CN116286894A - 一种催化稀有人参皂苷生物合成的糖基转移酶及其编码基因与应用 - Google Patents
一种催化稀有人参皂苷生物合成的糖基转移酶及其编码基因与应用 Download PDFInfo
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- CN116286894A CN116286894A CN202310150439.1A CN202310150439A CN116286894A CN 116286894 A CN116286894 A CN 116286894A CN 202310150439 A CN202310150439 A CN 202310150439A CN 116286894 A CN116286894 A CN 116286894A
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Abstract
本发明通过聚合酶链式反应克隆得到三七中糖基转移酶UGT33基因全长并成功在大肠杆菌中表达重组蛋白,首次鉴定确认该转移酶UGT33其可以催化人参皂苷上C20和C3位糖链的延伸的发生,能够分别催化人参皂苷CK,人参皂苷Rh2、人参皂苷F1和人参皂苷F2生成相应的加糖产物,具有极高的经济价值和应用前景。
Description
技术领域
本发明涉及基因工程领域,更为具体的,本发明涉及植物体外稀有人参皂苷的生物合成。
背景技术
三七[Panax notoginseng(Burk.)F.H.Chen],又名田七、山漆,为五加科(Araliceae)人参属多年生草本植物,是我国传统的名贵中药。三七主要以根和根茎入药,具有散瘀止血、消肿定痛之功效,应用历史悠久,收载于历年《药典》。三七中含有的皂苷类化合物为其主要的活性成分,包括以含量较高的人参皂苷Rb1、Rg1和三七皂苷R1为主的三七总皂苷对防治心脑血管疾病有显著作用。除含量较高的这些皂苷类化合物之外,三七中还含有较多稀有皂苷,由于其含量较少,因此限制了对稀有皂苷的研究和应用,并且三七植物生长年限较长、连作障碍现象严重等问题也大大限制了其应用。合成生物学应运而生,利用生物技术设计和改造微生物菌株来生产天然活性成分已经成为一种绿色可持续发展的新方法。
三七皂苷的主要成分为达玛烷型四环三萜类化合物。已有研究表明植物中达玛烷型四环三萜皂苷主要通过乙酸/甲羟戊酸途径合成,其生物合成途径包含了200多步连续的酶促反应,一般可划分为3个阶段:①合成异戊烯基焦磷酸(IPP)和二甲基烯丙基焦磷酸(DMAPP);②由异戊烯基转移酶和萜类环化酶催化IPP和DMAPP形成2,3-氧化鲨烯;③2,3-氧化鲨烯依次经过环化、羟基化、糖基化修饰后最终形成三七皂苷。生物合成途径中含有的关键酶包括3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutary-coenzyme Areductase,HMGR)、法呢基焦磷酸合成酶(farnesyl pyrophaophate synthase,FPS)、鲨烯合成酶(squalene synthase,SS)、鲨烯氧化酶(squalene epoxidase,SE)、达玛烯二醇合成酶(dammarenediol synthase,DS)、细胞色素P450单加氧酶(cytochrome P450monooxygenases,CYP450)和糖基转移酶(glycosyltransferase,GT)。
糖基转移酶可在植物体内催化单糖集团从活化的核苷酸供体与不同的受体结合,与受体分子的氧、氮、硫或碳原子形成糖苷键。三七中含有的糖基转移酶主要为以UDP-戊糖、UDP-己糖和UDP-木糖为供体底物的UDP-糖基转移酶(UGTs)。UGTs的C端附近有个由44个氨基酸组成的高度保守区,是植物次生代谢糖基转移酶(plant secondary productglycosyltransferase,PSPG)保守结构域,它能够识别和结合UDP糖供体。然而目前国内外对于能够用于催化人参皂苷生物合成的UGT酶报道较少。
发明内容
为了填补现有技术的空白,本发明提供一种UGT33基因,其具有催化人参皂苷糖链延伸的生物学功能,能够分别催化人参皂苷CK,人参皂苷Rh2、人参皂苷F1和人参皂苷F2生成相应的加糖产物。为了实现所述效果,本发明提供如下的技术方案:
本发明的第一个方面,提供一种UGT33基因,所述基因的核苷酸序列如SEQ IDNo.1所示。
