本文選題：甜葉懸鉤子 + 明日葉��； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：在植物中很多具有活性的天然產(chǎn)物以糖苷的形式存在,其糖苷化由糖基轉(zhuǎn)移酶(glycosyltransferase,GT),主要是UDP-糖基轉(zhuǎn)移酶(UGT)催化產(chǎn)生。研究這些酶對(duì)于認(rèn)識(shí)植物中功能性天然產(chǎn)物糖苷的形成和利用有積極意義。相比于傳統(tǒng)的植物提取天然產(chǎn)物的方式,生物合成通常具有高效、安全、經(jīng)濟(jì)等特點(diǎn)。隨著生物技術(shù)的發(fā)展,利用生物方法合成有價(jià)值的有效成分已經(jīng)成為提高天然產(chǎn)物產(chǎn)量和改善天然產(chǎn)物品質(zhì)的重要途徑。利用高通量轉(zhuǎn)錄組測(cè)序的方式挖掘關(guān)鍵酶基因,克隆以及在合適的系統(tǒng)中進(jìn)行體外高效表達(dá),為研究天然產(chǎn)物生物合成提供了重要的平臺(tái)。甜菊糖苷(steviol glycosides)是一類以貝殼杉烯(kaurane)為母核的四環(huán)二萜類糖苷化合物,共同的苷元為甜菊醇(steviol),其中的一些成分具有極高的甜度,尤其是菊科植物甜葉菊中提取的甜葉菊糖苷(stevioside),因其高甜度、低熱量、安全無(wú)毒等特性而作為代糖添加劑廣泛應(yīng)用于食品、飲料等工業(yè)生產(chǎn)中[1-2],被譽(yù)為繼蔗糖和甜菜糖之后的“世界第三類糖源”[3-4]。甜菊糖苷迄今僅在四個(gè)植物物種中被報(bào)道過(guò),分別是菊科甜菊屬的甜葉菊(Stevia rebaudiana)和Stevia phlebophylla A.Gray[5]、薔薇科懸鉤子屬甜葉懸鉤子(Rubus suavissimus S.Lee)[6]以及傘形科當(dāng)歸屬明日葉(Angelica keiskei(Miq.)Koidz.)[7]。本研究選擇甜葉懸鉤子和明日葉兩種植物作為研究對(duì)象,以兩種植物的葉片高通量測(cè)序獲得的轉(zhuǎn)錄組數(shù)據(jù)為基礎(chǔ),分別從轉(zhuǎn)錄組數(shù)據(jù)分析、酶基因的挖掘與克隆、基因的異源表達(dá)、酶功能測(cè)定等方面研究甜葉懸鉤子和明日葉中涉及貝殼杉烯型四環(huán)二萜化合物生物合成的糖基轉(zhuǎn)移酶。本文的研究?jī)?nèi)容主要包括以下幾個(gè)方面:1)甜葉懸鉤子(Rubus suavissimus S.Lee),又名甜茶、甜葉覆盆子,屬于薔薇科懸鉤子屬,是廣西特有的野外珍稀天然植物[8-10]。甜葉懸鉤子能生產(chǎn)與甜葉菊中結(jié)構(gòu)類似的貝殼杉烯型四環(huán)二萜糖苷類化合物,但涉及其合成途徑的關(guān)鍵UGT尚不明確。本研究基于甜葉懸鉤子葉片轉(zhuǎn)錄組數(shù)據(jù),對(duì)其中注釋的191條推測(cè)UGT基因序列進(jìn)行生物信息學(xué)分析,選擇部分序列(氨基酸數(shù)250~600)與已知的植物UGT構(gòu)建進(jìn)化樹,篩選出62條可能和甜菊糖苷型化合物合成有關(guān)的具有完整全長(zhǎng)的糖基轉(zhuǎn)移酶基因,成功克隆了其中的40條,將其甜菊醇類底物共同孵育后篩選出具有相應(yīng)底物糖基化活性的UGT六條,其中四條具有類似UGT74G1酶活性,催化甜菊醇母核的19位羧基O的糖基化,一條具有類似UGT85C2酶活性,催化甜菊醇的13位糖基化,另有一條具有類UGT91D2活性,可能催化steviol-19-O-β-D-glucoside的雙糖基化。研究完成了甜葉懸鉤子中從甜菊醇到甜茶素(rubusoside)合成路徑中兩步糖基化所需的UGT的鑒定。此外,測(cè)定了RsUGT41轉(zhuǎn)化甜菊醇生成steviol-19-O-β-D-glucoside的酶動(dòng)力學(xué)參數(shù)。2)明日葉(Angelica keiskei(Miq.)Koidz.),屬傘形科當(dāng)歸屬多年生草本植物。本研究針對(duì)明日葉葉片轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行分析,獲得151條注釋為UGT序列,對(duì)這些推測(cè)的UGT序列進(jìn)行系統(tǒng)進(jìn)化分析,對(duì)明日葉UGT(AkUGT)進(jìn)行家族歸類和分組,對(duì)A、D、E、G、H及L六組中的82條UGT序列進(jìn)行進(jìn)一步分析,臨時(shí)編號(hào)為AkUGT1~82。篩選以上82條序列中具有完整ORF和基因全長(zhǎng)序列23條,對(duì)其進(jìn)行全長(zhǎng)克隆和重組質(zhì)粒構(gòu)建,最終克隆得到序列21條,90%的克隆序列與原cDNA序列匹配度99%。將重組質(zhì)粒轉(zhuǎn)入大腸桿菌表達(dá)宿主中表達(dá),表達(dá)粗酶液分別與甜菊醇(steviol)、瑞鮑迪苷A(Rebaudioside A)、甜菊單糖苷(steviolmonoside)、甜菊雙糖苷(steviolbioside)以及steviol-19-O-β-D-glucoside底物共同孵育,測(cè)定明日葉UGT轉(zhuǎn)化活性。最終得到兩條與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,生成產(chǎn)物鑒定為steviol-19-O-β-D-glucoside。3)通過(guò)對(duì)轉(zhuǎn)錄組數(shù)據(jù)的發(fā)掘,以及基因的克隆和重組酶的酶活性鑒定,我們最終從甜葉懸鉤子和明日葉中篩選到了八條具有貝殼杉烯型系列底物活性的UGT,我們對(duì)這八條序列進(jìn)行了理化性質(zhì)、序列多重比對(duì)以及蛋白功能域預(yù)測(cè)等初步分析,為進(jìn)一步研究其功能特性奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;O629

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