CN118028320A - 一种人参PgGUAT252645基因及其应用 - Google Patents
一种人参PgGUAT252645基因及其应用 Download PDFInfo
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Abstract
本发明首次在人参中发现直接参与人参皂苷Ro生物合成的尿苷二磷酸葡萄糖醛酸转移酶基因，PgGUAT252645，基因序列如SEQ ID No.1所示。该基因是人参中在人参皂苷Ro合成过程中特异性糖醛酸化修饰齐墩果酸C3位置的尿苷二磷酸葡萄糖醛酸转移酶的mRNA。在其开放阅读框622bp处能够引起氨基酸替换的SNP突变可显著影响人参皂苷Ro含量，未突变的PgGUAT252645基因能增加人参皂苷Ro的含量，突变的PgGUAT252645基因会降低人参皂苷Ro的含量，此发现对人参育种具有重要意义。
Description
技术领域
本发明属于生物技术领域，具体涉及一种人参皂苷Ro合成相关基因PgGUAT252645及其应用。
背景技术
人参(Panax ginseng C.A.Meyer)，一种五加科人参属多年生草本植物，是传统名贵中草药，因其极具药用价值在世界范围内被广泛应用于多种疾病的临床治疗以及保健食品等商品的生产。人参皂苷是一类人参属植物的次生代谢产物，也是人参的主要药理成分。各种人参皂苷的分子结构中的三萜分子种类、糖基化位置、糖基种类以及糖基数量的不同，决定了它们多样的药理活性。
人参皂苷Ro是迄今为止唯一一种被报道的、来源于人参的齐墩果烷型五环三萜人参皂苷，目前已知的药理活性有抑制肿瘤生长、抗血栓、抗炎、改善肥胖等。此外有研究指出，如同人参皂苷Rb1、Re可参与调控人参不定根的生长，人参皂苷Ro对人参不定根生长具有低浓度促进、高浓度抑制的作用。人参皂苷Ro的合成途径与四环三萜型人参皂苷的合成途径在前体beta-amyrin之前完全相同，皆是以由MVA、MEP途径的终产物IPP经催化生成的2，3氧化鲨烯为底物进行苷元骨架的合成。人参皂苷Ro是由beta香树脂醇经齐墩果酸合酶催化生成的齐墩果酸的糖基化修饰产物。这些糖基化包括修饰齐墩果酸C3位置的葡萄糖醛酸基，修饰该葡萄糖醛酸基C2位置的葡萄糖基以及修饰姜状三七皂苷R1或竹节参皂苷IVaC28位置的葡萄糖基。
然而，关于人参皂苷Ro合成相关基因的研究仍然较少，目前仅包括β-香树脂醇、齐墩果酸等前体的合成酶基因的克隆表征，以及来源于人参，姜状三七，三七和竹节参的一组属于不同基因家族的糖基转移酶，它们被证实参与人参皂苷Ro合成过程中的糖基化修饰步骤。然而由于代谢网络的复杂性，与这一合成途径相关的许多关键基因及调控因子仍未被揭示。因此，继续深度挖掘人参皂苷Ro合成相关基因对丰富人参基因资源以及进一步揭示人参皂苷合成机制具有重大意义。
发明内容
本发明的的目的在于提供一种人参PgGUAT252645基因。
本发明的另一目的在于提供一种人参PgGUAT252645基因在人参皂苷Ro合成中应用。
为了实现上述目的，本发明采用的技术方案如下：
一种人参皂苷Ro合成相关基因PgGUAT252645，所述基因开放阅读框碱基序列如SEQ ID No.1所示。
一种人参PgGUAT252645基因在人参皂苷Ro合成中应用，具体方法如下：
PgGUAT252645是人参中在人参皂苷Ro合成过程中特异性糖醛酸化修饰齐墩果酸C3位置的尿苷二磷酸葡萄糖醛酸转移酶的mRNA；在其开放阅读框622bp处能够引起氨基酸替换的SNP突变可显著影响人参皂苷Ro含量，如图1所示。
本发明具有如下有益效果：
对于人参中人参皂苷合成途径中的糖基化修饰步骤，不仅存在某一种糖基转移酶可催化多种底物糖基化修饰的混杂催化现象，还存在着由多种糖基转移酶同工酶共同催化某一底物糖基化修饰的现象，且这些糖基转移酶同工酶可能来自不同的基因家族。类似于对底物偏好性的研究可阐明伴随着混杂催化现象的人参皂苷糖基化修饰代谢网络中的主要途径，对于各个同工酶之间的主次或补充关系的探索以及其它同工酶成员的深度挖掘将进一步解析人参皂苷的合成过程。