本发明的第二个方面,提供上述UGT33基因UGT33基因的编码蛋白,所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。
本发明的第三个方面,提供上述UGT33基因和/或其编码蛋白在催化原人参二醇类化合物人参皂苷CK、Rh2和F2中的应用。
在一种实施方式中,上述应用为UGT33基因和/或其编码蛋白催化人参皂苷CK、Rh2和F2的C3位和C20位糖链延伸。
在一种实施方式中,上述应用为催化人参皂苷CK生成多糖苷Gypenoside LXXV、催化人参皂苷Rh2生成人参皂苷Rg3以及Gypenoside XVII,催化人参皂苷F2生成人参皂苷Rd和人参皂苷Rb1。
本发明的第四个方面,提供上述UGT33基因和/或其编码蛋白在催化原人参二醇类化合物人参皂苷F1中的应用。
在一种实施方式中,上述应用为催化原人参三醇类化合物人参皂苷F1的C20位糖链延伸。
在一种实施方式中,上述应用为催化原人参三醇类化合物人参皂苷F1生成双糖苷Yesanchinoside U。
本发明的第五个方面,提供上述UGT33基因和/或其编码基因在用于制备多糖苷Gypenoside LXXV、人参皂苷Rg3、Gypenoside XVII、人参皂苷Rd、人参皂苷Rb1和/或双糖苷Yesanchinoside U中的应用。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1 UGT33在三七植物不同年生不同组织部位的基因表达分析;
图2 pET28a-UGT33重组蛋白胶图。BlueV Protein Marker(10-190kDa)自上而下的顺序为:190kDa,140kDa,95kDa,70kDa,55kDa;甬道1为空载pET28a的对照蛋白,甬道2为pET28a-UGT33的重组蛋白,甬道3为纯化后空载pET28a的对照蛋白,甬道4为纯化后pET28a-UGT33的重组蛋白;
图3 UGT33分别催化人参皂苷CK和人参皂苷Rh2的酶促反应HPLC图谱。检测波长为203nm;
图4 UGT33催化人参皂苷F1的酶促反应TOF/MS图谱。A:UGT33催化人参皂苷F1生成Yesanchinoside U的反应式;B:反应产物TOF检测的TIC色谱图和质谱图。K-F1表示pET28a空载蛋白的催化产物结果;UGT33-F1表示UGT33重组蛋白的催化产物结果;图5UGT33催化人参皂苷F2的酶促反应TOF/MS图谱。A:UGT33催化人参皂苷F2生成相应加糖产物的反应式;B:反应产物TOF检测的TIC色谱图和质谱图。K-F2表示pET28a空载蛋白的催化产物结果;UGT33-F2表示UGT33重组蛋白的催化产物结果;
图6人参皂苷Gypenoside LXXV的1H NMR谱图(CD3OD-d4,800MHz)。1H NMR(800MHz,MeOD)δ5.14(t,J=6.7Hz,1H),4.67(d,J=7.7Hz,1H),4.43(d,J=7.3Hz,1H),3.84(dd,J=27.6,11.6Hz,2H),3.65(dd,J=11.9,5.4Hz,1H),3.61(dd,J=11.8,6.2Hz,1H),3.59-3.51(m,3H),3.35(t,J=9.0Hz,1H),3.30-3.23(m,3H),3.23-3.18(m,3H),2.18-2.12(m,1H),2.05-1.98(m,3H),1.91-1.81(m,2H),1.73(dd,J=22.3,11.5Hz,3H),1.68(s,3H),1.62(s,3H),1.60-1.50(m,5H),1.50-1.44(m,2H),1.39(td,J=12.9,5.2Hz,1H),1.36-1.23(m,10H),1.15(s,3H),1.09-1.03(m,5H),1.01(s,3H),0.92(s,3H),0.91(s,3H),0.90(s,1H),0.86(s,3H),0.79(d,J=11.8Hz,1H);
图7人参皂苷Gypenoside LXXV的13C NMR谱图(CD3OD-d4,200MHz)。13C NMR(200MHz,MeOD)δ:130.58,124.78,104.01,103.12,89.89,79.69,77.11,76.95,76.48,76.30,74.91,73.00,70.74,70.52,70.20,61.72,61.45,56.13,53.73,51.18,49.95,47.92,47.81,47.70,47.60,47.49,47.38,47.28,39.57,39.18,38.84,36.54,34.91,34.52,30.63,30.60,26.96,25.95,25.85,25.11,24.49,21.88,17.84,16.29,15.71,15.36,15.31,14.76;
