长春花MT基因克隆、原核表达及其功能分析
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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CdCl_2 1.6 mM and ZnCl_2 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。
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 CdCl_2 and 1.6 mM ZnCl_2. The metal-ion (Cd~(2+) and Zn~(2+)) 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.
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CdCl_2 1.6 mM and ZnCl_2 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。
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 CdCl_2 and 1.6 mM ZnCl_2. The metal-ion (Cd~(2+) and Zn~(2+)) 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.
引文
[1] Margoshes M and Vallee B L. A cadmium protein from equine kidney cortex[J]. J Am Chem Soc, 1957, 79(1): 4813-4814.
[2] Vallee B L, and Wolfgang Maret. The function potential and potential functions of metallothioneins: a personal perspective [J]. Metallothionein Ⅲ, 1993, 52(12): 1-28.
[3] A H Robbins, D E McRee, M Williamson, et al. Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution[J]. Journal of Molecular Biology, 1991,221 (4): 1269-1293.
[4] Lane B G, Kajioka R and Kennedy T D. The wheat germ Ec protein is a zinc containing metallothionein [J]. Biochem Cell Biol, 1987, 65(3): 1001-1005.
[5] P Meuwly, P Thibault and W E Rauser. Gamma-Glutamylcysteinylglutamic acid-a new homologue of glutathione in maize seedlings exposed to cadmium[J]. FEBS Lett, 1993, 336(3): 472-476.
[6] M B Jelena, T S I Jelena, S T Gordana, et al. Expression analysis of buckwheat (Fagopyrum esculentum Moench) Metallothionein-like gene (MT3) under different stress and physiological conditions [J]. Journal of Plant Physiology, 2004, 161(6): 741-746.
[7] A Zhigang, L Cuijie, Z Yuangang, et al. Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings [J]. J Exp Bot, 2006, 57(14): 3575-3582.
[8] M. E. Katherine, A. G. John, P. L. William, et al. Expression of the pea metallothionein-like gene PsMTA in Escherichia coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: Implications for PsMTA function [J]. Plant Molecular Biology, 1992, 20(6): 1019-1028.
[9] G Mir, J Domenech, G Huguet, et al. A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress [J]. J Exp Bot, 2004, 55(408): 2483-2493.
[10] K E Rigby and M J Stillman. Structural studies of metal-free metallothionein[J]. Biochem Biophys ResCommun, 2004, 325(4): 1271-1278.
[11] A J Morgan, S R Sturzenbaum, C Winters, et al. Differential metallothionein expression in earthworm (Lumbricus rubellus)tissues[J]. Ecotoxicology and Environmental Safety, 2004, 57(1): 11-19.
[12] K Hirose, B Ezaki, T Liu, et al. Diamide stress induces a metallothionein BmtA through a repressor BxmR and is modulated by Zn-inducible BmtA in the cyanobacterium Oscillatoria brevis[J]. Toxicol Lett, 2006, 163(3): 250-256.
[13] G K Andrews. Regulation of metallothionein gene expression by oxidative stress and metal ions [J]. Biochem Pharmacol, 2000, 59(1): 95-104.
[14] M J Chung, P A Walker, R W Brown, et al. Zinc-mediated gene expression offers protection against H_2O_2-inducedcytotoxicity [J]. Toxicol Appl Pharmacol, 2005, 205(3): 225-236.
[15] E Mocchegiani, L Costarelli, R Giacconi, et al. Zinc-binding proteins (metallothionein and alpha-2 macroglobulin) and immunosenescence [J]. Exp Gerontol, 2006, 41(11): 1094-1107.
[16] E Freisinger. Spectroscopic characterization of a fruit-specific metallothionein: M acuminata MT3 [J]. InorganicaChimicaActa, 2007, 360(1): 369-380.
[17] R W Olafson, W D McCubbin and C M Kay. Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium [J]. Biochem J, 1088, 251(3): 691-699.
[18] Hamer D H. Metallothionein[J]. AnnRev Biochem, 1986, 55(6): 913-951.
[19] W E Rauser. Phytochelatins[J]. AnnuRevBiochem, 1990, 59(10): 61-86.
[20] Yutaka K. Definitions and nomenclature of metallothionein [J]. Methods in Enzymology, 1991, 205(1): 8-10.