本发明首次在人参中发现直接参与人参皂苷Ro生物合成的尿苷二磷酸葡萄糖醛酸转移酶基因，PgGUAT252645，其开放阅读框碱基序列如SEQ ID No.1所示。该基因是人参中在人参皂苷Ro合成过程中特异性糖醛酸化修饰齐墩果酸C3位置的尿苷二磷酸葡萄糖醛酸转移酶的mRNA。在其开放阅读框622bp处能够引起氨基酸替换的SNP突变可显著影响人参皂苷Ro含量，未突变的PgGUAT252645基因能增加人参皂苷Ro的含量，突变的PgGUAT252645基因会降低人参皂苷Ro的含量，此发现对人参育种具有重要意义。
附图说明
图1：PgUGAT252645开放阅读框622bp处SNP突变对人参皂苷Ro的影响。
图2：SDS-PAGE电泳检测蛋白的诱导表达(泳道1和泳道4是未诱导表达的细胞破碎液上清样品，通道2和5是通过IPTG诱导表达的样品细胞破碎液上清样品，泳道3是蛋白质分子量标记；*标出的即为特异性表达蛋白条带的位置)。
图3：液相色谱-质谱联用检测催化反应体系提取物(A.标准品及提取物样品的液相色谱分析。红色曲线为含有齐墩果酸(OA)和金盏花皂苷E(CE)的标准品溶液，绿色和蓝色曲线分别代表不同催化反应体系提取物样品；B.标准品质谱检测结果)。
图4：人参发状根的诱导和培养(A.人参不定根；B.预培养；C.侵染后除菌培养；D.人参发状根的发育；E.人参发状根增殖传代；F.人参发状根液体培养)。
图5：转基因人参发状根中目的基因表达量以及人参皂苷Ro皂苷含量(A、B中蓝色柱代表样品中人参皂苷Ro含量和PgUGAT252645ORF相对表达量；C、D中橙色柱代表样品中人参皂苷Ro含量和PgUGAT252645ORF622M相对表达量；灰色柱则分别代表对照组中人参皂苷Ro含量和内参基因的表达量)。
具体实施方式
实施例1PgGUAT252645基因原核表达及催化活性检测
PgGUAT252645开放阅读框序列(PgUGAT252645ORF)如SEQ ID No.1所示。。该序列及其622bp处突变序列(PgUGAT252645ORF622M)由人工合成后分别连接至质粒载体pET-28a的酶切位点BamH I以及Hind III之间以构建原核表达质粒。将构建的重组质粒分别转化大肠杆菌BL21(DE3)化学感受态细胞，以构建重组表达菌株。通过筛选得到的阳性菌株在37℃、180rpm的条件下培养至OD600达到0.8后加入终浓度为0.1mM的异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)，在16℃、180rpm的条件下持续诱导蛋白表达12h。随后在4℃、12000rpm的离心条件下离心15min收集菌体细胞，并用HEPES缓冲液重悬菌体细胞，再利用超声波细胞破碎仪对菌体细胞进行破碎。最后在4℃、12000rpm的离心条件下离心45min，收集上清液，用于SDS-PAGE蛋白电泳检测以及催化活性检测。
体积为100μL的催化反应体系包括85μL上述上清液，0.5mM齐墩果酸作为受体底物，5mM尿苷二磷酸葡萄糖醛酸(uridine-5'-diphosphoglucuronic acid,UDPGA)作为糖基供体底物。30℃孵育12h后加入等体积水饱和正丁醇终止反应，随后分离有机相并蒸干。残留物溶于色谱甲醇，通过如表1所示的检测条件进行液相色谱-质谱联用检测分析。
SDS-PAGE蛋白电泳检测结果如图2所示，，相较于未添加IPTG进行诱导表达的对照组，实验组细胞破碎上清液泳道中出现特异性蛋白条带，表明成功诱导表达。催化活性检测结果如图3所示，在含有PgUGAT252645ORF蛋白的反应体系中，检测到了齐墩果酸C3葡萄糖醛酸化修饰产物金盏花皂苷E，而在含有PgUGAT252645ORF622M蛋白的反应体系中组未检测到相应的糖醛酸化修饰产物。
