甜叶悬钩子和明日叶来源UGT基因的发掘、克隆及功能鉴定

发布时间：2018-04-25 12:38

本文选题：甜叶悬钩子 + 明日叶　；参考：《上海海洋大学》2017年硕士论文

【摘要】：在植物中很多具有活性的天然产物以糖苷的形式存在,其糖苷化由糖基转移酶(glycosyltransferase,GT),主要是UDP-糖基转移酶(UGT)催化产生。研究这些酶对于认识植物中功能性天然产物糖苷的形成和利用有积极意义。相比于传统的植物提取天然产物的方式,生物合成通常具有高效、安全、经济等特点。随着生物技术的发展,利用生物方法合成有价值的有效成分已经成为提高天然产物产量和改善天然产物品质的重要途径。利用高通量转录组测序的方式挖掘关键酶基因,克隆以及在合适的系统中进行体外高效表达,为研究天然产物生物合成提供了重要的平台。甜菊糖苷(steviol glycosides)是一类以贝壳杉烯(kaurane)为母核的四环二萜类糖苷化合物,共同的苷元为甜菊醇(steviol),其中的一些成分具有极高的甜度,尤其是菊科植物甜叶菊中提取的甜叶菊糖苷(stevioside),因其高甜度、低热量、安全无毒等特性而作为代糖添加剂广泛应用于食品、饮料等工业生产中[1-2],被誉为继蔗糖和甜菜糖之后的“世界第三类糖源”[3-4]。甜菊糖苷迄今仅在四个植物物种中被报道过,分别是菊科甜菊属的甜叶菊(Stevia rebaudiana)和Stevia phlebophylla A.Gray[5]、蔷薇科悬钩子属甜叶悬钩子(Rubus suavissimus S.Lee)[6]以及伞形科当归属明日叶(Angelica keiskei(Miq.)Koidz.)[7]。本研究选择甜叶悬钩子和明日叶两种植物作为研究对象,以两种植物的叶片高通量测序获得的转录组数据为基础,分别从转录组数据分析、酶基因的挖掘与克隆、基因的异源表达、酶功能测定等方面研究甜叶悬钩子和明日叶中涉及贝壳杉烯型四环二萜化合物生物合成的糖基转移酶。本文的研究内容主要包括以下几个方面:1)甜叶悬钩子(Rubus suavissimus S.Lee),又名甜茶、甜叶覆盆子,属于蔷薇科悬钩子属,是广西特有的野外珍稀天然植物[8-10]。甜叶悬钩子能生产与甜叶菊中结构类似的贝壳杉烯型四环二萜糖苷类化合物,但涉及其合成途径的关键UGT尚不明确。本研究基于甜叶悬钩子叶片转录组数据,对其中注释的191条推测UGT基因序列进行生物信息学分析,选择部分序列(氨基酸数250~600)与已知的植物UGT构建进化树,筛选出62条可能和甜菊糖苷型化合物合成有关的具有完整全长的糖基转移酶基因,成功克隆了其中的40条,将其甜菊醇类底物共同孵育后筛选出具有相应底物糖基化活性的UGT六条,其中四条具有类似UGT74G1酶活性,催化甜菊醇母核的19位羧基O的糖基化,一条具有类似UGT85C2酶活性,催化甜菊醇的13位糖基化,另有一条具有类UGT91D2活性,可能催化steviol-19-O-β-D-glucoside的双糖基化。研究完成了甜叶悬钩子中从甜菊醇到甜茶素(rubusoside)合成路径中两步糖基化所需的UGT的鉴定。此外,测定了RsUGT41转化甜菊醇生成steviol-19-O-β-D-glucoside的酶动力学参数。2)明日叶(Angelica keiskei(Miq.)Koidz.),属伞形科当归属多年生草本植物。本研究针对明日叶叶片转录组数据进行分析,获得151条注释为UGT序列,对这些推测的UGT序列进行系统进化分析,对明日叶UGT(AkUGT)进行家族归类和分组,对A、D、E、G、H及L六组中的82条UGT序列进行进一步分析,临时编号为AkUGT1~82。筛选以上82条序列中具有完整ORF和基因全长序列23条,对其进行全长克隆和重组质粒构建,最终克隆得到序列21条,90%的克隆序列与原cDNA序列匹配度99%。将重组质粒转入大肠杆菌表达宿主中表达,表达粗酶液分别与甜菊醇(steviol)、瑞鲍迪苷A(Rebaudioside A)、甜菊单糖苷(steviolmonoside)、甜菊双糖苷(steviolbioside)以及steviol-19-O-β-D-glucoside底物共同孵育,测定明日叶UGT转化活性。最终得到两条与UGT74G1功能相似的AkUGT:AkUGT49和AkUGT50,均可催化甜菊醇19位羧基的糖基化,生成产物鉴定为steviol-19-O-β-D-glucoside。3)通过对转录组数据的发掘,以及基因的克隆和重组酶的酶活性鉴定,我们最终从甜叶悬钩子和明日叶中筛选到了八条具有贝壳杉烯型系列底物活性的UGT,我们对这八条序列进行了理化性质、序列多重比对以及蛋白功能域预测等初步分析,为进一步研究其功能特性奠定了基础。
[Abstract]:Many natural products in plants exist in the form of glucoside, which are catalyzed by glycosyltransferase (GT) and mainly by UDP- glycosyltransferase (UGT). These enzymes are positive for understanding the formation and utilization of glycosides of functional natural products in plants. The biosynthesis of natural products is usually characterized by high efficiency, safety and economy. With the development of biotechnology, the use of biological methods to synthesize valuable effective components has become an important way to improve the production of natural products and improve the quality of natural products. And the efficient expression in the suitable system in vitro provides an important platform for the study of the biosynthesis of natural products. Steviol glycosides is a class of four ring two terpenoid glycosides with kaurane as the parent nucleus, and the common glycoside is steviol (steviol), some of which have high sweetness. Especially, stevioside, which is extracted from the Compositae, is widely used in food, beverage and other industrial production for its high sweetness, low calorie, safety and non-toxic properties. [1-2] is known as the "world third sugar source" [3-4]. stevioside, followed by sucrose and beet sugar, so far only four plants have been planted. The species are reported in the species of Chrysanthemum (Stevia rebaudiana) and Stevia phlebophylla A.Gray[5], the Rubus of the Rosaceae (Rubus suavissimus S.Lee) [6] and the umbel belonging to the day leaf (Angelica keiskei) (Angelica keiskei (Miq.)). As the research object, based on the transcriptional data obtained from the high throughput sequencing of the leaves of two plants, the biosynthesis of the