[21] Fowler B A et al. Nomenclature of metallothionein: Metallothionein Ⅱ proceedings of the Second International Meeting on Metallothionein and other low molecular weight[D]. USA: Second International Meeting, 1985: 19-22.
[22] I Bertini, H J Hartmann, T Z Klein, et al. High resolution solution structure of the protein part of CuT metallothionein[J]. Eur J Biochem, 2000, 267(4): 1008-1018.
[23] J H Kagi, M Vasak, K Lerch, et al. Structure of mammalian metallothionein[J]. Environ Health Perspect, 1984, 54(12): 93-103.
[24] M Ebadi, P L Iversen, R Hao, et al. Expression and regulation of brain metallothionein [J]. NeurochemInt, 1995, 27(1): 1-22.
[25] Yoko Uchida, et al. The growth in hibitory factor that is deficient in the Alzhemer's disease brain is a 68 amino acid metallothionein-like protein[J]. Neuron, 1991, 7(6): 337-347.
[26] Kagi H R. Evolution. structure and chemical activity of class Ⅰ Metallothionein: An overview [J]. MetallothioneinⅢ, 1993, 53(20): 29-56.
[27] I Kawashima, T D Kennedy, M Chino, et al. Wheat Ec metallothionein genes-Like mammalian Zn~(2+) metallothionein genes, wheat Zn~(2+) metallothionein genes are conspicuously expressed during embryogenesis[J]. EurJBiochem, 1992, 209(3): 971-976.
[28] Grill E, Winnacker E L, and Zenk M H. Phytochelations: The principal heavy metal complexing peptides of higher plant [J]. Science, 1985, 230(44): 674-676.
[29] Bernhard W R. Purification cadimium-binding polypeptides from and characterization of a typical zeamays[J]. MetallothioneinⅢ, 1987, 52(10): 5-12.
[30] 田丽萍．中国明对虾(Fenneropenaeus chinensis)金属硫蛋白基因和启动子的克隆与鉴定[D]．北京：中国科学院研究生院，2005：3-4．
[31] Shen Z G, Zhao F J and M S P. Uptake and transport of zinc in hyperaccumulator Thlaspi caerulescens and non-hyperaccumulator Thlaspvi ochroleucum [J]. Plant Cell and Environment, 1997, 7(20): 898-906.
[32] Rupp Happroach. Circular dichroism of metallothionein. a structural approach[J]. Biochem BiophysActa, 1978, 533(1): 209-226.
[33] A Murphy, J Zhou, P B Goldsbrough, et al. Purification and immunological identification of metallothioneins 1 and 2 from Arabidopsis thaliana [J]. Plant Physiol, 1997, 113(4): 1293-1301.
[34] 朱春明，吕暾，张日清等．猕猴桃金属硫蛋白的结构建模[J]．科学通报，2000，45(6)：602-607．
[35] 茹炳根，潘爱华，黄秉乾等．金属硫蛋白[J]．生物化学与生物物理进展，1991，18(4)：254-259．
[36] Stiliman M J. Metallothioneins[J]. Coordination Chemistry Review, 1995, 144(1): 461-511.
[37] 魏欣，茹炳根．二价铅离子与金属硫蛋白相互作用的研究[J]．中国生物化学与分子生物学学报，1999，15(2)：289-295．
[38] E Tokuda, S Ono, K Ishige, et al. Metallothionein proteins expression, copper and zinc concentrations, and lipid peroxidation level in a rodent model for amyotrophic lateral sclerosis[J]. Toxicology, 2007, 229(1-2): 33-41.
[39] F Yang, M Zhou, Z He, et al. High-yield expression in Escherichia coli of soluble human MT2Awith native functions[J]. Protein Expr Purif, 2007, 53(1): 186-194.
[40] Masao S et al. Induction of metallothionein synthesis by oxidative stress and possible role in acute phase response [J]. Metallothionein Ⅲ, 1993, 56(3): 125-140.
[41] S Labbe, L Larouche, D Mailhot, et al. Purification of mouse MEP-1, a nuclear protein which binds to the metal regulatory elements of genes encoding metallothionein[J]. Nucleic Acids Res, 1993, 21(7): 1549-1554.
