CN117867058A - 枇杷黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因及其编码蛋白和应用 - Google Patents
枇杷黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因及其编码蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种黄酮醇3‑O‑半乳糖基转移酶EjUGT78T4基因,该基因从枇杷果实中分离获得,核苷酸序列如SEQ:NO.1所示,其编码蛋白的氨基酸序列如SEQ:NO.2所示。本发明首次克隆并验证了枇杷黄酮醇3‑O‑半乳糖苷合成相关的EjUGT78T4基因的功能。本发明通过构建重组质粒,实现了EjUGT78T4基因在大肠杆菌中的重组表达,提供转基因工程菌。在体外,EjUGT78T4重组蛋白可将黄酮醇转化为黄酮醇3‑O‑半乳糖苷。本发明可应用于基于基因工程的植物基因组改造,定向改良植物黄酮醇组分,增加食物的保健功能;也可应用于基于代谢工程的黄酮醇糖苷工业化生产。
Description
技术领域
本发明属于植物分子生物技术和基因工程领域,涉及一种枇杷黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因及其编码蛋白和应用。
背景技术
枇杷(Eriobotrya japonica)是蔷薇科枇杷属的常绿果树,原产中国,西汉司马迁所作的《上林赋》最早记载了我国枇杷栽培,迄今已有2000多年的历史。枇杷秋冬开花,春夏果实成熟,是开春之后较早应市的水果。由于其果肉酸甜适度、风味好,颇受消费者欢迎,加之枇杷叶片又是传统的中草药,使得枇杷在我国南方都有一定的种植规。枇杷的叶、花、果具有多种药理功效,如止咳化痰平喘、抗炎、治疗糖尿病、护肝、抗肿瘤、抗氧化、抗病毒、增强免疫功能等。
枇杷花、叶、果中均富含丰富的黄酮醇3-O-半乳糖苷,包括槲皮素3-O-半乳糖苷和山奈酚3-O-半乳糖苷。现代研究证明,黄酮醇3-O-半乳糖苷具有广泛的药理学活性,例如,槲皮素3-O-半乳糖苷具有抗癌、抗炎、抗菌、抗病毒、抗抑郁等活性,这些药理特性为其用于治疗多种疾病,如败血症、关节炎、结肠炎、糖尿病和癌症等奠定基础。黄酮醇通常以糖苷形式存在于植物细胞的液泡中,黄酮醇糖基化在细胞质中由糖基转移酶(glycosyltransferase,GT,EC2.4.x.y)催化发生。糖基化是植物中广泛存在的化合物修饰方式,也是许多次生代谢产物合成反应的最后一步。糖基化可以改变黄酮醇化合物的亲水性,增加其溶解度和化学稳定性,影响其生物活性,有助于其在细胞内和生物体内的储存和转运等。
枇杷中含有大量槲皮素3-O-半乳糖苷,是决定枇杷生物学活性的重要组成成分。尿苷二磷酸糖依赖的糖基转移酶(UDP-glycosyltransferases,UGTs)是参与枇杷中黄酮醇3-O-半乳糖苷合成的关键酶,因此鉴别相关基因,阐明枇杷黄酮醇糖苷生物合成途径具有重要意义。可应用于基于基因工程的植物基因组改造,定向改良黄酮醇组分,增加食物的保健功能;也可应用于基于代谢工程的黄酮醇糖苷工业化生产,对提高黄酮醇产量,提高药品生物安全性奠定基础。
发明内容
本发明的目的是提供一种参与枇杷黄酮醇3-O-半乳糖苷生物合成的黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因及其编码蛋白,所述半乳糖基转移酶至少具有下列1)~2)特征之一:
1)所述EjUGT78T4基因的核苷酸序列如SEQ:NO.1所示;编码序列全长为1440个核苷酸;
2)所述EjUGT78T4基因编码蛋白的氨基酸序列如SEQ:NO.2所示;可编码一个含479个氨基酸的蛋白。
