CN117551627A - 黑龙骨中黄酮糖苷类糖基转移酶PfUGT92A8及其编码基因与应用 - Google Patents
黑龙骨中黄酮糖苷类糖基转移酶PfUGT92A8及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了黑龙骨中黄酮糖苷类糖基转移酶PfUGT92A8及其编码基因与应用。所述糖基转移酶PfUGT92A8的氨基酸序列如SEQ ID NO:2所示。所述糖基转移酶PfUGT92A8的编码基因的核苷酸序列如SEQ ID NO:1所示。本发明旨在揭示黑龙骨中参与黄酮糖苷类成分生物合成的糖基转移酶,经本发明实验证明,糖基转移酶PfUGT92A8能够催化芹菜素生成芹菜素‑7‑O‑葡萄糖苷,以及催化木犀草素生成木犀草素‑7‑O‑葡萄糖苷,为芹菜素‑7‑O‑葡萄糖苷和木犀草素‑7‑O‑葡萄糖苷的体外合成及大规模应用奠定基础。
Description
技术领域
本发明涉及生物技术领域,特别地涉及黑龙骨中黄酮糖苷类糖基转移酶PfUGT92A8及其编码基因与应用。
背景技术
黄酮苷类化合物广泛存在于植物界,在植物胁迫响应以及生长发育调控中具有关键作用,同时黄酮苷类成分较多存在于食品和药品中,具有良好的生物活性。药用植物黑龙骨(Periploca forrestii Schltr)为夹竹桃科(Apocynaceae)杠柳属(Periploca L.)物种,其干燥根或全株可入药(黑骨藤),药用历史悠久,具有舒筋活络、祛风除湿、清热解毒等作用。此外,黑骨藤追风活络胶囊、黑骨藤液等中成药也广泛应用于临床。已有研究表明黄酮类成分是黑龙骨中重要活性成分之一,但其合成机制尚未有研究。
芹菜素和木犀草素C-7位羟基的葡萄糖基化可以形成芹菜素-7-O-葡萄糖苷和木犀草素-7-O-葡萄糖苷。现代药理学研究证明芹菜素-7-O-葡萄糖苷具有体外抗氧化及抗炎等活性,木犀草素-7-O-葡萄糖苷能够减轻心肌损伤,具有较好的药理价值和应用前景。然而,植物来源的芹菜素-7-O-葡萄糖苷和木犀草素-7-O-葡萄糖苷含量低,限制了其发展与应用,同时不利于中药资源的可持续发展。
植物中黄酮类成分的糖基化反应由尿苷二磷酸活化的糖基转移酶(UDP-glycosyltransferase,UGT)家族参与完成,目前已被鉴定功能的糖基转移酶仍不到UGT总数量的1%。UGT92家族是UGT中分布较少的一个家族,已报道大豆中UGT92G1和UGT92G2对槲皮素等黄烷醇类化合物具有糖基化活性,七叶树中UGT92G7可催化香豆素类化合物的糖基化。
发明内容
有鉴于此,本发明提出黑龙骨中黄酮糖苷类糖基转移酶PfUGT92A8及其编码基因与应用。
本发明的技术方案如下:
一种蛋白,所述蛋白的氨基酸序列如SEQ ID NO:2所示。
本发明还提供了所述蛋白的编码基因,所述编码基因的核苷酸序列如SEQ ID NO:1所示。
将该基因命名为PfUGT92A8,将其编码的蛋白命名为PfUGT92A8。具体信息如下:PfUGT92A8基因序列如序列表中序列1所示,序列1含有1581个核苷酸,其编码序列表中序列2所示的蛋白,序列2由526个氨基酸组成。
含有所述编码基因的表达盒、重组表达载体或重组菌也属于本发明的保护范围。
扩增所述编码基因全长的引物对也属于本发明的保护范围,所述引物对中,一条引物序列如SEQ ID NO:3所示,另一条引物序列如SEQ ID NO:4所示。
所述蛋白在作为糖基转移酶中的应用也属于本发明的保护范围。
所述糖基转移酶为催化芹菜素生成芹菜素-7-O-葡萄糖苷的糖基转移酶或催化木犀草素生成木犀草素-7-O-葡萄糖苷的糖基转移酶。
