转金属硫蛋白基因(MT1)烟草抗Cd2+胁迫的生理特性分析

将柽柳(Tamarixsp.)金属硫蛋白基因(MT1,GenBank登录号:AB298390)插入植物表达载体pBI121,取代花椰菜病毒35S启动子下游的gus基因,利用农杆菌(Agrobactrium tumefaciens)介导法将MT1导入烟草(Nicotiana tobacum)基因组。对具有卡那霉素抗性,且经PCR-Southern检测和Northern杂交表现阳性的转基因株系进行抗Cd2 分析表明,金属硫蛋白基因的过量表达提高了转基因植株的抗Cd2 能力,在含200μmol L-1和400μmol L-1Cd2 的MS培养基上,转基因植株的株高和鲜重均明显优于非转基因株系;在生理性状上表现为转基因植株MDA含量及POD活性明显低于非转基因株系,叶绿素含量和SOD活性比非转基因株系明显增加。     

二色补血草中2个金属硫蛋白基因的克隆及分析

从二色补血草中分离出2个金属硫蛋白基因的cDNA全序列,分别命名为LbMT1和LbMT2。LbMT1全长为569bp,其中5′非编码区23bp,3′非编码区300bp,开放读码框(ORF)长246bp,编码含81个氨基酸的蛋白,相对分子质量为8070,等电点为4.7;LbMT2全长为523bp,其中5′非编码区61bp,3′非编码区216bp,开放读码框长246bp,编码81个氨基酸,编码蛋白的相对分子质量为8000,等电点为5.0。这2个基因编码的氨基酸都含有14个半胱氨酸Cys,分布在蛋白质的N端和C端,呈CC、CXC和CXXC形式排列。疏水性分析表明:这2个蛋白都存在35~48个氨基酸之间较强的疏水区。通过对多种植物的MT蛋白序列比对分析表明,金属硫蛋白家族在氨基酸数目上保守性差,但半胱氨酸残基(Cys)的位置和数目保守性达到100%,揭示了Cys对金属硫蛋白的功能十分重要。这2个基因已经在Genbank上注册,LbMT1基因的登录号为EF103574,LbMT2基因的Genbank登录号为EF103575。     

金属硫蛋白及其在养殖业中的应用前景

金属硫蛋白是一类广泛存在于生物中的低分子量、富含金属和半胱氨酸的金属结合蛋白，具有重金属解毒、调节机体应激反应以及必需微量元素代谢等功能，因而它成为了近年来的研究热点。本文简要综述了金属硫蛋白的理化性质、生理功能及其在动物营养代谢和维持畜禽健康中的作用，并展望了金属硫蛋白在现代养殖业中的应用前景。     

金属硫蛋白在大鼠全脑缺血再灌注损伤中的神经保护作用

采用股动脉放血并双侧颈总动脉夹闭的全脑缺血再灌注模型,测定缺血10min再灌注后6h、1d、3d、5d脑组织中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH Px)活力及丙二醛(MDA)含量,以探讨金属硫蛋白-1(MT 1)对实验性脑缺血再灌注的神经保护作用。结果表明:在对应的各时间相,老年组SOD及GSH Px的活性均显著低于青年组(P<0 05);MDA水平显著高于青年组(P<0 05);而MT-1可显著提高SOD及GSH Px的活性、降低MDA的水平(P<0 01)。本实验证明MT 1对脑缺血再灌注有明显神经保护作用。     

柽柳金属硫蛋白基因的克隆及分析

应用生物信息学技术从柽柳cDNA文库中分离出了一种金属硫蛋白全长cDNA序列,去除PolyA后该基因全长517bp。其中,5′非翻译区31bp,3′非翻译区267bp,开放读码框(ORF)长219bp,编码72个氨基酸,含13个半胱氨酸Cys,分布在蛋白质的N端和C端,呈CXC形式排列,基因编码蛋白的分子量为7.1kD,等电点为4.71。疏水性分析表明,蛋白的30～50个氨基酸之间有较强的疏水性,经过Pfam15.0(St.Louis(USA))分析证明,为金属硫蛋白家族基因。金属硫蛋白基因参与了植物的发育、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。此基因的Genbank登录号为AY620987(基因),AAT39518(蛋白)。     

Metallothioneins and regulation of mitochondrial function

Metallothioneins (MTs) are small, cysteine-rich and metal ions binding proteins, which are widely distributed in various mammalian organs. A large number of studies have found that MTs regulate metal ion homeostasis, detoxify the toxicity of heavy metal, scavenge oxygen free radicals, inhibit inflammatory cytokines and aging body, store zinc, prevent cells to become cancerous and protect against radiation and apoptosis. Recent studies suggest that metallothionein, as a multifunctional protein, may be involved in direct or indirect regulation of mitochondrial function. In particular, metallothionein plays an important role in maintaining mitochondrial function and repairing mitochondrial damage under oxidative stress. This review focus on the effects and mechanisms of metallothioneins on the regulation of mitochondrial function.     