本发明提供的一种黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因,是从枇杷果实中分离获得,为一种尿苷二磷酸半乳糖(UDP-半乳糖)依赖的黄酮醇3-O-半乳糖基转移酶。本发明的另一个目的是提供所述黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因及其编码蛋白在合成黄酮醇3-O-半乳糖苷中的应用。将上述黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因连接到pET32a载体的多克隆位点中构建重组质粒,命名为pET32a-EjUGT78T4。在大肠杆菌中表达重组质粒pET32a-EjUGT78T4,得到EjUGT78T4重组蛋白,重组蛋白可利用UDP-半乳糖作为糖基供体将底物黄酮醇转化成产物黄酮醇3-O-半乳糖苷。
本发明提供了一种黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因及其编码蛋白和应用,进一步的所述的黄酮醇3-O半乳糖苷选自槲皮素3-O-半乳糖苷和山奈酚3-O-半乳糖苷,首次克隆并验证了枇杷黄酮醇3-O-半乳糖苷生物合成相关糖基转移酶EjUGT78T4基因的功能,在体外,EjUGT78T4重组蛋白可将槲皮素和山奈酚分别转化成槲皮素3-O-半乳糖苷和山奈酚3-O-半乳糖苷。
本发明还提供一种重组质粒及其在生物合成黄酮醇3-O半乳糖苷中的应用,所述重组质粒包含3-O-半乳糖基转移酶EjUGT78T4基因,所述EjUGT78T4基因的核苷酸序列如SEQ:NO.1所示。
本发明还提供一种转基因工程菌及其在生物合成黄酮醇3-O半乳糖苷中的应用,所述转基因工程菌包含3-O-半乳糖基转移酶EjUGT78T4基因,所述半乳糖基转移酶至少具有下列1)~2)特征之一:
1)所述EjUGT78T4基因的核苷酸序列如SEQ:NO.1所示;
2)所述EjUGT78T4基因编码蛋白的氨基酸序列如SEQ:NO.2所示。
进一步的,所述重组质粒或转基因工程菌可通过代谢工程方法大量合成黄酮醇3-O-半乳糖苷。本发明提供了一种能大量合成黄酮醇3-O-半乳糖苷的途径,为进一步开展黄酮醇糖苷生物合成调控研究奠定基础。
附图说明
图1为枇杷EjUGT78T4蛋白与其他植物UGT系统发育树分析图。
AcF3GT1(GU079683),AcUFGT3a(A0A2R6Q8R5),AtF5GlcT(AAM91686),AtF7GlcT(AT4G34138),AtUGT78D1(At1g30530),AtUGT78D2(At5g17050),AtUGT78D3(At5g17030),AtUGT79B1(At5g54060),AtUGT79B6(At5g54010),CaUGT3(AB443870),Cm1,2RhaT(AY048882),Cs1,6RhaT(DQ119035),CsF7GlcT(ALO19892),CsUGT78A14(KP682360),CsUGT78A15(KP682361),FaGT1(AAU09442),FaGT7(ABB92749),FeF3G6RhaT(LC312144),GeIF7GlcT(BAC78438),GhA5GlcT(BAA36423),GmF3G2Gt(NM_001359019),GmF3G6RhaT(AB828193),Gt5GT7(BAG32255),Ip3GGT(AB192315),MrUFGT(KAB1205527),MrUGT78R1(KAB1224450),MrUGT78R2(KAB1224448),MrUGT78W1(MZ727195),PfA5GlcT(BAA36421),PhA5GlcT(BAA89009),PhF3GalT(AAD55985),PpUGT78A2(Prupe.1g091000),PpUGT78T3(Prupe.2g324700),PpUGT91AK6(Prupe.2g175000),SbF7GlcT(BAA83484),ThA5GlcT(BAC54093),Va5GT(KF996717),VvGT1(AB047092),VvGT5(AB499074),VvGT6(AB499075).