图8人参皂苷Yesanchinoside U的1H NMR谱图(CD3OD-d4,800MHz)。1H NMR(800MHz,MeOD)1H NMR(800MHz,MeOD)δ5.16(s,1H),4.61(d,J=7.7Hz,1H),4.37(d,J=7.7Hz,1H),4.06(d,J=13.4Hz,2H),3.88(d,J=11.8Hz,1H),3.81(dd,J=11.5,5.4Hz,1H),3.76(dd,J=15.3,10.3Hz,1H),3.70-3.64(m,1H),3.46(s,1H),3.36(d,J=8.6Hz,4H),3.31-3.27(m,2H),3.23(t,J=8.5Hz,1H),3.14(t,J=11.8Hz,2H),2.31(q,J=9.9Hz,1H),2.19(dd,J=17.1,10.5Hz,1H),2.06(dd,J=12.3,6.0Hz,1H),1.96-1.90(m,1H),1.82(dd,J=25.2,12.2Hz,2H),1.74(t,J=10.1Hz,2H),1.71(s,3H),1.69-1.64(m,5H),1.62(t,J=9.5Hz,2H),1.56(t,J=10.4Hz,2H),1.47(d,J=13.2Hz,1H),1.40-1.34(m,5H),1.31(s,4H),1.24(dd,J=24.3,12.1Hz,1H),1.11-1.03(m,5H),0.98(s,9H),0.92(d,J=10.6Hz,1H);
图9人参皂苷Yesanchinoside U的13C NMR谱图(CD3OD-d4,200MHz)。13C NMR(200MHz,MeOD)δ:130.86,130.22,127.70,126.86,124.62,103.59,96.71,83.52,78.16,77.15,76.55,76.53,75.40,73.91,73.74,70.27,70.19,70.12,68.83,67.53,61.41,60.73,57.09,50.87,49.10,47.92,47.82,47.71,47.60,47.50,47.39,47.28,40.60,39.10,38.77,38.69,35.33,30.08,30.05,29.30,26.36,25.81,24.54,22.48,21.06,16.62,16.34,16.22,15.97,14.72。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例1三七UGT33全长cDNA序列的克隆
1.三七叶片总RNA提取及cDNA第一链的获得
自云南省文山市三七道地药材的种植产区采集一至四年生三七新鲜植株,分为花、叶、茎、根茎、须根、周皮、木质部和韧皮部,分别用清水和蒸馏水清洗干净后滤纸擦干,液氮速冻后保存于-80℃冰箱。使用植物总RNA提取试剂盒,按照说明书进行三七叶片的总RNA提取。使用Fast Quant cDNA第一链合成试剂盒(天根生化科技北京有限公司),按照说明书将提取的质量较好的总RNA反转为cDNA。cDNA保存于-20℃冰箱备用。
2.引物设计
根据三七基因组数据注释筛选得到基因ORF序列片段,使用primer primer 5.0软件设计UGT33基因的5′和3′端特异性引物,引物序列如下:
UGT33-F:ATGGATATCGAGAAAGGTAGAATCA
UGT33-R:ATATTGTGCGTCTTTCTTCATCTTA
3.PCR扩增
DNA聚合酶采用高保真DNA聚合酶(Phusion High-Fidelity PCR Master Mi×)。
以步骤1获得的cDNA为模板,以UGT33-F/R为引物,利用Phusion DNA高保真酶进行PCR扩增,得到PCR扩增产物。
(1)PCR扩增体系
PCR反应程序:98℃预变性30s;98℃10s,60℃15s,72℃2min,35个循环;72℃延伸5min。
(2)切胶回收基因片段
反应结束后,1%凝脂糖凝胶电泳,目的条带用目的条带用Thermo公司胶回收试剂盒的方法切胶回收,步骤如下:a.在紫外灯下切胶装入1.5mL离心管中,加入1mg:1μL的Binding Buffer,50℃水浴10min,使凝胶完全溶解后离心,完全转移至吸附管中,12000g离心1min,倒掉收集管中的废液;b.向吸附管中加入700μL Wash Buffer,12000g离心1min,弃废液;12000g离心1min,弃废液;c.用50μL ddH2O洗脱晾干的柱子,5分钟后12000g离心1min,收集PCR扩增的DNA溶液。