表1.原核表达产物催化活性液相色谱-质谱联用检测分析条件
实施例2PgUGAT252645ORF以及PgUGAT252645ORF622M过表达人参发状根的诱导、培养
PgUGAT252645ORF和PgUGAT252645ORF622M由人工合成后分别连接至质粒载体pCAMBIA1300-35S的酶切位点Sac I和Sal I之间，以构建过表达质粒。以携带pCAMBIA1300-35S空白质粒的农杆菌C58C1所诱导的发状根样品作为对照。将构建的重组质粒分别转化农杆菌C58C1感受态细胞，以构建用于侵染的重组菌株。通过筛选得到的阳性菌株在28℃，170rpm的条件下培养至OD600达到0.4后在4℃、12000rpm的离心条件下离心15min收集菌体细胞，并用含有乙酰丁香酮(AS)的1/2MS液体培养基重悬菌体细胞，细胞浓度调节至OD600＝0.5。然后将预培养的人参不定根加入菌液中进行侵染。经过侵染的不定根先在含有20μMAS的1/2MS固体培养基上培养48h，然后转移到含有头孢菌素的1/2MS固体培养基上继续培养，直到发状根发育。选取在传代过程中生长状态稳定的发状根无性单根系在22℃，110rpm的条件下用液体1/2MS培养基进行扩大培养。诱导及培养过程如图4所示。
实施例3转基因人参发状根中目的基因表达量以及人参皂苷Ro皂苷含量的检测
各转基因发状根无性单根系的干燥粉碎样品用于人参皂苷提取。每个单根系设置3个生物学重复，分别使用ODS法对各样品水煎液中的人参皂苷进行提取，然后通过如表2所示的检测条件进行液相色谱-质谱联用检测分析。
各转基因发状根无性单根系的低温冻存样品用于总RNA提取。每个单根系设置3个生物学重复，分别使用TRIpure总RNA提取试剂盒提取总RNA，然后使用HiFiScript快速去基因组cDNA第一链合成试剂盒去除基因组DNA并反转录成cDNA。随后以cDNA为模板，使用UltraSYBR Mixture(Low ROX)，通过实时荧光定量PCR检测各个样品中PgUGAT252645ORF或PgUGAT252645ORF622M的表达量。其中每个样品设置3个技术重复，选择肌动蛋白基因Actin1为内参基因。将内参基因表达量归一化处理后，通过2-ΔΔCt法计算目标基因表达量。
各样品中人参皂苷Ro含量及PgUGAT252645ORF或PgUGAT252645ORF622M的表达量的检测结果如图5所示。与作为对照的由携带pCAMBIA1300-35S空白质粒的农杆菌C58C1所诱导的发状根样品相比，5个PgUGAT252645ORF过表达发状根无性单根系中PgUGAT252645ORF的表达量均有不同程度的增加，范围为1.509-3.967倍(图5A)。同时，有4个样品中的人参皂苷Ro含量显著提高，且与表达增量成一定相关性(图5B)。虽然部分PgUGAT252645ORF622M过表达发状根无性单根系样品中人参皂苷Ro含量也有所提高，但增幅总体上远小于过表达PgUGAT252645ORF的样品，且与表达增量无关(图5C、D)。
表2.人参发状根人参皂苷Ro含量液相色谱-质谱联用检测分析条件
Claims (3)
1.一种人参PgGUAT252645基因，其特征在于：所述基因开放阅读框碱基序列如SEQ IDNo.1所示。
2.一种人参PgGUAT252645基因在人参皂苷Ro合成过程中应用。
3.如权利要求2所述的应用，其特征在于，具体应用方法如下：
PgGUAT252645基因在人参皂苷Ro合成过程中特异性糖醛酸化修饰齐墩果酸C3位置的尿苷二磷酸葡萄糖醛酸转移酶的mRNA；在其开放阅读框622bp处能够引起氨基酸替换的SNP突变可显著影响人参皂苷Ro含量，如图1所示。
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