu Val Arg Pro Val Pro Ser Val
245 250 255
Val Ser Asp Asp Glu Glu Glu Glu Glu Lys Asp Gly Cys Leu Gln Trp
260 265 270
Leu Asp Lys His Lys Pro Ala Ser Val Ala Tyr Ile Ser Phe Gly Ser
275 280 285
Val Gly Ala Leu Pro Pro Ile Glu Val Ala Ala Leu Ala Glu Ala Leu
290 295 300
Glu Glu Gly Gly Phe Pro Phe Leu Trp Ser Phe Arg Gly Asn Leu Glu
305 310 315 320
Asp Phe Pro Lys Gly Phe Ile Glu Arg Thr Ser Ile Gly Lys Val Val
325 330 335
Pro Trp Val Pro Gln Val Gln Ile Leu Asn His Ala Ser Ile Gly Val
340 345 350
Phe Val Thr His Cys Gly Trp Asn Ser Val Leu Glu Ser Val Thr Tyr
355 360 365
Gly Val Pro Met Ile Gly Arg Pro His Phe Ala Asp Gln Asn Leu Asn
370 375 380
Met Arg Ser Val Glu Val Val Trp Lys Ile Gly Met Arg Ile Glu Gly
385 390 395 400
Gly Val Phe Thr Lys Thr Gly Ala Ile Lys Ala Leu Glu Gln Ala Leu
405 410 415
Ser Leu Glu Gln Gly Lys Glu Met Arg Arg Arg Val Gly Val Leu Lys
420 425 430
Gln Leu Ala Gln Glu Ala Val Gly Pro Asn Gly Arg Ser Thr Gln Asp
435 440 445
Leu Lys Ala Leu Val Gln Ile Ile Lys Ser
450 455
<210> 3
<211> 20
<212> DNA
<213> 3
<400> 3
atgagcccca aacttgctgc 20
<210> 4
<211> 34
<212> DNA
<213> 4
<400> 4
tcatgatttg atgatctgta ccaaagcttt caag 34
<210> 5
<211> 29
<212> DNA
<213> 5
<400> 5
actgaccggt atgagcccca aacttgctg 29
<210> 6
<211> 48
<212> DNA
<213> 6
<400> 6
cgtgattcag gatttgcact tgaggaaccc agggaactac ttttccaa 48
<210> 7
<211> 48
<212> DNA
<213> 7
<400> 7
ttggaaaagt agttccctgg gttcctcaag tgcaaatcct gaatcacg 48
<210> 8
<211> 39
<212> DNA
<213> 8
<400> 8
tacactctcc aaaactgaat tccaaccgca atgtgtcac 39
<210> 9
<211> 39
<212> DNA
<213> 9
<400> 9
gtgacacatt gcggttggaa ttcagttttg gagagtgta 39
<210> 10
<211> 48
<212> DNA
<213> 10
<400> 10
catggtcgac tagtgatggt gatggtgatg tgatttgatg atctgtac 48
<210> 11
<211> 27
<212> DNA
<213> 11
<400> 11
actgaccggt atgagcccca aacttgc 27
<210> 12
<211> 58
<212> DNA
<213> 12
<400> 12
catggtcgac tagtgatggt gatggtgatg tgatttgatg atctgtacca aagctttc 58
Claims (5)
1.黑莓糖基转移酶突变基因，其特征在于，所述突变基因为mUGT78H2基因，其编码的氨基酸序列如SEQ ID No.2所示，所述mUGT78H2基因为将UGT78H2基因编码蛋白的PSPG结构域中第3位天冬酰胺突变为脯氨酸，第23位赖氨酸突变为天冬酰胺的基因；所述UGT78H2基因编码的氨基酸序列如SEQ ID No.1所示。
2.基于权利要求1所述突变基因的重组蛋白，其特征在于，所述重组蛋白为mUGT78H2蛋白，其氨基酸序列如SEQ ID NO.2所示。
3.基于权利要求1所述基因的应用，其特征在于，用于体外获取重组蛋白。
4.基于权利要求2所述重组蛋白的应用，其特征在于，用于生产槲皮素糖苷。
5.基于权利要求3所述基因的应用，其特征在于，包括以下步骤：
步骤A，克隆得到黑莓UGT78H2基因；
步骤B，设计特异引物，采用OE-PCR技术，将UGT78H2基因中用于UGT78H2蛋白PSPG结构域中第3位表达为天冬酰胺的基因片段突变为表达为脯氨酸的基因片段，第23位表达为赖氨酸的基因片段突变为表达为天冬酰胺的基因片段，从而获得mUGT78H2基因；
步骤C，将mUGT78H2基因通过表达载体转入宿主进行表达获取重组蛋白。
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Non-Patent Citations (2)
| Title |
|---|
| Plant secondary metabolism glycosyltransferases: the emerging functional analysis;Claire M.M. Gachon 等;《TRENDS in Plant Science》;20051130;第542-549页 * |
| 黑莓糖基转移酶基因UGT78H2的分离鉴定及与类黄酮化合物的分子对接;陈清 等;《植物研究》;20150315;第35卷(第2期);摘要，表2，图2，第271页左栏第1段，第273页左栏第1段-右栏第1段，第277页左栏第2段 * |
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