| 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). |
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