[42] G K Zhou, Y F Xu and J Y Liu. Characterization of a rice class Ⅱ metallothionein gene: tissue expression patterns and induction in response to abiotic factors[J]. Plant Physiol, 2005, 162(6): 686-696.
[43] A M Scheuhammer and M G Cherian. The influence of manganese on the distribution of essential trace elements Ⅱ. The tissue distribution of manganese, magnesium, zinc, iron and copper in rats after chronic manganese exposure[J]. Toxicol Environ Health, 1983, 12(2-3): 361-370.
[44] S H Oh, J T Deagen, P D Whanger, et al. Biological function of metallothionein V. Its induction in rats by various stresses[J]. Am J Physiol, 1978, 234(3): E282-285.
[45] N J Robinson, A M Tommey, C Kuske, et al. Plant metallothioneins[J]. Biochem, 1993, 295 (Pt 1): 1-10.
[46] C Cobbett and P Goldsbrough. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis[J]. Annu Rev Plant Biol, 2002, 53(5): 159-182.
[47] Casterline J L. Isolation and characterization of cadmium-binding components in Soybean [J]. Plant Physiol, 1997, 59(6): 124.
[48] Zhou J M and Goldsbrough P B. Functional homologs of fungal metallothionein genes from Arabidopsis[J]. Plant Cell and Environment, 1994, 6(33). 875-884.
[49] J Zhou and P B Goldsbrough. Structure, organization and expression of the metallothionein gene family in Arabidopsis[J]. Mol Gen Genet, 1995, 248(3). 318-328
[50] H M Hsieh, W K Liu, A Chang, et al. RNA expression patterns of a type 2 metallothionein-like gene from rice[J]. Plant Mol Biol, 1996, 32(3): 525-529.
[51] S E Ledger and R C Gardner. Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa) [J]. Plant MolBiol, 1994, 25(5): 877-886.
[52] W J Guo, W Bundithya and P B Goldsbrough. Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper[J]. New Phytologist, 2003, 159(2): 369-381
[53] L H Yu, M Umeda, J Y Liu, et al. A novel MT gene office plants is strongly expressed in the node portion of the stem [J]. Gene, 1998, 206(1): 29-35.
[54] M Garcia-Hemandez, A Murphy and L Taiz. Metallothioneins 1 and 2 have Distinct but Overlapping Expression Pattems in Arabidopsis [J]. Plant Physiol, 1998, 118(2): 387-397.
[55] R C Foley, Z M Liang and K B Singh. Analysis of type 1 metallothionein cDNAs in Vicia faba[J]. PlantMolBiol, 1997, 33(4): 583-591.
[56] A M Tommey, J Shi, W P Lindsay, et al. Expression of the pea gene PSMTA in E. coli. Metal-binding properties of the expressed protein[J]. FEBS Lett, 1991, 292(1-2): 48-52.
[57] N H Roosens, C Bernard, R Leplae, et al. Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens[J]. FEBS Lett, 2004, 577(1-2): 9-16.
[58] H R Nancy, L Raphael, B Catherine, et al. Variations in plant metallothioneins: the heavy metal hyperaccumulator Thlaspi caerulescens as a study case [J]. Planta, 2005, 222(4): 716-729.
[59] 全先庆，张洪涛，单 雷等．植物金属硫蛋白及其重金属解毒机制研究进展[J]．遗传HEREDITAS，2006，28(3)：375-382．
[60] Z Haiyan, X Wenzhong, D Wentao, et al. Functional characterization of cadmium-responsive garlic gene AsMT2b: A new member ofmetallothionein family [J]. Chinese Science Bulletin, 2006, 51(4): 409-416.
[61] 陈虹，姜廷波，丁宝建等．转柽柳金属硫蛋白基因(MT_1)烟草的获得及对重金属镉的抗性分析[J]．农业生物技术学报，2007，15(2)：247-256．
[62] E Himelblau and R M Amasino. Delivering copper within plant cells[J]. Current Opinion in Plant Biology, 2000, 3(6): 205-210.
[63] E Himelblau, H Mira, S-J Lin, et al. Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATXI from Arabidopsis[J]. PLANT PHYSIOLOGY, 1998, 117(4): 1227-1234.
[64] H M Hsieh, W K Liu and P C Huang. A novel stress-inducible metallothionein-like gene from rice[J]. PlantMolBiol, 1995, 28(3): 381-389.
