甜菊UDPG糖基转移酶基因的克隆和功能分析

甜菊（Steviarebaudiana）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量不同，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...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...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：B19992600

MOLECULAR CLONING AND CHARACTERIZATION OF STEVIA REBAUDIANA UDP-GLUCOSYLTRANSFERASE

本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析。获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53.2K Da。与常见的糖基转移酶基因比较,相似性达44%以上,且具有糖基转移酶的保守序列。体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性。在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成。结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色。We report here the cloning and characterization of a UDP-glucose flavonoid glucosyltransferase (srUFGT) in Stevia rebaudiana. The isolated cDNA was 1419bp in length encoding 473 deduced amino acids with a predicted molecular mass of 53. 2 kDa. The products of in vitro translation from an expression vector had anthocyanidins and steviol glucosyltransferase activity. Comparison of the activity of the recombinant UDP-glucosyltransferase 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 a broad substrate specificity,may be not only involved in flavonoid glucosylation but also play a role in producing the water-soluble steviol-glycosides in S. rebaudiana

一类具有抗肿瘤活性的新型紫杉烷卤代衍生物

一类具有抗肿瘤活性的新型紫杉烷卤代衍生物，其特征在于：所述为具有如结构通式的新型紫杉烷卤代衍生物及以其作为活性成分的抗肿瘤药物如式I，其中R为H或Ac；R’具有下右式结构，其中R”为选自-OMe，-OH的取代基，X为选自Cl，Br中的卤素。本发明中所示的化合物对于白血病细胞(K562)，非小细胞肺癌细胞(A549)，结肠癌细胞 (HT-29)，卵巢癌细胞(OVCAR-3)，人成骨肉瘤细胞(Saos-2)，乳腺癌细胞 (MCF-7)等多种肿瘤细胞系均表现出与紫杉醇相似的细胞毒性，具有作为抗癌药物开发的价值。带填

Cloning and Sequencing of the cDNA of UDPG-glucosyltransferase from Stevia rebaudiana

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 .根据获得的基因片段设计引物GSTr和GSTf,分别与cDNA文库的T3 和T7通用引物扩增获得全长核苷酸序列的甜菊糖基转移酶基因 ,定名为sudpt 2 ,该基因全长 16 6 2bp ,包括poly(A)和一个 136 2bp的开放阅读框 ,编码 45 4个氨基酸 .与常见的糖基转移酶基因的相似性达 44 %～ 77% ,且具有UDPG糖基转移酶的特征性保守序列 .分子发育进化分析表明 ,此基因为类黄酮糖基转移酶基因家族成员By using two homologous degenerated primers, DNA fragments encoding steviol UDPG glucosyltransferase were amplified from the leaves of Stevia rebaudiana. The DNA fragment with strong homology to known flavonoid glucosyltransferases was used to design two primers to amplify upstream and downstream cDNA of stevia glucosyltransferase. The cDNA of stevia glucosyltransferase sudpg-2 was 1 662 bp in length, with a poly(A) tail and a 1 362 bp open reading frame which encoded 454 deduced amino acid residues. The deduced amimo acids sequence has 40%～60% identity to other plant glucosyltransferase and includes a part of the glucosyltransferase signature sequence. Molecular phylogenic relations of the deduced amino acids sequences with other plant glucosyltransferase suggested that the steviol UDPG glucosyltransferase belongs to family of flavonoid glucosyltransferases

蟾蜍内酯类3-表异构体的立体特异性葡萄糖醛酸化研究

Bufadienolides, a class of natural products with a unique steroidal skeleton were separated and identified from traditional Chinese medicine Chansu, has been reported with Na+-K+-ATPase inhibitory effects and outstanding antitumor activities. In the current study, the glucuronidation of three pairs of bufadienolide epimers including Cinobufagin (CB), deacetylcinobufagin (DCB) and bufalin (BF), as well as their corresponding 3-epimers (ECB, EDCB and EBF), were investigated by using human liver microsomes (HLM). Interestingly, among these investigated compounds, only EDCB and EBF can form corresponding glucuronidated metabolite when the substrate was incubated with HLM in the presence of UDPGA, while no metabolites of CB, DCB, BF or ECB were detected under the same conditions. The EDCB mono-glucuronide and EBF mono-glucuronide were characterized by using LC-MS/MS, and these two glucuronidated metabolites were also confirmed by hydrolysis with β-glucuronidase. Further investigation on EDCB glucuronidation in human liver microsomes was carried out, due to the detection limitation for quantification of EBF glucuronide. A combination of assays with a panel of recombinant human UGT isoform(s) and chemical inhibition study were used to explore the involved isoform(s) in the EDCB glucuronidation, only UGT2B7 exhibited the catalytic activity toward EDCB glucuronidation, while the inhibitory effect of mefanamic acid (a potent inhibitor of UGT2B7) for EDCB glucuronidation in HLM was similar to its in recombinant UGT2B7. These results revealed that human UGT2B7 was the major isoform involved in the glucuronidation of EDCB. The formation of EDCB glucuronide in pooled human liver microsomes can be well fitted to a substrate inhibition model and the kinetic parameters of EDCB glucuronidation were as follows: Km=48.4 ± 12.6 µM, Ksi=342.2 ± 95.3 µM, and Vmax=1.6 ± 0.3 nmol/min/mg of protein. In summary, this study revealed a stereoselective glucuronidation pathway to bufadienolides, and our results indicated that the structure variants of these epimers including the C-16 ester and the configuration of C-3 hydroxyl may influence the glucuronidation of bufadienolides

Optimizing recovery and cloning of positive bands in AFLP analysis

通过将AFLP聚丙烯酰胺变性胶切小、适当曝光过量、以聚丙烯酰胺凝胶电泳代替琼脂糖电泳检测克隆片段大小的方法 ,增加了回收克隆AFLP阳性带的准确性。该方法对于从变性和非变性大page胶上回收克隆目的片段具有普遍意义