4.克隆载体构建
测定胶纯化回收后的质量和浓度,将质量浓度符合要求的产物连接到B-zero载体上,连接体系如下:
将反应体系在25℃放置15min进行连接,连接产物放置在冰上。将连接产物转化到Trans-T1大肠杆菌感受态细胞中,转化过程如下:a.连接好的载体加入到TransT1感受态细胞中,冰上放置30min;b.42℃热激30s,取出放于冰上2min;c.加液体LB培养基500μL,37℃的摇床上180r/min震荡培养1h,在含氨苄抗性的固体培养基上37℃培养过夜,挑选单斑送北京睿博兴科生物技术有限公司完成测序。
测序结果表明:PCR扩增产物的序列如序列1所示,将序列1所示的基因命名为UGT33,其编码由446个氨基酸残基组成的蛋白质,氨基酸序列如SEQ ID NO.2所示。该克隆载体命名为pEASY-Blunt-UGT33质粒,存于-80℃冰箱。
实施例2、UGT33基因组织表达分析
1、三七不同组织部位总RNA的提取及RNA-seq法测定基因表达量
将保存于-80℃冰箱的不同组织部位样品在液氮环境下粉碎,采用改良CTAB法(CTAB Buffer:2%CTAB(W/V);100mm0l·L-1Tris-HCl(pH 8.0);25mmol·L-1EDTA;2.0mol·L-1NaCl;0.5g·L-1亚精胺)提取样品的RNA,根据转录组测序,获得基因在不同组织部位的表达量FPKM值(图1)。
实施例3、三七UGT33生物学功能研究
1.原核表达载体构建
将测序结果正确的菌株和pET28a载体菌株分别接种到含相应抗性的液体LE培养基中,37℃、250r/min条件下振荡培养12~16h,用质粒小提试剂盒(天根生化科技北京有限公司)提取质粒。
设计带有同源臂的目的基因特异性的引物,送睿博兴科生物技术有限公司合成。
pET28a-UGT33-F:TTCTGTTCCAGGGGCCCGAAATGGATATCGAGAAAGGTAG
pET28a-UGT33-R:TGGTGGTGGTGCTCGAGTGCATATTGTGCGTCTTTCTTCA
(1)线性化空载体制备:采用NEB公司限制性内切酶Not I和EcoR I对提取的pET28a(His-MBP)空载体进行双酶切,37℃孵育2h,切胶回收酶切产物。
(2)带有同源臂的PCR产物(目的基因)的制备:以含有三七UGT33基因全长cDNA的载体pEASY-Blunt-UGT33质粒为模板,使用pET28a-UGT33-F/R为引物,采用Phusion DNA高保真酶进行PCR扩增基因编码区。PCR反应程序:98℃预变性30s;98℃10s,60℃15s,72℃2min,35个循环;72℃延伸5min;4℃维持。
(3)切胶回收线性载体及基因片段:取PCR产物与6×DNA loading buffer预混合,在1.5%琼脂糖凝胶上以低电压(约5Vcm-1)电泳30min;用解剖刀或剃刀片切割含有DNA片段的凝胶,尽可能的靠近DNA片段切割,以减小凝胶的含量,将胶片放在事先称重的1.5mL离心管并称重。将凝胶按照Gene JET Gel Extraction Kit琼脂糖凝胶回收试剂盒按照说明书操作回收。
(4)表达载体连接:使用pEASY-Uni Seamless Cloning and Assembly Kit试剂盒,按照说明书将线性化载体与PCR产物的轻轻混合,50℃,20min反应,体系如下:
线性化空载体与目的基因片段的摩尔比为1:2,其中线性化空载体的加入量为0.01-0.02pmols,pmol的计算方式为pmols=ng/(片段长度bp×0.65kDa)
体系配制完成后,用移液器上下轻轻吹打几次混匀各组分,50℃反应25min,待反应完成后,立即将反应物置于冰水浴中冷却5min之后,将反应产物进行转化到Trans-T1大肠杆菌感受态细胞中,在含Kana抗性的固体培养基上37℃培养过夜,挑选单菌落送天一辉远生物科技有限公司完成测序。测序结果正确的菌株接种至10mL含有100mg/L Kana的LB固体培养基中摇培过夜,利用质粒小提试剂盒(天根生化科技北京有限公司)提取质粒,质粒存放于-20℃冰箱保存。
(5)转化表达载体:将提取的pET28a-UGT33(Trans-T1)质粒转化入BL21(DE3)表达感受态细胞中,在含Kana抗性的固体培养基上37℃培养过夜,挑选单菌落送天一辉远生物科技有限公司完成测序。测序结果正确的菌株摇培后加入20%体积的甘油,保存于-80℃冰箱。