[65] J Shumei, C Yuxiang, G Qingjie, et al. A metallothionein-like protein of rice (rgMY) functions in E. coli and its gene expression is induced by abiotic stresses[J]. Biotechnology Letters, 2006, 28(21): 1749-1753.
[66] K Akashi, N Nishimura, Y Ishida, et al. Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon[J]. Biochem Biophys Res Commun, 2004, 323(1): 72-78.
[67] 唐中华，郭晓瑞，张洋洋等．拟南芥MT-Ⅱ过量表达提高抗旱性[J]．植物研究，2005，25(4)：415-418．
[68] 全先庆，高翔．植物金属硫蛋白及其在自由基清除中的作用[J]．临沂师范学院学报，2003，25(6)：64-66．
[69] N Gorinova, M Nedkovska, E Todorovska, et al. Improved phytoaccumulation ofcadmium by genetically modified tobacco plants (Nicotiana tabacum L.). Physiological and biochemical response of the transformants to cadmium toxicity [J]. Environ Pollut, 2007, 145(1): 161-170.
[70] N Futakawa, M Kondoh, S Ueda, et al. Involvement of oxidative stress in the synthesis of metallothionein induced by mitochondrial inhibitors[J]. Biol Pharm Bull, 2006, 29(10): 2016-2020.
[71] J Domenech, G Mir, G Huguet, et al. Plant metallothionein domains: functional insight into physiological metal binding and protein folding [J]. Biochimie, 2006, 88(6): 583-593.
[72] Carstens C P and Waeshe A. Codon bias-adjusted BL21 derivatives for protein expression[J]. Strategies, 1999, 129(10): 49-51.
[73] 周宇荀，魏东芝，王二力．融合蛋白表达载体pGEX及其应用[J]．生命科学，1998，10(3)：122-124．
[74] C L Chyan, T T Lee, C P Liu, et al. Cloning and expression of a seed-specific metallothionein like protein from sesame [J]. Biosci Biotechnol Biochem, 2005, 69(12): 2319-2325.
[75] Chalfie M, Tu Y, Euskirchen G, et al. Green flurescent protein as a marker for gene expression [J]. Science, 1994, 263(5148): 802-805.
[76] Cheng L, Fu J, Ysukamoto A, et al. Use of green fluorescent protein variants to monito gene transfer and expression in mammalian cells [J]. Nat Biotechnol, 1996, 14(5): 606-609.
[2] Vallee B L, and Wolfgang Maret. The function potential and potential functions of metallothioneins: a personal perspective [J]. Metallothionein Ⅲ, 1993, 52(12): 1-28.
[3] A H Robbins, D E McRee, M Williamson, et al. Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution[J]. Journal of Molecular Biology, 1991,221 (4): 1269-1293.
[4] Lane B G, Kajioka R and Kennedy T D. The wheat germ Ec protein is a zinc containing metallothionein [J]. Biochem Cell Biol, 1987, 65(3): 1001-1005.
[5] P Meuwly, P Thibault and W E Rauser. Gamma-Glutamylcysteinylglutamic acid-a new homologue of glutathione in maize seedlings exposed to cadmium[J]. FEBS Lett, 1993, 336(3): 472-476.
[6] M B Jelena, T S I Jelena, S T Gordana, et al. Expression analysis of buckwheat (Fagopyrum esculentum Moench) Metallothionein-like gene (MT3) under different stress and physiological conditions [J]. Journal of Plant Physiology, 2004, 161(6): 741-746.
[7] A Zhigang, L Cuijie, Z Yuangang, et al. Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings [J]. J Exp Bot, 2006, 57(14): 3575-3582.
[8] M. E. Katherine, A. G. John, P. L. William, et al. Expression of the pea metallothionein-like gene PsMTA in Escherichia coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: Implications for PsMTA function [J]. Plant Molecular Biology, 1992, 20(6): 1019-1028.
[9] G Mir, J Domenech, G Huguet, et al. A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress [J]. J Exp Bot, 2004, 55(408): 2483-2493.
[10] K E Rigby and M J Stillman. Structural studies of metal-free metallothionein[J]. Biochem Biophys ResCommun, 2004, 325(4): 1271-1278.
