【作者】 郭志雄；

【导师】 吕柳新；

【作者基本信息】 福建农林大学 ， 果树学， 2006， 博士

【摘要】 在龙眼离体胚胎发生中发现存在某种类型的脱氢酶，初步的研究显示该组蛋白质可能是NAD依赖型的类糖醇脱氢酶。比较了该类糖醇脱氢酶在龙眼离体胚胎发生过程中的变化，发现随着胚发育进程，其活性逐步降低；同工酶电泳显示，虽然其酶谱各谱带均出现于胚性愈伤组织、球形胚和子叶胚中，但随着胚的发育其染色强度不断降低，显示龙眼离体培养材料存在的该组酶与其离体胚的发生、发育有关。为了明确该组类糖醇脱氢酶的属性，以揭示其在龙眼胚胎发生、发育过程中的生理作用，本试验进行了该酶的分离、纯化、生物质谱鉴定及其cDNA克隆。主要研究结果如下： 1．龙眼胚性愈伤组织类糖醇脱氢酶的分离纯化。采用低浓度的DTT以及加入10％的甘油有利于该组类糖醇脱氢酶在体外溶液环境的稳定。在此基础上，通过研究其在不同层析分离介质中的分离特性，建立了适用于该组类糖醇脱氢酶的分离纯化体系。经过80％饱和度的（NH₄）₂SO₄盐析、Sephadex G-25柱脱盐，然后经过DEAE-Sepharose F．F．、DEAE-52、Phenyl Sepharose和Superdex 200柱层析，从龙眼胚性愈伤组织中分离纯化得到类糖醇脱氢酶的2个同工酶组分Lc．A和Lc．B。比较非变性PAGE和SDS-PAGE的结果，显示Lc．A和Lc．B均为同型二体蛋白，其亚基的表观分子量大小相同，为47.4kD。Lc．A和Lc．B具不同的等电点。 2．龙眼胚性愈伤组织类糖醇脱氢酶的生物质谱鉴定。应用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）进行肽质量指纹分析，显示Lc．A和Lc．B均能较好地与其他植物的乙醇脱氢酶（ADH）相匹配。应用电喷雾解离—四极杆—飞行时间串连质谱（ESI-Q-TOF2）技术解析了Lc．B的4个胰酶水解肽段的氨基酸序列，分别是：GTFFGNYKPR、KFGVTEFVNPK、AFEYMLGGDGR和FITHEVPFSEINK；解析了Lc．A的4个胰酶水解肽段的氨基酸序列，分别是：FGVTEFVNPK、GTFFGNYKPR、FITHEVPFSEINK和DYDKPVQEVIAEMTDGGVDR（或者DYDKPVKEV LAEMTDNVDR）。数据库检索和序列比对分析均表明Lc．A和Lc．B均显著匹配于ADH。因此，在肽质量指纹的基础上，鉴定从龙眼胚性愈伤组织中纯化得到的类糖醇脱氢酶Lc．A和Lc．B分别为ADH同工酶的2个组分。 3．龙眼离体胚胎发生、发育过程中ADH的活性变化测定。试验发现，从胚性愈伤组织至球形胚和子叶胚阶段，ADH的活性呈下降趋势，同工酶谱分析进一步显示了相同的结果，表明高ADH活性可能与龙眼的体胚早期发生或与维持愈伤组织的高再生能力有关。 4．龙眼胚性愈伤组织ADH cDNA克隆。采用同源克隆的方法获得了龙眼ADHcDNA的保守片段，并在此基础上，通过3′和5′-RACE，获得了龙眼ADH的cDNA全序列，该序列与其他植物ADH cDNA有很高的同源性。该cDNA全长1386bp，更多还原

【Abstract】 A certain type of dehydrogenase from longan embryogenic calli （EC） was found and the preliminary studies indicated that the proteins were probably NAD⁺-dependent putative sugar alcohol dehydrogenases （SADHs）. The activity of the enzymes decreased gradually with the development of embryoids, the zymogram pattern did not change in the embryogenic callus, globular embryo and cotyledonary embryo but the staining intensity decreased during somatic embryogenesis, which indicated that the enzymes located in embryogenic calli of longan were correlated to somatic embryogenesis. In order to identify the characteristics of these putative sugar alcohol dehydrogenases and reveal their involvement during somatic embryogenesis, further studies including separation, purification, identification and cDNA cloning of the enzymes were performed, and the main results showed as follows:1. Separation and purification of the putative SADHs. The putative SADHs were sensitive to 0.5mmol/L DTT and the stability of the enzymes increased when added with 10% glycerol. After separation experiments performed on different column media, a protocol for purification of the putative SADHs was developed. After homogenization and centrifugation, the crude enzyme extract was fractionated with 0～80% saturation of ammonium sulphate [（NH₄）₂SO₄]. The precipitate was redissolved and then desalted by gel filtration on Sephadex G-25. Two isozymes Lc.A and Lc.B of putative sugar alcohol dehydrogenase in longan embryogenic calli were purified when chromatographed on DEAE-Sepharose F.F., DEAE-52, Phenyl Sepharose HP and Superdex 200 subsequently. The Lc.A and Lc.B were homodimer and carried different isoelectric points when compared with the results of native linear gradient PAGE and SDS-PAGE, and the apparent molecular weight of subunit estimated was the same of 47.4kD.2. Identification of the putative SADHs by biology MS. Matrix-assisted laser desorption/ ionization time of flight-mass spectrometry （MALDI-TOF MS） was applied to the analysis of peptide mass fingerprinting （PMF） in Lc.A and Lc.B respectively. The PMF data of Lc.A and Lc.B were used in Mascot search, and the Lc.A and Lc.B were identified as alcohol dehydrogenase （ADH） preliminarily. Then, the technique of electrospray ionization quadrupole time-of-flight tandem mass spectrometry （ESI-Q-TOF2 MS） was used in Lc.A and Lc.B identification. Four tryptic digested peptide fragments in Lc.B, GTFFGNYKPR, KFGVTEFVNPK, AFEYMLGGDGR and FITHEVPF SEINK, were sequenced by ESI-MS/MS; another four fragments in Lc.A, FGVTEFVNPK, GTFFGN YKPR, FITHEVPFSEINK and DYDKPVQEVIAEMTDGGVDR （or DYDKPVKEVLAEMTDNVD R）, were obtained by ESI-MS/MS sequencing. The results of database search and multiple alignments showed that Lc.A and Lc.B matched to the ADH protein significantly. Therefore, the putative sugar alcohol dehydrogenases Lc.A and Lc.B were identified as two isozymes of ADH from longan EC.3. Measurement of the activity and zymogram pattern of ADH during somatic embryogenesis in longan. The results showed that the activity of ADH were high in embryogenic calli and decreased in globular embryo and cotyledonary embryo; the zymogram pattern showed that the staining intensity更多还原