图2为EjUGT78T4氨基酸序列SEQ:NO.2与其他植物UDP-鼠李糖基转移酶氨基酸比对结果图。
图3为枇杷EjUGT78T4重组蛋白的SDS-PAGE分析图。
图4为重组蛋白EjUGT78T4对槲皮素和山奈酚体外酶活性分析HPLC色谱图。
图5为黄酮醇3-O-半乳糖基转移酶EjUGT78T4催化模式图;以UDP-半乳糖为糖基供体,槲皮素和山奈酚为糖基受体,经EjUGT78T4催化生成槲皮素3-O-半乳糖苷和山奈酚3-O-半乳糖苷。
具体实施方式
下面对本发明的实施例和附图作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:枇杷RNA提取及EjUGT78T4基因克隆
一、实验方法
1、材料:以‘硬条白沙’枇杷果实为材料,设置三个生物学重复,每个重复4-6个果实,取果实果皮组织,迅速用液氮冻透,放至-80℃冰箱中保存。
2、利用CTAB法提取枇杷果皮的RNA,按照PrimeScriptTMRT reagent Kit withgDNA Eraser(Takara)试剂说明书操作合成cDNA。以反转录产物cDNA为模板,用SEQ:NO.3和SEQ:NO.4所示引物进行PCR扩增,PCR反应体系为50μL,成分分别为:2×Phanta Max Buffer25μL,dNTP Mix(10mM each)1μL,DNApolymerse(1U/μL)1μL,上下游引物(10μM)各2μL,cDNA1μL,H2O 18μL。PCR程序为:95℃预变性3min,35个循环的95℃15s,58℃15s和72℃1min30s,72℃5min,4℃hold。得到扩增产物。
3、将PCR扩增产物连接到T-easy载体,转化大肠杆菌DH5α,挑取单菌落进行菌落PCR验证,获得阳性菌落进行测序。
二、实验结果
1、测序结果返还之后经过比对分析得到与枇杷基因组预测编码序列相匹配的EjUGT78T4基因序列SEQ:NO.1,含有1440个核苷酸,编码479个氨基酸的蛋白质,如SEQ:NO.2所示。将SEQ:NO.2与已报道其他植物UGT进行系统发育树分析,得到图1所示结果。图中红色圆点标注的即为EjUGT78T4。
2、利用EjUGT78T4氨基酸序列与部分已发表具有半乳糖基转移功能的糖基转移酶比对,结果如图2所示。他们共同含有UDP糖基转移酶保守序列PSPG-box。
实施例2:EjUGT78T4基因的原核表达
一、实验方法
1、设计带有表达载体pET32a载体的多克隆酶切位点的特异引物,其引物序列如SEQ:NO.5和SEQ:NO.6所示。
2、以测序正确T-easy载体为模板,用SEQ:NO.5和SEQ:NO.6所示引物进行PCR扩增,PCR反应体系为50μL,成分分别为:2×Phanta Max Buffer 25μL,dNTP Mix(10mM each)1μL,DNApolymerse(1U/μL)1μL,上下游引物(10μM)各2μL,模板1μL,H2O 18μL。PCR程序为:95℃预变性3min,35个循环的95℃15s,58℃15s和72℃1min30s,72℃5min,4℃hold。
3、将PCR扩增产物连接到用限制性内切酶BamHI和XhoI双酶切过的线性pET32a载体,获得pET32a-EjUGT78T4重组质粒。
4、将pET32a-EjUGT78T4重组质粒转化到大肠杆菌BL21(DE3)PlysS表达宿主菌中,经菌落PCR验证,挑取阳性菌落接种到500mL LB(Amp+)液体培养基,37℃培养,直至OD600为0.6~1.0,获得转基因工程菌。