兔肝金属硫蛋白提取工艺研究

研究提取液Tris-HCl的浓度、pH、加热温度和时间对兔肝金属硫蛋白提取的影响,并用正交试验优选出最适提取条件,为兔肝金属硫蛋白的研究提供一种新的提取技术。结果表明,Tris-HCl的浓度对金属硫蛋白的提取效果影响最大,pH和加热时间次之,温度对其影响最小。金属硫蛋白的合适提取条件为Tris-HCl的浓度0.01mol/L,pH 8.2,热处理温度80℃,加热时间8min。     

Metallothionein protects rat hepatic nuclear nucleoside triphosphatase from hydroxyl radical-induced suppression

AIM:Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The present study was designed to investigate whether MT can directly protect NTPase on nuclear envelope from damage induced by hydroxyl radical.METHODS:Isolated hepatic nuclei from rat liver were exposed to Fe²⁺/H₂O₂ with or without MT, and the NTPase activity on nuclei was assayed using ATP and GTP as substrate, respectively.RESULTS:Incubation of rat hepatic nuclei with the Fe²⁺/H₂O₂ (in μmol·L^-1/μmol·L^-1 : 0.1/0.5, 0.5/2.5, 1/5, 5/25) resulted in a concentration-dependent decrease in nuclear NTPase activities (P＜0.01). Incubation of hepatic nuclei with different concentrations of MT (10^-9-10^-4mol·L^-1)and Fe²⁺/H₂O₂ (1 μmol·L^-1/5 μmol·L^-1) for 10 min, nuclear NTPase activities were increased in a MT concentration-dependent fashion as compared with that of incubation with Fe²⁺/H₂O₂(1 μmol·L^-1/5 μmol·L^-1) alone. When MT was at 10^-4 mol·L^-1, TNPase activities reversed to (102±10) nmol·mg^-1 protein·min^-1(for ATP as substrated) and (131±12) μmol·g^-1 protein·min^-1(for GTP as substrate), which had no significant defferences from that of the controls (112±8 and 142±10 μmol·g^-1 protein·min^-1, respectively) (P＞0.05). In addition, incubation of hepatic nuclei with only MT had no effect on nuclear NTPase activity. CONCLUSION:These data demonstrate that hydroxyl radical generated from Fe²⁺/H₂O₂ might attack nuclear NTPase. MT antagonistically reduces toxicity of Fe²⁺/H₂O₂ system to the NTPase.     

Metallothionein involvement in myocardial protection of basic fibroblast growth factor

AIM: To observe whether metallothionein plays a role in cardiac protective effect of basic fibroblast growth factor on anoxic/reperfusion (A/R) injury in cultured cardiomyocytes and study the possible mechanism of cardiac protection by bFGF. METHODS: The present study made the model of myocyte A/R injury after having a 24 h incubation by bFGF (10^-10, 10^-9 and 10^-8 mol/L) and bFGF (10^-9 mol/L) +PD₀₉₈₀₅₉, respectively. We measured the levels of MT and MDA in myocytes, and the changes of LDH and protein in cultured medium. We also counted the number of viable cell in groups. RESULTS: The contents of myocardial MT were significantly increased after treatment by bFGF. The levels of MT in 10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L bFGF treated groups increased 54%, 62% and76%, respectively, compared with the A/R group, and the number of viable cell were also greatly increased, LDH and protein leakage in cultured medium and MDA contents in myocyte were dramatically decreased in bFGF treated groups. All the protection were completely disappeared with the inhibition of MT production with PD₀₉₈₀₅₉, the inhibitor of mitogen-activated protein kinase (MAPK). CONCLUSION: MT involves in the protection of bFGF in cultured cardiomyocytes. It might be related with activation of MAPKase.     

转金属硫蛋白基因(MT1)烟草抗Cd2+胁迫的生理特性分析

将柽柳（Tamarix sp.）金属硫蛋白基因（MT₁，GenBank登录号：AB298390）插入植物表达载体pBI121，取代花椰菜病毒35S启动子下游的gus基因，利用农杆菌（Agrobactrium tumefaciens）介导法将MT1导入烟草（Nicotiana tobacum）基因组。对具有卡那霉素抗性，且经PCR-Southern检测和Northern杂交表现阳性的转基因株系进行抗Cd²⁺分析表明，金属硫蛋白基因的过量表达提高了转基因植株的抗Cd²⁺能力，在含200 µmol L^-1和400 µmol L^-1 Cd²⁺的MS培养基上，转基因植株的株高和鲜重均明显优于非转基因株系；在生理性状上表现为转基因植株MDA含量及POD活性明显低于非转基因株系，叶绿素含量和SOD活性比非转基因株系明显增加。