[11] A J Morgan, S R Sturzenbaum, C Winters, et al. Differential metallothionein expression in earthworm (Lumbricus rubellus)tissues[J]. Ecotoxicology and Environmental Safety, 2004, 57(1): 11-19.
[12] K Hirose, B Ezaki, T Liu, et al. Diamide stress induces a metallothionein BmtA through a repressor BxmR and is modulated by Zn-inducible BmtA in the cyanobacterium Oscillatoria brevis[J]. Toxicol Lett, 2006, 163(3): 250-256.
[13] G K Andrews. Regulation of metallothionein gene expression by oxidative stress and metal ions [J]. Biochem Pharmacol, 2000, 59(1): 95-104.
[14] M J Chung, P A Walker, R W Brown, et al. Zinc-mediated gene expression offers protection against H_2O_2-inducedcytotoxicity [J]. Toxicol Appl Pharmacol, 2005, 205(3): 225-236.
[15] E Mocchegiani, L Costarelli, R Giacconi, et al. Zinc-binding proteins (metallothionein and alpha-2 macroglobulin) and immunosenescence [J]. Exp Gerontol, 2006, 41(11): 1094-1107.
[16] E Freisinger. Spectroscopic characterization of a fruit-specific metallothionein: M acuminata MT3 [J]. InorganicaChimicaActa, 2007, 360(1): 369-380.
[17] R W Olafson, W D McCubbin and C M Kay. Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium [J]. Biochem J, 1088, 251(3): 691-699.
[18] Hamer D H. Metallothionein[J]. AnnRev Biochem, 1986, 55(6): 913-951.
[19] W E Rauser. Phytochelatins[J]. AnnuRevBiochem, 1990, 59(10): 61-86.
[20] Yutaka K. Definitions and nomenclature of metallothionein [J]. Methods in Enzymology, 1991, 205(1): 8-10.
[21] Fowler B A et al. Nomenclature of metallothionein: Metallothionein Ⅱ proceedings of the Second International Meeting on Metallothionein and other low molecular weight[D]. USA: Second International Meeting, 1985: 19-22.
[22] I Bertini, H J Hartmann, T Z Klein, et al. High resolution solution structure of the protein part of CuT metallothionein[J]. Eur J Biochem, 2000, 267(4): 1008-1018.
[23] J H Kagi, M Vasak, K Lerch, et al. Structure of mammalian metallothionein[J]. Environ Health Perspect, 1984, 54(12): 93-103.
[24] M Ebadi, P L Iversen, R Hao, et al. Expression and regulation of brain metallothionein [J]. NeurochemInt, 1995, 27(1): 1-22.
[25] Yoko Uchida, et al. The growth in hibitory factor that is deficient in the Alzhemer's disease brain is a 68 amino acid metallothionein-like protein[J]. Neuron, 1991, 7(6): 337-347.
[26] Kagi H R. Evolution. structure and chemical activity of class Ⅰ Metallothionein: An overview [J]. MetallothioneinⅢ, 1993, 53(20): 29-56.
[27] I Kawashima, T D Kennedy, M Chino, et al. Wheat Ec metallothionein genes-Like mammalian Zn~(2+) metallothionein genes, wheat Zn~(2+) metallothionein genes are conspicuously expressed during embryogenesis[J]. EurJBiochem, 1992, 209(3): 971-976.
[28] Grill E, Winnacker E L, and Zenk M H. Phytochelations: The principal heavy metal complexing peptides of higher plant [J]. Science, 1985, 230(44): 674-676.
[29] Bernhard W R. Purification cadimium-binding polypeptides from and characterization of a typical zeamays[J]. MetallothioneinⅢ, 1987, 52(10): 5-12.
[30] 田丽萍．中国明对虾(Fenneropenaeus chinensis)金属硫蛋白基因和启动子的克隆与鉴定[D]．北京：中国科学院研究生院，2005：3-4．
[31] Shen Z G, Zhao F J and M S P. Uptake and transport of zinc in hyperaccumulator Thlaspi caerulescens and non-hyperaccumulator Thlaspvi ochroleucum [J]. Plant Cell and Environment, 1997, 7(20): 898-906.
[32] Rupp Happroach. Circular dichroism of metallothionein. a structural approach[J]. Biochem BiophysActa, 1978, 533(1): 209-226.
