【作者】 房思良；

【导师】 祖元刚； 聂明珠；

【作者基本信息】 东北林业大学 ， 生药学， 2007， 硕士

【摘要】 金属硫蛋白是一类分子量较小、富含Cys的金属结合蛋白，广泛分布于生物界。近年来研究表明，金属硫蛋白参与体内微量元素的储存、转运和代谢，拮抗电离辐射，清除自由基以及对重金属的解毒作用。植物MT的发现相对较晚，研究表明，植物金属硫蛋白可以通过其大量的Cys残基螯合重金属并清除活性氧，使植物免受氧化损伤，参与了植物的发育、胚胎发生、抵抗逆境胁迫等多种生理过程，是一种重要的功能基因。但其蛋白功能尚未完全清楚。为了充分研究植物金属硫蛋白的功能，更多有关于植物的MTs基因克隆和功能分析的研究将对进一步研究和开发植物金属硫蛋白具有重要的科学价值。本论文首次从长春花cDNA文库中克隆了长春花金属硫蛋白（CrMT）全长基因序列，采用基因重组技术，将长春花CrMT基因重组入原核表达载体pGEX-6P-1中，该载体含有一个谷胱甘肽-S-转移酶（GST）基因，便于融合蛋白表达后的纯化。将重组质粒转入E coli BL21中进行体外表达，SDS-PAGE分析表明，经IPTG诱导后成功地表达了目的融合蛋白GST-CrMT。通过研究了温度、诱导时间以及诱导剂IPTG终浓度等不同条件对GST-CrMT融合蛋白表达量的影响，优化了原核表达条件，获得了较高浓度的GST-CrMT融合蛋白，为制备抗体进行Western杂交打下基础。利用Anti-GST抗体进行Western-blotting检测，鉴定了表达蛋白发生特异免疫反应。通过对MT的重金属离子耐受性进行了研究，既能抑制细菌的生长，又不至于使细菌死亡的适合金属离子浓度分别为CdCl₂ 1.6 mM and ZnCl₂ 1.0 mM。转化MT的大肠杆菌BL21与对照相比，对重金属耐受性都得到了增加。本论文通过使用SignalP 3.0 Server对来自长春花的CrMT进行了信号肽分析，将CrMT基因与绿色荧光蛋白GFP基因融合，构建到酵母表达载体pYES2，通过电转化法将pYES2-CrMT-GFP质粒转入酵母（Saccharomyce，scerevisiae）INUScl中，利用激光共聚焦显微镜（Confocal Laser Scanning Biological Microscope，Nikon）观察，从而对CrMT基因编码的蛋白在酵母细胞中进行定位，结果证明CrMT基因编码的蛋白存在于酵母的细胞质中。本研究结果为进一步了解植物金属硫蛋白的表达调控机理、结构和功能具有重要的科学参考价值，对开发植物金属硫蛋白产品提供重要的科学研究基础。更多还原

【Abstract】 Metallothioneins, which widely distribute in eukaryotic and prokaryotic organisms, are a class of cystein-rich and heavy metal-binding proteins with low molecular weights. Recently, many study demonstrated that metallothionein played a role in storing and carting trace element, scavenging of free radicals and detoxifying of heavy metals. The study on plant MTs is just started because of the discovery of plant MTs is relatively later. Many study demonstrated that the plant metallothionein may function in both metal chaperoning and scavenging of reactive oxygen species with their large number of cysteine residues to protect plant from oxidative damage and participated in many physiology processes, such as development of plant, embryogenesis and stress resistance, and was regarded as an important functional gene. But, the function of plant MTs remains unclear. To demonstrate the function（s） of plant MTs, more studies on the cloning and functional analysis of plant MTs genes would be of important scientific value for further researching and developing plant metallothionein.In this paper, the Catharanthus roseus metallothionein （CrMT） gene was first isolated from the cDNA library of Catharanthus roseus. CrMT gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1, the vector pGEX-6p-1 has a giutathione-S-transferase （GST） gene, which was in favor of the purification of the fusion protein by affinity chromatography subsequently. The recombinant plasmids were successfully introduced into E. coli BL21, SDS-PAGE analysis showed that the fusion protein GST-CrMT was successfully expressed by the induction of IPTG. We optimized the prokaryotic expression conditions through studing the effects of various factors on the protein expression, which included culture temperature, inducing time and the final concentration of inductor IPTG. Finally, we produced high concentration of GST-MT fusion protein and lay the foundation of producing antibody for Western-blotting. The Western-blotting test showed that the fusion protein had immunological reactive activity by using Anti-GST. Through analyzing the metal-ion tolerance of CrMT, concentrations of various metal ions were suitable for comparing metal-ion tolerance of transformed and non-transformed E. coli BL21were 1.0 mM CdCl₂ and 1.6 mM ZnCl₂. The metal-ion (Cd²⁺ and Zn²⁺) tolerance of E. coli cells expressing GST-CrMT fusion protein increased. These results suggest that CrMT functions in E. coli cells.In this paper, we analyzed the Signal Peptide through using SignalP 3.0 Server and applying the CrMT gene as a heterogeneous gene. The CrMT gene and GFP were together cloned into yeast expression vector pYES2, then the plasmid pYES2-CrMT-GFP was transformed into Saccharo cerevisiae INVScl by electric impulse transformation, observing the cell by Confocal Laser Scanning Biological Microscope （Nikon）, so, the gene coding CrMT was studied on the localization of CrMT gene encoding protein in yeast. The results demonstrated that the intracellular localization sites was in cytoplasm.The results have important scientific value for further researching on regulation mechanism, structure and function of plant metallothionein expression and will provide important scientific basis for developing the products of plant metallothionein.更多还原