5、在上述转基因的工程菌中加入IPTG至终浓度为0.5mM,16℃诱导24h,收集菌体,500mL收集到1管中,加入20mL 1×PBS缓冲液,充分重悬浮菌体,-80℃放置12h以上。将菌体置于30℃水浴锅解冻后,用超声破碎仪破碎5min。在4℃温度下,10000rpm离心30min,收集上清液。用Clontech HisTALON重力纯化试剂盒进一步纯化,获得目的蛋白。
二、实验结果
利用SDS-PAGE方法检测蛋白表达和纯化效果,结果如图3所示。图中可看出,加上重组标签后在70.75kDa左右有明显的重组蛋白条带,重组蛋白条带大小与预测的一致。纯化的蛋白可用于进一步的酶学分析。
实施例3:EjUGT78T4重组蛋白的酶学活性检测分析
一、实验方法
1、对于糖基供体和糖基受体溶液配制,糖基供体UDP-半乳糖用纯净水溶解为10mg/mL,糖基受体槲皮素和山奈酚用色谱级纯甲醇溶解为1mg/mL。
2、对于黄酮醇底物的酶活性检测,是在总体积50μL,0.1M pH 7.5的Tris-HCl缓冲液中进行,缓冲液包含1μL UDP-半乳糖作为糖基供体,2μL黄酮醇作为糖基受体,1μg纯化后的重组蛋白。
3、酶反应体系在30℃反应5min后加入等量甲醇停止反应,反应均以空载蛋白作为对照,获得酶反应产物。
4、酶反应产物经HPLC进行检测鉴定,所述HPLC检测条件如下:Waters 2695-2996DAD检测器,ODS C18柱(4.6×250mm)色谱柱。以含0.1%甲酸水溶液(溶液A)和含0.1%甲酸的100%乙腈(溶液B)为流动相,洗脱梯度为:0-7min,10%-50%B;7-10min,50%B;10-15min,50%-100%B;15-16min,100%-10%B;16-20min,10%B。检测波长为370nm,柱温为25℃,流速为1ml/min,进样体积为10μl。
二、实验结果
结果如图4所示,EjUGT78T4重组蛋白以UDP-半乳糖作为糖基供体,可选择性地催化槲皮素、山奈酚3-OH糖基化,生成与标准品一致的槲皮素3-O-半乳糖苷、山奈酚3-O-半乳糖苷,催化流程如图5所示,说明EjUGT78T4重组蛋白具有黄酮醇3-O-半乳糖基转移酶活性。
Claims (6)
1.一种3-O-半乳糖基转移酶EjUGT78T4基因在生物合成黄酮醇3-O半乳糖苷中的应用,其特征在于,所述半乳糖基转移酶至少具有下列1)~2)特征之一:
1)所述EjUGT78T4基因的核苷酸序列如SEQ:NO.1所示;
2)所述EjUGT78T4基因编码蛋白的氨基酸序列如SEQ:NO.2所示。
2.一种重组质粒在生物合成黄酮醇3-O半乳糖苷中的应用,其特征在于,所述重组质粒包含3-O-半乳糖基转移酶EjUGT78T4基因,所述EjUGT78T4基因的核苷酸序列如SEQ:NO.1所示。
3.一种转基因工程菌在生物合成黄酮醇3-O半乳糖苷中的应用,其特征在于,所述转基因工程菌包含3-O-半乳糖基转移酶EjUGT78T4基因,所述半乳糖基转移酶至少具有下列1)~2)特征之一:
1)所述EjUGT78T4基因的核苷酸序列如SEQ:NO.1所示;
2)所述EjUGT78T4基因编码蛋白的氨基酸序列如SEQ:NO.2所示。
4.根据权利要求1-3任一项所述的应用,其特征在于,所述的黄酮醇3-O半乳糖苷选自槲皮素3-O-半乳糖苷和山奈酚3-O-半乳糖苷。
5.一种黄酮醇3-O半乳糖苷的制备方法,其特征在于,将所述黄酮醇3-O-半乳糖基转移酶EjUGT78T4基因构建重组质粒pET32a-EjUGT78T4,通过在大肠杆菌中表达重组质粒pET32a-EjUGT78T4,得到EjUGT78T4重组蛋白,重组蛋白将底物黄酮醇转化成产物黄酮醇3-O-半乳糖苷。
6.一种黄酮醇3-O半乳糖苷的制备方法,其特征在于,所述重组质粒或转基因工程菌通过代谢工程方法合成黄酮醇3-O-半乳糖苷。
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