[33] A Murphy, J Zhou, P B Goldsbrough, et al. Purification and immunological identification of metallothioneins 1 and 2 from Arabidopsis thaliana [J]. Plant Physiol, 1997, 113(4): 1293-1301.
[34] 朱春明，吕暾，张日清等．猕猴桃金属硫蛋白的结构建模[J]．科学通报，2000，45(6)：602-607．
[35] 茹炳根，潘爱华，黄秉乾等．金属硫蛋白[J]．生物化学与生物物理进展，1991，18(4)：254-259．
[36] Stiliman M J. Metallothioneins[J]. Coordination Chemistry Review, 1995, 144(1): 461-511.
[37] 魏欣，茹炳根．二价铅离子与金属硫蛋白相互作用的研究[J]．中国生物化学与分子生物学学报，1999，15(2)：289-295．
[38] E Tokuda, S Ono, K Ishige, et al. Metallothionein proteins expression, copper and zinc concentrations, and lipid peroxidation level in a rodent model for amyotrophic lateral sclerosis[J]. Toxicology, 2007, 229(1-2): 33-41.
[39] F Yang, M Zhou, Z He, et al. High-yield expression in Escherichia coli of soluble human MT2Awith native functions[J]. Protein Expr Purif, 2007, 53(1): 186-194.
[40] Masao S et al. Induction of metallothionein synthesis by oxidative stress and possible role in acute phase response [J]. Metallothionein Ⅲ, 1993, 56(3): 125-140.
[41] S Labbe, L Larouche, D Mailhot, et al. Purification of mouse MEP-1, a nuclear protein which binds to the metal regulatory elements of genes encoding metallothionein[J]. Nucleic Acids Res, 1993, 21(7): 1549-1554.
[42] G K Zhou, Y F Xu and J Y Liu. Characterization of a rice class Ⅱ metallothionein gene: tissue expression patterns and induction in response to abiotic factors[J]. Plant Physiol, 2005, 162(6): 686-696.
[43] A M Scheuhammer and M G Cherian. The influence of manganese on the distribution of essential trace elements Ⅱ. The tissue distribution of manganese, magnesium, zinc, iron and copper in rats after chronic manganese exposure[J]. Toxicol Environ Health, 1983, 12(2-3): 361-370.
[44] S H Oh, J T Deagen, P D Whanger, et al. Biological function of metallothionein V. Its induction in rats by various stresses[J]. Am J Physiol, 1978, 234(3): E282-285.
[45] N J Robinson, A M Tommey, C Kuske, et al. Plant metallothioneins[J]. Biochem, 1993, 295 (Pt 1): 1-10.
[46] C Cobbett and P Goldsbrough. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis[J]. Annu Rev Plant Biol, 2002, 53(5): 159-182.
[47] Casterline J L. Isolation and characterization of cadmium-binding components in Soybean [J]. Plant Physiol, 1997, 59(6): 124.
[48] Zhou J M and Goldsbrough P B. Functional homologs of fungal metallothionein genes from Arabidopsis[J]. Plant Cell and Environment, 1994, 6(33). 875-884.
[49] J Zhou and P B Goldsbrough. Structure, organization and expression of the metallothionein gene family in Arabidopsis[J]. Mol Gen Genet, 1995, 248(3). 318-328
[50] H M Hsieh, W K Liu, A Chang, et al. RNA expression patterns of a type 2 metallothionein-like gene from rice[J]. Plant Mol Biol, 1996, 32(3): 525-529.
[51] S E Ledger and R C Gardner. Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa) [J]. Plant MolBiol, 1994, 25(5): 877-886.
[52] W J Guo, W Bundithya and P B Goldsbrough. Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper[J]. New Phytologist, 2003, 159(2): 369-381
[53] L H Yu, M Umeda, J Y Liu, et al. A novel MT gene office plants is strongly expressed in the node portion of the stem [J]. Gene, 1998, 206(1): 29-35.
[54] M Garcia-Hemandez, A Murphy and L Taiz. Metallothioneins 1 and 2 have Distinct but Overlapping Expression Pattems in Arabidopsis [J]. Plant Physiol, 1998, 118(2): 387-397.
[55] R C Foley, Z M Liang and K B Singh. Analysis of type 1 metallothionein cDNAs in Vicia faba[J]. PlantMolBiol, 1997, 33(4): 583-591.