所述蛋白在催化芹菜素生成芹菜素-7-O-葡萄糖苷中的应用也属于本发明的保护范围。
所述蛋白在催化木犀草素生成木犀草素-7-O-葡萄糖苷中的应用也属于本发明的保护范围。
所述编码基因在催化芹菜素生成芹菜素-7-O-葡萄糖苷中的应用也属于本发明的保护范围。
所述编码基因在催化木犀草素生成木犀草素-7-O-葡萄糖苷中的应用也属于本发明的保护范围。
本发明旨在揭示黑龙骨中参与黄酮糖苷类成分生物合成的糖基转移酶。经本发明实验证明,糖基转移酶PfUGT92A8能够催化芹菜素生成芹菜素-7-O-葡萄糖苷,以及催化木犀草素生成木犀草素-7-O-葡萄糖苷,为芹菜素-7-O-葡萄糖苷和木犀草素-7-O-葡萄糖苷的生物合成提供基因元件,同时为芹菜素-7-O-葡萄糖苷和木犀草素-7-O-葡萄糖苷的绿色高效合成和大规模应用奠定基础。
附图说明
为了说明而非限制的目的,现在将根据本发明的优选实施例、特别是参考附图来描述本发明,其中:
图1为基于黑龙骨转录组的糖基转移酶(UGTs)家族成员鉴定及系统进化分析。
图2为黑龙骨中99个UGTs编码基因在其不同组织部位中的差异表达。
图3为黑龙骨PfUGT92A8体外催化芹菜素的超高效液相色谱鉴定结果。
图4为黑龙骨PfUGT92A8体外催化木犀草素的超高效液相色谱鉴定结果。
图5为黑龙骨PfUGT92A8体外催化芹菜素产物峰1A的质谱鉴定结果。
图6为黑龙骨PfUGT92A8体外催化木犀草素产物峰2L的质谱鉴定结果。
具体实施方式
以下结合实例详细说明本发明。实施例是为更好的理解本发明,但不限定于本发明。以下实施方法中的实验方法均为常规方法,所涉及的实验试剂均为常规生化试剂。
实施例1、基于黑龙骨全长转录组和二代转录组数据UGTs基因的筛选及系统进化分析
1.1实验方法
利用PacBio Sequel平台对黑龙骨根茎叶混合样品进行全长转录组建库测序,通过全长序列识别、isoform水平聚类得到一致序列和一致序列校正最终得到非冗余转录本序列。使用Trans Decoder软件从转录本序列中识别可靠的潜在编码区序列。基于黑龙骨三代全长转录组信息,从TAIR数据库中提取拟南芥的UGTs蛋白序列对黑龙骨进行BLASTP,寻找同源基因,将所得到的序列提交至Pfam数据库验证UGT的保守结构域,同时提交MEME网站验证PSPG-box,没有UGT保守结构与和PSPG-box的氨基酸序列将被排除。使用MAFFT完成多序列比对。采用IQtree和最大似然(Maximum likelihood)方法构建系统进化树,bootstrap选择重复1000次。同时,通过Illumina RNA-seq高通量测序平台,对黑龙骨根、茎、叶进行二代测序。使用三代全长转录本为参考,利用HiSAT2将二代转录组获得的Clean Reads与三代转录本进行序列比对,获取转录本位置信息。利用RSEM软件对转录本在不同组织部位表达水平进行定量分析,计算FPKM值。采用DESeq2软件进行差异表达分析,进一步筛选出表达量较高的PfUGTs基因。
1.2结果与分析
基于黑龙骨三代全长转录组数据,共筛选出了99个UGTs具有PSPG-box的保守结构域,系统进化分析将99个UGTs聚类于17个亚组(图1)。基于二代转录组分析,发现PfUGT92A8在黑龙骨根、茎、叶中均较高表达(图2),推测可能具有糖基转移酶活性。
实施例2、候选PfUGT92A8基因的克隆与表达载体构建
2.1实验方法
