【作者】 雷红灵；

【导师】 吴永尧；

【作者基本信息】 湖南农业大学 ， 植物生物化学与分子生物学， 2010， 博士

【摘要】 硒在有益和毒害水平之间非常狭小的浓度范围内对人和动物起着重要生理作用,而硒在地球上的分布却极不均匀,导致硒污染和硒缺乏在不同的地区危害人和动物的健康,因此治理硒污染和补硒受到人们的共同关注。植物在这两方面的关系中起着枢纽作用,特别是一些超富硒能力的植物,它们可以从富硒地区吸收大量的硒,对硒毒土壤或水域进行修复,在缺硒地区作为硒释放系统把体内积累的硒归还土壤或供给人和动物。在恩施高硒区(恩施双河乡鱼塘坝)发现了一种富硒能力极强的十字花科碎米荠属(Cardamine L)植物碎米荠(Cardamine sp.),其苗期叶片含硒量可以超过国际上的超富硒植物的含硒临界标准1000 mg/kg。为了了解硒对该植物的作用机理及植物中含硒蛋白的分布、性质及其生物活性,为深度开发和利用该富硒植物资源提供理论依据,进行了以下几方面的研究：(1)以亚硒酸钠作为硒源,设计含硒0、15、30、45、60、75、90m/L的完全营养液培养恩施碎米荠幼苗,探讨了营养液中不同硒质量浓度对恩施碎米荠的光合作用、有效成分及抗氧化系统的影响。(2)利用双向电泳等蛋白质组学技术鉴定了该植物种子和叶片蛋白质的组成分布；并对不同硒质量浓度的营养液培养的植物叶片中蛋白质的表达差异进行了分析；利用质谱结合数据库检索鉴定了3个差异蛋白。(3)以恩施高硒地区生长的野生碎米荠为材料,采用SDS-PAGE电泳,电洗脱回收电泳分离的蛋白条带,经氢化物发生原子荧光光度法测硒,鉴定了叶片中含硒蛋白的分布；建立了一个基于盐析和凝胶分离的蛋白质纯化流程；并对纯化的蛋白质进行了理化性质的初步分析；利用傅里叶红外光谱(FTIR)及圆二色谱对纯化的蛋白质进行了二级结构的表征。(4)模拟机体产生超氧阴离子的体系研究了纯化的含硒蛋白抗自由基的活性；以昆明小鼠为研究对象,探讨了纯化含硒蛋白的体外抗氧化活性；通过胃饲小鼠一月,研究了含硒蛋白对小鼠肝脏和血清中抗氧化酶的影响。以上研究显示：(1)恩施碎米荠能有效地富集外源硒,幼苗叶片中硒的质量分数最高可达到1651.22 mg/kg。(2)营养液中硒质量浓度在15-75 m/L可以通过促进碎米荠叶绿素b的合成而提高光合效率,促进植物叶片中可溶性糖、氨基酸、蛋白质的合成；硒质量浓度在30-75mg/L,可以抑制碎米荠脂质过氧化,减少叶片和根系的MDA生成,促进叶片及根系的抗氧化物质(GSH和Vc)的合成,提高抗氧化酶(GSH-Px、APX、CAR、POD、SOD)的活性,不同的酶的促进效应略有不同。(3)碎米荠种子蛋白质主要分布在pH5-10,而叶片中蛋白质主要分布在pH4-7；在含硒45、90 mg/L的营养液中生长的碎米荠叶片蛋白质表达与对照相比,有3个蛋白点无限下调,12个蛋白点无限上调,5个表达下调大于3倍差异,4个表达上调大于3倍；三个点的质谱鉴定表明,1,5二磷酸核酮糖羧化酶在硒处理组中表达明显增加,而新表达的两个蛋白点在数据库中无已知蛋白可匹配。(4)叶片可溶性蛋白经SDS-PAGE电泳分离后检测出8条含硒蛋白带,其相对分子质量分别为15.506、21.416、27.529、29.051、31.212、57.435、62.825、82.220 kD。(5)35%-55%饱和度硫酸铵沉淀的蛋白质经Sephadex G-25脱盐,Sephadex G-100纯化,DEAE Sepharose fast flow离子交换,可以得到纯的蛋白质,等电点pH 4.8左右,高效液相分离检测含硒氨基酸的摩尔百分数为0.28%；二级结构中,a-螺旋17.8%,p-折叠31.8%,p回角20.3%,无规则卷曲30.1%。(6)纯化的恩施碎米荠含硒蛋白具有清除自由基和抗脂质过氧化的能力,含硒蛋白溶液的硒浓度为0.95μg/mL时,对羟自由基和超氧阴离子的清除率超过50%,含硒5.96μg/mL的蛋白溶液对MDA生成的抑制率达到62.27%；在体外,对小鼠线粒体和肝脏MDA的生成具有明显的抑制作用,硒质量浓度为5.96μg/mL的含硒蛋白溶液可以保护红细胞免受过氧化损伤。(7)小鼠胃饲含硒2.25、4.45、8.90、17.79μg/mL的含硒蛋白溶液(相当于每天补硒0.45、0.89、1.78、3.46μg),可以促进小鼠的生长发育,提高血硒水平,增强小鼠血清及肝脏的总抗氧化能力、GSH-Px以及SOD的活力,减少肝脏和血清中MDA的生成。结果表明,恩施碎米荠具有强的富硒和耐硒能力,营养液中的硒质量浓度在30-60mg/L范围内可以明显促进植物的生长发育；硒可以影响叶片蛋白质的表达；叶片中的含硒蛋白具有稳定的抗氧化活性。更多还原