2.诱导融合蛋白表达
(1)将已转化入表达载体的pET28a-UGT33阳性单克隆菌落,于37℃和250r/min的摇床中过夜培养,再按1千分之一的比例扩大培养到200mL含50mg/mL Kana的液体培养基中,37℃摇培到OD600约为0.6-0.8后,加入异丙基硫代半乳糖苷(IPTG)至终浓度为1mM,低温16℃诱导表达20h;
(2)低温诱导的菌液在4℃8000g条件下离心3min收集菌体,加入5mLResuspension buffer(100mM Tris-HCI pH7.5,150mM NaCl,1mM DTT,0.1mM EDTA,5%甘油)重悬菌体;
(3)加入Chicken white lysozyme(50mg/mL),于重悬菌体中至终浓度为0.5mg/mL,混均后冰上静置20min。
(4)加入10%Triton X-100至终浓度为0.1%,加入1/10体积的5mol/L NaCl溶液,超声破碎30min(超声5s,暂停5s),4℃12000g离心30min。
(5)取1mL的Amylose Resin加入到柱子中(Amylose Resin提前用5倍柱体积的Wash Buffer清洗)(Wash Buffer:50mM Tris-HCl pH7.5,150mM NaCl,1mM DTT,0.1mMEDTA,5%甘油),将破碎的菌液上清加到含有Amylose Resin的15mL离心管中,小摇床高转速冰上晃动2h;
(6)15倍柱体积Wash buffer清洗后再用15倍柱体积Resuspension buffer清洗;
(7)加入1倍柱体积Elution buffer A洗脱一次。加入两倍柱体积Elution bufferB,4℃摇晃10min后洗脱,重复两次;(Elution Buffer:50mM Tris-HCl pH7.5,150mM NaCl,1mM DTT,0.1mM EDTA,5%甘油;麦芽糖A:2mM,麦芽糖B:10mM)
(8)将Elution buffer B洗脱液分别用10KD和30KD的超滤管浓缩空载对照蛋白和糖基转移酶融合蛋白,浓缩蛋白于-80℃保存。
3.蛋白检测:十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)
(1)凝胶配制:参考碧云天SDS-PAGE凝胶配制试剂盒配制8%分离胶(下层胶)和5%的浓缩胶;
(2)样品制备:将蛋白质样品与6X Protein Loading Buffer在PCR小管中混合,放入100℃加热10min,常温12000g离心5min,取上清点样;
(3)电泳过程:组装好电泳系统,加入1×电泳缓冲液,上样5-10μL,低电压90V跑20min至样品分离至分离胶层,改变电压至高电压120V,当溴酚蓝跑出分离胶停止电泳。卸下胶板,分离凝胶,One step blue Protein gel strain染色,照胶(图2)。
使用NanoDrop One核酸/蛋白定量仪(Thermo Scientific)测量蛋白浓度。
结果显示,蛋白浓度为9.8ng/μL。
4.酶促反应
100μL Tris-HCI(100mM,pH=7.5)的反应体系中,加入1mM UDP-葡萄糖(UDPG),底物100μM,纯化蛋白50mg,以重组质粒pET28a-UGT33表达蛋白为实验组,以空载质粒pET28a表达蛋白为对照组,35℃培养箱中反应24h后,加100μL甲醇终止反应,0.22μm滤膜过滤,分别用液相色谱(HPLC1260,Waters)和超高效液相色谱/离子淌度/四级杆-飞行时间质谱(SYNAPT G2-Si,Waters)分析反应结果。
液相检测方法与条件:色谱柱为Agilent ZORBAX SB-C18柱(4.6x250mm,5-Micron),进样量为52μL,扫描波长为203nm。洗脱梯度为:A为0.1%甲酸水,B相为乙腈,Omin80%A,5min 60%A,7min 55%A,12min 40%A,15min 10%A,20min 0%A,25min 80%A。后运行时间5min。