biosynthesis of the four ring two terpene compounds in the leaf Rubus and the leaf of the Sequoia is studied from the analysis of the transcriptional data, the mining and cloning of the enzyme gene, the heterologous expression of the gene and the determination of the enzyme function. The main contents of this study include the following aspects: 1) Rubus suavissimus S.Lee, also known as sweet tea, sweet leaf raspberry, and raspberry of Rosaceae. It is a rare natural wild plant of Guangxi, [8-10]. sweet leaf Rubus, which can produce four ring two terpene glycosides similar to the sweet Ye Juzhong structure. Compounds, but the key UGT involved in the synthesis pathway is not clear. Based on the data of the leaf raspberry leaf transcriptome, 191 of the annotated UGT gene sequences were analyzed by bioinformatics, and a partial sequence (amino acid number 250~600) was selected to construct an evolutionary tree with the known plant UGT, and 62 possible and stevioside types were screened. A complete and fully long glycosyltransferase gene related to compound synthesis was successfully cloned, and 40 of them were cloned successfully. After CO incubation of their steviool substrates, six UGT were screened with corresponding substrate glycosylation activity, of which four had a glycosylation of 19 bit carboxyl O, which had similar activity to the parent of steviol, one of which was similar. The activity of UGT85C2 enzyme catalyzes the 13 glycosylation of steviol, and another with UGT91D2 like activity and may catalyze the biglycylation of steviol-19-O- beta -D-glucoside. The study completed the identification of the UGT for glycosylation of the sweet leaf Rubus from steviol to rubusoside (rubusoside) synthesis path. In addition, the RsUGT41 conversion of steviol was determined. The enzyme kinetic parameter.2 of steviol-19-O- beta -D-glucoside).2 (Angelica keiskei (Miq.) Koidz.), which belongs to the perennial herb of umbelgus congener. In this study, the data of the leaf leaf transcriptome were analyzed and 151 notes were annotated as UGT sequences. UGT) family classification and grouping, further analysis of 82 UGT sequences in the six groups of A, D, E, G, H and L, and temporarily numbered 23 of the 82 sequences with complete ORF and gene sequence in the 82 sequences of AkUGT1~82. screening. The whole long clones and recombinant plasmids were constructed, and 21 sequences, 90% cloned sequences and the original cDNA sequences were cloned. The recombinant plasmid was transferred into the Escherichia coli expression host and expressed in the host, and the crude enzyme solution was incubated with steviol (steviol), baubodi A (Rebaudioside A), Stevia monoglycoside (steviolmonoside), stevia double glycoside (steviolbioside) and steviol-19-O- beta -D-glucoside substrate, respectively, to determine the activity of UGT transformation in the leaves of 99%.. Two AkUGT:AkUGT49 and AkUGT50, similar to UGT74G1 functions, can catalyze the glycosylation of the 19 bit carboxyl of steviol, and the product is identified as steviol-19-O- beta -D-glucoside.3). Through the discovery of the data of the transcriptional group, and the cloning of the gene and the identification of the enzyme activity of the recombinant enzyme, we were finally screened from the sweet leaf Rubus and the leaf of the bright leaf. Eight UGT with the substrate activity of a series of beta cells, we have made a preliminary analysis on the physical and chemical properties of the eight sequences, the multiple alignment of the sequence and the prediction of the functional domain of the protein, which laid the foundation for further study of its functional properties.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;O629

【参考文献】