[56] A M Tommey, J Shi, W P Lindsay, et al. Expression of the pea gene PSMTA in E. coli. Metal-binding properties of the expressed protein[J]. FEBS Lett, 1991, 292(1-2): 48-52.
[57] N H Roosens, C Bernard, R Leplae, et al. Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens[J]. FEBS Lett, 2004, 577(1-2): 9-16.
[58] H R Nancy, L Raphael, B Catherine, et al. Variations in plant metallothioneins: the heavy metal hyperaccumulator Thlaspi caerulescens as a study case [J]. Planta, 2005, 222(4): 716-729.
[59] 全先庆，张洪涛，单 雷等．植物金属硫蛋白及其重金属解毒机制研究进展[J]．遗传HEREDITAS，2006，28(3)：375-382．
[60] Z Haiyan, X Wenzhong, D Wentao, et al. Functional characterization of cadmium-responsive garlic gene AsMT2b: A new member ofmetallothionein family [J]. Chinese Science Bulletin, 2006, 51(4): 409-416.
[61] 陈虹，姜廷波，丁宝建等．转柽柳金属硫蛋白基因(MT_1)烟草的获得及对重金属镉的抗性分析[J]．农业生物技术学报，2007，15(2)：247-256．
[62] E Himelblau and R M Amasino. Delivering copper within plant cells[J]. Current Opinion in Plant Biology, 2000, 3(6): 205-210.
[63] E Himelblau, H Mira, S-J Lin, et al. Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATXI from Arabidopsis[J]. PLANT PHYSIOLOGY, 1998, 117(4): 1227-1234.
[64] H M Hsieh, W K Liu and P C Huang. A novel stress-inducible metallothionein-like gene from rice[J]. PlantMolBiol, 1995, 28(3): 381-389.
[65] J Shumei, C Yuxiang, G Qingjie, et al. A metallothionein-like protein of rice (rgMY) functions in E. coli and its gene expression is induced by abiotic stresses[J]. Biotechnology Letters, 2006, 28(21): 1749-1753.
[66] K Akashi, N Nishimura, Y Ishida, et al. Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon[J]. Biochem Biophys Res Commun, 2004, 323(1): 72-78.
[67] 唐中华，郭晓瑞，张洋洋等．拟南芥MT-Ⅱ过量表达提高抗旱性[J]．植物研究，2005，25(4)：415-418．
[68] 全先庆，高翔．植物金属硫蛋白及其在自由基清除中的作用[J]．临沂师范学院学报，2003，25(6)：64-66．
[69] N Gorinova, M Nedkovska, E Todorovska, et al. Improved phytoaccumulation ofcadmium by genetically modified tobacco plants (Nicotiana tabacum L.). Physiological and biochemical response of the transformants to cadmium toxicity [J]. Environ Pollut, 2007, 145(1): 161-170.
[70] N Futakawa, M Kondoh, S Ueda, et al. Involvement of oxidative stress in the synthesis of metallothionein induced by mitochondrial inhibitors[J]. Biol Pharm Bull, 2006, 29(10): 2016-2020.
[71] J Domenech, G Mir, G Huguet, et al. Plant metallothionein domains: functional insight into physiological metal binding and protein folding [J]. Biochimie, 2006, 88(6): 583-593.
[72] Carstens C P and Waeshe A. Codon bias-adjusted BL21 derivatives for protein expression[J]. Strategies, 1999, 129(10): 49-51.
[73] 周宇荀，魏东芝，王二力．融合蛋白表达载体pGEX及其应用[J]．生命科学，1998，10(3)：122-124．
[74] C L Chyan, T T Lee, C P Liu, et al. Cloning and expression of a seed-specific metallothionein like protein from sesame [J]. Biosci Biotechnol Biochem, 2005, 69(12): 2319-2325.
[75] Chalfie M, Tu Y, Euskirchen G, et al. Green flurescent protein as a marker for gene expression [J]. Science, 1994, 263(5148): 802-805.
[76] Cheng L, Fu J, Ysukamoto A, et al. Use of green fluorescent protein variants to monito gene transfer and expression in mammalian cells [J]. Nat Biotechnol, 1996, 14(5): 606-609.