利用同源重组法获得pET32a-PfUGT92A8表达载体,选用BamHI和SalI为双酶切位点,设计引物序列(见表1),以黑龙骨叶片的cDNA为模板,利用KOD oneTM PCR master mix高保真酶(购自东洋纺上海生物科技有限公司,产品编号为KMM-101)克隆到PfUGT92A8基因片段(克隆体系程序总体积为50μL:25μL KOD oneTM PCR master mix,5μL模板,1.5μL正向引物,1.5μL反向引物和17μL水,程序见表2),采用诺唯赞同源重组试剂盒(购自南京诺唯赞生物科技有限公司,产品编号为C112)将获得的基因片段连接到pET32a载体(购自北京启研生物科技有限公司,产品编号为QYV0649),连接体系直接转化至TransT1感受态(购自北京全式金生物技术有限公司,产品编号为CD501),挑选阳性克隆测序(菌落PCR体系总体积为20μL:10μL 2×Taq PCR Mix,1μL模板,1μL正向引物,1μL反向引物和7μL水,程序见表3)。
表1构建pET32a-PfUGT92A8表达载体引物序列
表2KOD高保真酶PCR反应程序
表3菌落PCR反应程序
2.2结果与分析
PfUGT92A8基因序列如序列表中序列1所示,序列1含有1581个核苷酸,其编码序列表中序列2所示的蛋白,序列2由526个氨基酸组成,将该蛋白命名为PfUGT92A8。
实施例3、候选UGTs编码基因的功能验证
3.1实验方法
将上述获得的pET32a-PfUGT92A8表达载体转化至BL21(DE3)大肠杆菌表达菌株(购自天根生化科技(北京)有限公司,产品号为CB105)中。取过夜诱导的菌液2mL加入50mLLB液体培养基中(含有50μg/mL氨苄青霉素),37℃,200rpm活化至OD600值为0.6-0.8,加入终浓度为0.3mM的IPTG进行诱导,16℃,110rpm诱导24h,离心取菌体沉淀进行蛋白纯化。
将纯化的蛋白在体外按照以下反应体系进行功能验证(200μL):1×磷酸盐缓冲液(PBS,pH 7.4),50μg纯化的蛋白,1mM UDP-Glucose(购自默克Sigma-Aldrich公司,产品编号为670120),40μM的芹菜素或木犀草素(均购自武汉琼格生物科技有限公司)。用甲醇终止反应后混匀过0.22μm滤膜,利用UPLC检测其化学成分。仪器型号:Nexera UPLC LC-20Asystem,SHIMADZU,Japan。进样量3μL,色谱柱:Waters AcquityBEH C18column(1.7μm,100×2.1mm),柱温:40℃。色谱条件:UV 330nm,流动相:(A):水(含0.1%甲酸),(B):乙腈,流速:0.25mL/min,洗脱程序:0-2min,5% B,2-10min,线性增长至18% B;10-12min,18%B线性增长至95% B;12-14min,95% B;14-14.5min,95% B回到起始状态5% B。利用Agilent Technologies 1290InfinityⅡ和6530Q-TOF液质联用仪器检测产生的反应提取出来的产物,进行定性分析。
3.2结果与分析
体外催化结果显示PfUGT92A8能够将芹菜素转化产生2个产物峰(1A、2A),其中1个产物峰(1A)与芹菜素-7-O-葡萄糖苷标准品具有相同的保留时间(图3);PfUGT92A8能够将木犀草素转化产生6个产物峰(1L、2L、3L、4L、5L、6L),其中产物峰2L与木犀草素-7-O-葡萄糖苷标准品具有相同的保留时间(图4)。
以上描述均在液相色谱检测基础上判断,产物的定性分析需要四级杆串联飞行时间质谱仪(Q-TOF)检测进一步确定。
实施例4、催化产物的定性分析