超高效液相检测方法与条件:色谱柱为ACQUITY UPLCHSS T3柱(2.1mm 100mm,1.8μm,USA),进样量2μL,扫描波长为190-450nm,采用负离子模式电离化合物。检测条件为:毛细管电压1.0kV,锥孔电压40V,离子源温度120℃,脱溶剂温度450℃,锥孔气流50L/h,脱溶剂气流800L/h,TOF-MS设置为50~1200m/z,扫描时间为0.2秒。使用Masslynx软件收集和处理样品数据。反应结果如图3-5所示,显示PnUGT33能够催化原人参二醇类化合物人参皂苷CK、Rh2和F2的C3位和C20位糖链延伸,分别生成多糖苷Gypenoside LXXV、人参皂苷Rg3以及Gypenoside XVII,人参皂苷Rd和人参皂苷Rb1。PnUGT33能够催化原人参三醇类化合物人参皂苷F1的C20位糖链延伸,生成双糖苷Yesanchinoside U。
5.制备产物反应
(1)挑取含有重组质粒pET28a-UGT33单个阳性克隆E.coli BL21(DE3)菌落接种到10mL的LB液体培养基(含有50mg/mL Kana)中,37℃,250r/min培养过夜;
(2)按1‰的比例各扩大培养至10L,37℃250r/min摇培至OD600约为0.6-0.8后,加入IPTG至终浓度为1mM,低温16℃诱导表达20h;
(3)低温诱导的菌液在高速离心机中离心3min(4℃,10000g)收集菌体,弃去培养基。蒸馏水清洗后重悬于300mL含有1mM DTT的Tris-HCl(100mM,pH=7.5)缓冲液中。
(4)利用ATS匀质机高压破碎重悬菌液,破碎30min。将破碎菌体与高速离心机中离心20min(4℃,10000g)收集上清液。
(5)300mL上清液中加入UDPG至终浓度为1mM和雷酚内酯至终浓度为100μM,35℃水浴中反应24h。
(6)反应液用等体积的正丁醇萃取3次,将萃取液减压蒸发除去正丁醇,用10mL甲醇溶解产物。过0.22μm滤膜后,进样制备得到反应产物Gypenoside LXXV和YesanchinosideU。
(7)制备液相色谱条件
仪器:安捷伦制备液相(Agilent 1260);流动相A:水;流动相B:乙腈;10mL·min-1;进样量5mL;色谱柱:Waters C18 OBD(19×50mm,5μm);检测波长:203nm;0-5min:80%(A)-80%(A);5-17min:80%(A)-60%(A);17-30min:60%(A)-50%(A);30-35min:50%(A)-0%(A)。收集相应馏分,减压旋蒸除去水和乙腈,准确称量生成所得产物的质量。
(8)将制备得到的反应产物溶解于氘代甲醇至终浓度为6mg/mL,利用800M核磁检测人参皂苷Gypenoside LXXV和Yesanchinoside U的1H NMR、13C NMR谱图,通过与相应底物的核磁数据进行比较,分别确定Gypenoside LXXV为在人参皂苷CK的C20位糖链上延伸一个葡萄糖的糖基化产物,Yesanchinoside U为在人参皂苷F1的C20位糖链上延伸一个葡萄糖的糖基化产物,化合物相对应的碳谱和氢谱结果如图6-9所示。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (9)
1.一种UGT33基因,所述基因的核苷酸序列如SEQ ID No.1所示。
2.如权利要求1所述的UGT33基因的编码蛋白,所述编码蛋白的氨基酸序列如SEQ IDNO.2所示。
3.一种如权利要求1所述的UGT33基因和/或如权利要求2所述的编码蛋白在用于催化原人参二醇类化合物人参皂苷CK、Rh2和F2中的应用。
4.如权利要求3所述的应用,其特征在于,所述应用为UGT33基因和/或其编码蛋白催化人参皂苷CK、Rh2和F2的C3位和C20位糖链延伸。
5.如权利要求3或4所述的应用,其特征在于,所述应用为催化人参皂苷CK生成多糖苷Gypenoside LXXV、催化人参皂苷Rh2生成人参皂苷Rg3以及Gypenoside XVII,催化人参皂苷F2生成人参皂苷Rd和人参皂苷Rb1。
6.一种如权利要求1所述的UGT33基因和/或如权利要求2所述的编码蛋白在用于催化原人参二醇类化合物人参皂苷F1中的应用。
7.如权利要求6所述的应用,其特征在于,所述应用为催化原人参三醇类化合物人参皂苷F1的C20位糖链延伸。
8.如权利要求6或7所述的应用,其特征在于,所述应用为催化原人参三醇类化合物人参皂苷F1生成双糖苷Yesanchinoside U。
9.一种如权利要求1所述的UGT33基因和/或如权利要求2所述的编码蛋白在用于制备多糖苷Gypenoside LXXV、人参皂苷Rg3、Gypenoside XVII、人参皂苷Rd、人参皂苷Rb1和/或双糖苷Yesanchinoside U中的应用。
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