海带GDP-甘露糖脱氢酶基因及磷酸甘露糖变位酶基因的研究

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	海带GDP-甘露糖脱氢酶基因及磷酸甘露糖变位酶基因的研究
	张朋艳
学位类型	硕士
导师	段德麟
	2015-05-23
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	海带褐藻酸 Gdp-甘露糖脱氢酶磷酸甘露糖变位酶
摘要	GDP-甘露糖脱氢酶（GMD）是褐藻酸合成途径的限速酶，海带中GMD基因的功能及特性尚未解析。本研究在前期海带转录组数据分析的基础上，通过RACE法克隆得到两条海带GMD基因（Sjgmd1, Sjgmd2）全长序列。同源比对分析表明，两者都属于GDP-甘露糖脱氢酶/UDP-葡萄糖脱氢酶家族的成员。Sjgmd1和Sjgmd2 的ORF序列长度分别为963 bp 和948 bp，两者之间表现70.06%的一致性。SjGMD1和SjGMD2的主要二级结构均为无规则卷曲和α-螺旋。多序列比对及三维结构模拟预测表明，海带中GMD可能存在与细菌GMD不同的结合及催化机制。酶动力学分析结果表明，SjGMD1的最适温度和最适pH值分别为30℃，8.0；而SjGMD2的最适温度和最适pH值分别为20℃和8.25。SjGMD1对GDP-甘露糖和NAD⁺ 的Km值分别为289 µM和139 µM；SjGMD2对GDP-甘露糖和NAD⁺ 的K值分别为177 µM和195 µM。此外，我们还对褐藻酸合成途径中的磷酸甘露糖变位酶（PMM）基因进行了分析，获得两条海带PMM基因序列（Sjpmm1、Sjpmm2）。其中，Sjpmm1为HAD超家族成员，ORF长759 bp，编码1个含有252个氨基酸序列，分子量约为28.51 kDa；而Sjpmm2 属于磷酸己糖变位酶超家族的成员，ORF长1866 bp，编码含621个氨基酸的蛋白，分子量约为66.49 kDa。海带PMM基因的二级结构均以α-螺旋为主。分子进化分析表明，Sjpmm1来自于原始真核生物，而Sjpmm2是通过基因水平转移从细菌中获得。qRT-PCR检测分析发现，Sjpmm1 和Sjpmm2在高温或者低温刺激时表达量均有上升趋势，表明海带受温度胁迫时， PMM活性提高，岩藻聚糖或褐藻酸的合成增加。此外，体外表达高浓度可溶性融合蛋白，为后续PMM功能分析打下基础。
其他摘要	GDP-mannose dehydrogenase (GMD) is the rate-limiting enzyme which involved in the synthesis of alginate, but no GMD gene from S. japonica has been characterized so far. In this study, two GMD genes from S. japonica (Sjgmd1, Sjgmd2), which belonged to GDP-mannose dehydrogenase/UDP-glucose dehydrogenase family, were cloned by RACE method. The open reading frame (ORF) length of Sjgmd1 and Sjgmd2 is 963 bp and 948 bp, respectively, which showed 70.06% identity. Random coil and α-helix were the common major secondary structure for Sjgmd1 and Sjgmd2. The multiple sequences alignment and three-dimensional structures analysis proposed a novel binding and catalysis mechnaism in SjGMD. The fusion protein (SjGMD) were expressed successfully and the enzyme assays results showed the optimal temperature were 30℃ (SjGMD1) and 20℃ (SjGMD2), and optimal pH value were 8.0 (SjGMD1) and 8.25 (SjGMD2). The Km for GDP-mannose were 289 µM (SjGMD1) and 177 µM (SjGMD2), respectively. In addition, two phosphomannomutase (PMM) genes of S. japonica (Sjpmm1、Sjpmm2) , which involved in the synthesis of alginate and fucoidan, were cloned by RACE method. The homologous analysis indicated that Sjpmm1 belonged to the HAD superfamily, and the ORF length was 759 bp, encoding 252 amino acids with the molecular weight (MV) of 28.51 kDa; while Sjpmm2 belonged to the phosphohexomutase superfamily, and the ORF length was 1866 bp, encoding 621 amino acids with the MV of 66.49 kDa. The α-helix was regarded as the commom major secondary structure to SjPMM1 and SjPMM2. The phylogenetic analysis showed that Sjpmm1 was evolved from the ancient eukaryotes, while Sjpmm2 was from the bacteria by horizontal gene transfer. The qRT-PCR analysis indicated that temperature could increase the transcription of Sjpmm, which may cause the more synthesized of fucoidan. Furthermore, high-concentration fusion protein of SjPMM1 was expressed, which could be applied for further function analysis.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23250
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	张朋艳. 海带GDP-甘露糖脱氢酶基因及磷酸甘露糖变位酶基因的研究[D]. 北京. 中国科学院大学,2015.

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