【作者】 马凌波；

【导师】 陈睦传；

【作者基本信息】 厦门大学 ， 动物学， 2002， 博士

【摘要】 甜菊(Stevia rebaudiana)叶片中富集了约占干重10-30％的甜菊糖苷。以二萜类化合物甜菊醇(Steviol)为糖苷非糖配基，根据13位上的羟基和19位上的羧基上以O-糖苷键相连的糖基种类及数最不同，已有至少8种糖苷被发现。甜菊糖苷生物合成过程与赤霉素生物合成密切相关。已有研究表明赤霉素合成过程中的一些酶，在甜菊叶片细胞内的含量及其活性都比较高，表达方式也与其它植物有较大的差异，这将导致赤霉素和甜菊糖苷的共同前体物质内贝壳杉烯酸的大量积累。为了避免合成过量的赤霉素，这些前体物质被转化成为甜菊醇，并被糖基化形成甜菊糖苷。类黄酮UDPG糖基转移酶很有可能在甜菊糖苷的合成过程中起了重要作用，保证了赤霉素的代谢正常进行。为了证实这一点，实验克隆得到了甜菊类黄酮UDPG糖基转移酶基因，并对其功能进行了分析。 利用与植物次生代谢产物糖基化相关的UDPG糖基转移酶的特征性保守区域，设计了同源简并引物，以甜菊基因组DNA为模板，PCR扩增获得甜菊糖基转移酶基因片段GTASE-Ⅰ和GTASE-Ⅱ。基因片段包括了UDPG糖基转移酶基因的部分特征性序列。与其他同类UDPG糖基转移酶基因的氨基酸序列比较，具有较高的相似性，达53％至70％，约40％-50％氨基酸序列完全一致。表明获得的基因片段属于UDPG糖基转移酶。根据获得的基因片段设计引物，分别与cDNA文库的T3和T7通用引物配合，以cDNA文库为模板，扩增获得甜菊糖基转移酶基因Gt1和Gt2全长的核苷酸序列。获得的甜菊糖基转移酶Gt1基因全长1568bp(GenbankTM序列号：AF515727)，包括一个1419bp的开放阅读框，编码473个氨基酸。Gt2基因全长1662bp(GenbankTM序列号：AF548025)，包括一个1362bp的开放阅读框，编码454个氨基酸。Gt1和Gt2基因与常见的糖基转移酶基因的相似性分别达26-46％和44-74％。Gt1和Gt2都具有UDPG糖基转移酶的44个氨基酸的特征性保守序列，它被认为是UDP-glucose的结合区厦门大学博d二庵开究生学位创含文域。实验对Gtl基因进行了原核融合表达，获得的融合蛋白的大小约55kDa。功能分析表明，纯化后的Gtl融合蛋自以UDPG为糖基供体，能以甜菊醇(steviof)以及花青素(eyanidin)、飞燕草素(delphinidin)、芍药花素(伴on，din)和锦葵花素(malvidin)等花色素为糖基受体合成甜菊糖昔和花色素昔。其中，对花青素的催化活性最高，对甜菊醇的活性为花青素的33.5%。结果表明，Gtl作为甜菊体内的类黄酮糖基转移酶，具有广泛的底物特异性，它不仅参与类黄酮的糖基化，而且能够作用于甜菊醇，形成甜菊糖昔。在大量的GA前体物质被合成，使甜菊的正常代谢受到了威胁的情况下，甜菊很有可能利用了细胞内的类黄酮糖基转移酶基因合成惰性的甜菊糖苔。更多还原

【Abstract】 The pathway for steviol glycoside biosyntheses is shared with that of gibberellins. The highly activity nature and over expression of HMG-CoA reductase, CPS and KS would result in the production of a large amount of mevaloic acid, (-)-copalyl diphosphate and (-)-kaurene, the precursors of GAs and steviol in stevia rebaudiana leaves. To avoid the synthesis of an excess of GA, the precursor of which is also ent-kaurenoic acid must be converted to steviol, and then glucosylated into steviol glycosides, which accumulated in leaves at concentrations ranging from 10-30% of the leaf, dry weight.We report here the cloning and characterization of S. rebaudiana UDP-glucosyltransferase. Degenerate oligonucleotide primers were designed based on identical or highly conserved amino acid sequences of known flavonoid glucosyltransferase, and used to PCR amplify stevia glucosyltransferase gene fragments GTASE-I and GTASE-II. Two fragments with strong homology to known flavonoid glucosyltransferases were used to design other two primers to amplify 5’ and 3’-half cDNA of stevia glucosyltransferase Gtl and Gt2, respectively. The full-length Gtl gene is 1568bp in length, with an open reading frame encoding 473 amino acids (Genbank?accession numbers AF515727). The full-length Gt2 gene is 1662bp in length, with an open reading frame encoding 454 amino acids (GenbankTM accession numbers AF548025). The predicted amino acid sequences of Gtl and Gt2 were 26-46% and 44-74% identical to the known UDPG glucosyltransferase respectively. The deduced amino acid sequences of Gtl and Gt2 were comfirmed to have one conserved region of glucosyltransferases, which is the region for the UDPG-glucose binding domain. Plasmid was prepared from recombinant clones and transformed into the E. coli BL21 DE3 strain for production of recombinant Gtl. The Gtl products of in vitro translation, which were ~55 kDa recombinant protein, hadanthocyanidins and steviol glucosyltransferase activity. Incubation of the purified recombinant Gtl with steviol and other aglycones in the presence of UDP-glucose donor substrate resulted in the formation of the glucosides. Relative active against a range of acceptor substrates, including steviol, cyanidin, delphinidin, peonidin and malvidin, was determined by HPLC analysis. Cyanidin gave the highest rates of glucosylation. The average rate of steviol glucosylation was approximately 33.5% toward that observed for cyanidin. Comparison the activity of the recombinant UDP-glucosyltransferase Gtl toward a range of acceptor substrates, suggests that it may participate in the synthesis of steviol glycosides. The results support the hypothesis that the flavonoid glucosyltransferases, which have broad substrate specificity, may be not only involving in flavonoid glucosylation but also playing a role to produce the water-soluble steviol-glycosides in S. rebaudiana.更多还原