4.1实验方法
利用Agilent Technologies 1290InfinityⅡ和6530Q-TOF液质联用仪器检测产生的反应提取出来的产物,进行定性分析。进样量1μL,色谱柱:Waters AcquityBEH C18column(1.7μm,100×2.1mm),柱温:30℃。色谱条件:UV 330nm,流动相:(A):水(含0.1%甲酸),(B):乙腈,流速:0.25mL/min,洗脱程序:0-2min,5% B,2-10min,线性增长至18% B;10-12min,18% B线性增长至95% B;12-14min,95% B;14-14.5min,95% B回到起始状态5% B。Q-TOF质谱参数为:ESI离子源,负离子模式,50-1000m/z;干燥气体温度为300℃,流速6L/min;鞘气温度为300℃,流速11.0L/min;喷雾器为30psi;VCap为2000V;在Auto-MS/MS模式和Target-MS/MS模式下检测代谢物;利用30V碰撞能量获得离子碎片。
4.2结果与分析
基于实施例3的液相结果,对PfUGT92A8催化芹菜素和木犀草素的产物峰进行二级质谱分析。结果显示:PfUGT92A8催化芹菜素的产物峰中,与芹菜素-7-O-葡萄糖苷具有相同保留时间的产物峰1A,同时具有与芹菜素-7-O-葡萄糖苷相同的质谱裂解规律([M-H]-:m/z=431.0995、[M-H-Glu]-:m/z=269.0463)(图5)。此外,PfUGT92A8体外催化木犀草素的产物峰中,与木犀草素-7-O-葡萄糖苷具有相同保留时间的产物峰2L,与木犀草素-7-O-葡萄糖苷的质谱裂解规律一致([M-H]-:m/z=447.0928、[M-H-Glu]-:m/z=285.0406)(图6)。因此,结合实施例3的液相数据与实施例4的质谱结果,确定糖基转移酶PfUGT92A8具有催化芹菜素生成芹菜素-7-O-葡萄糖苷的糖基转移酶功能,以及催化木犀草素生成木犀草素-7-O-葡萄糖苷的糖基转移酶功能。
上述具体实施方式,并不构成对本发明保护范围的限制。本领域技术人员应该明白的是,取决于设计要求和其他因素,可以发生各种各样的修改、组合、子组合和替代。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明保护范围之内。
Claims (10)
1.一种蛋白,所述蛋白的氨基酸序列如SEQ ID NO:2所示。
2.权利要求1所述蛋白的编码基因,其特征在于:所述编码基因的核苷酸序列如SEQ IDNO:1所示。
3.含有权利要求2所述编码基因的表达盒、重组表达载体或重组菌。
4.扩增权利要求2所述编码基因全长的引物对,所述引物对中,一条引物序列如SEQ IDNO:3所示,另一条引物序列如SEQ ID NO:4所示。
5.权利要求1所述的蛋白在作为糖基转移酶中的应用。
6.根据权利要求5所述的应用,其特征在于:所述糖基转移酶为催化芹菜素生成芹菜素-7-O-葡萄糖苷的糖基转移酶或催化木犀草素生成木犀草素-7-O-葡萄糖苷的糖基转移酶。
7.权利要求1所述的蛋白在催化芹菜素生成芹菜素-7-O-葡萄糖苷中的应用。
8.权利要求1所述的蛋白在催化木犀草素生成木犀草素-7-O-葡萄糖苷中的应用。
9.权利要求2所述的编码基因在催化芹菜素生成芹菜素-7-O-葡萄糖苷中的应用。
10.权利要求2所述的编码基因在催化木犀草素生成木犀草素-7-O-葡萄糖苷中的应用。
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