【Abstract】 Between beneficial and toxic levels, selenium (Se) plays an important physiological role in humans and animals within a very narrow concentration range. However, as selenium is unevenly distributed on earth, selenium pollution and selenium deficiency have endangered the health of both humans and animals in different areas. Therefore, much attention has been given to abatement of pollution and supplement of Se. Plants play a vital role in these two aspects. The plants with a strong capacity of accumulating selenium can absorb a large amount of Se from Se -rich environment to decontaminate soil and water, and they can also serve as a selenium release system and return the accumulated Se to soil or provide it to humans and animals in Se -deficient areas.A species in Cardamine, which is found in Yutangba, Shuanghe Town (an area rich in Se) of Enshi, has strong capacity of accumulating Se. The Se content In seedling leaves exceeds 1000 mg/kg, which is the international standard of Se-hyperaccumulator. To know more about the mechanism of Se activity and the distributions, properties and biological activities of Se-containing proteins in this plant, as well as to provide a theoretical basis for making full use of this plant resource, the following studies were conducted:(1) Cardamine sp. was cultivated in hydroponic culture medium with different Na2SeO3 concentration (0,15,30,45,60,75 and 90 mgSe-L"1). The effects of different Se concentrations on photosynthesis, active ingredients and antioxidative systems in the plant were measured.(2) By Using proteomics technologies incuding two-dimensional (2D) electrophoresis, the protein composition and distribution in the seeds and leaves were identified, and the differences in the expression of proteins in leaves of this plants cultivated in culture medium with different Se concentration were analyzed. Moreover, three protein spots of differently-expressed protein were identified via mass spectrometry (MS) and protein database.(3) The proteins in its leaves were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands in the gel were reclaimed by electroelution, and Se content was detected with hydride generation atomic fluorescence spectrometry, the Se-containing proteins were identified in the wild Cardamine growing in Enshi City. The appropriate processes to separate and purify Se-containing proteins were developed. The physical and chemical properties of the purified proteins were analyzed, and the secondary structure was characterized by using Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD).(4) Anti-free radical activity of the purified proteins was studied by simulating the system of superoxide anion’s production. Taking Kunming mouse as the experimental animal, the antioxidant activity of purified selenoprotein was analyzed in vitro and in vivo by gastrogavage with Se-containing protein solution.The main results are as follows:(1) Cardamine sp. can absorb exogenous Se efficiently, and the mass fraction of Se in its seedling can reach as high as 1651.22 mg/kg.(2) When the concentration of Se ranges from 15 to 75 mg/L in hydroponic culture medium, the synthesis of chlorophyll b and the efficiency of photosynthesis, the synthesis of soluble sugars, amino acids, proteins and antioxidants such as glutathione (GSH) and Vitamin C(Vc) in the leaves of Cardamine sp. increased with concentration of selenium. When the concentration of Se range from 30 to 75 mg/L, lipid peroxidation was inhibited and the product of MDA was reduced in the leaves and roots, but the activities of anti-oxidative enzymes, such as glutathione peroxidase (GSH-Px), peroxidase (APX), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) were enhanced. The effects of selenium concentration on different enzymes varied sligtly.(3) The electrophoretogram showed that most of the proteins in the seeds distributed within pH 5 to 10, while the proteins in the leaves were mainly in the range of pH 4-7. By comparing the proteins in the leaves in hydroponic culture with Se concentration Ievels45 and 90mg/L with those in the control group,5 protein spots were up-regulated and 4 down-regulated (abundance changed more than 3 times),3 protein spots disappeared and 12 emerging in the 2D images. The identification on three-protein-spot via MS and protein database indicated that the upregulated protein matched to ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, and the other emerging two proteins did not match any known proteins.(4) Eight Se-containing protein bands were detected after SDS-PAGE electrophoresis, and the distribution of the molecular weights were 15.506,21.416,27.529,29.051,31.212, 57.435,62.825, and 82.220 kD respectively.(5) Water soluble proteins in the wild Cardamine sp. was purified by fractionation with SephadexG-25, G-100 filtration and DEAE Sepharose fast flow chromatography using 35%-55% ammonium sulfate, and the purified protein fraction was identified with the isoelectric point about pH4.8. The molar percent of Se-containing amino acid was 0.28%, measured by using high performance liquid chromatography (HPLC). FTIR and CD analysis showed that the purified protein hasα-helix (17.8%),β-fold (31.8%),β-turningangle (20.3%), and random coil (30.1%) in the secondary structure.(6) Purified Se-containing protein is capable to scavenge free radicals and resist to lipid peroxidation. When the concentration of Se is 0.95μg/mL in the Se-containing protein solution, it could scavenge more than 50% of the hydroxyl radical and superoxide anion. When the concentration reaches 5.96μg/mL, the inhibition rate of MDA could be up to 62.27%. In vitro, MDA in mitochondrial and liver of the mouse could be inhibited obviouslby by Se-containing protein. Moreover, when the selenium concentration reaches 5.96μg/mL, the Se-containing protein solution could protect the red blood cells from oxidative damage.(7) In vivo, after they were fed with different doses of Se-containing protein solution (Se concentration was 3.56,7.38,10.24, and 20.86μg/mL,respectively) by gastrogavage for a month, the mice grew up more quickly than those in the control group, and their blood selenium levels are also increased. Moreover, A-TOC were promoted, activities of GSH-Px and SOD were enhanced, and the generation of MDA were inhibited in the mouse’s serum and liver.It is concluded that Cardamine sp. is a kind of Se-hyperaccumulator and has a great tolerance to excessive selenium. When the selenium concentrations range from 30 to 60 mg/L in hydroponic culture, they can significantly promote the growth and development of the plant. Selenium can also affect the expression of the proteins in the leaves, and the selenium-containing proteins in the leaves have stable bioactivities (such as antioxidant activity).更多还原