Cd2+胁迫条件下椭圆小球藻的生理应答
PHYSICAL RESPONSES TO Cd2+ STRESS IN CHLORELLA ELLIPSOIDEA
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摘要: 研究了一株高重金属抗性的椭圆小球藻在Cd2+胁迫下的生理变化,结果显示,在10-240μmol/L浓度的Cd2+胁迫下,随着金属离子浓度提高,叶绿素的总量减少,光合放氧受到抑制.Cd2+离子浓度的提高,导致了氧自由基的大大增加,同时脯氨酸、SOD(超氧化物歧化酶)以及POD(过氧化物酶)水平均大大提高.结果提示这些与消除自由基有关的代谢产物的积累,有利于细胞减少胁迫造成的损伤.CAT(过氧化氢酶)对Cd2+离子是敏感的,其活性与胁迫浓度呈负相关.Abstract: A high heavy metal resistant Chlorella ellipsoidea strain was isolated from the electroplating waste water.Former researches had showed the alga had high tolerance to heavy metals and was able to eliminate Cu2+ ,Zn2+ ,Ni2+ and Cd2+ effectively from water.So it could be used for decreasing heavy metal pollution.To understand the physical mechanisms of the alga against the toxicities of heavy metals,we studied the physical responses of the alga to Cd2+ stress.In this research,we focused on the physical reactions to radical injuring in cells caused by Cd2+ . Methods:Photosynthesis O2 release was measured by membrane oxygen electrode;Superoxide anion radical was measured by xanthine oxidase and NBT system;Superoxide dismutase (SOD) activity is determined by hydroxylamine assay-developed from xanthine oxidase assay;Catalase(CAT)was measured by molybdic acid method;Peroxidase(POD)was determined by method of guaiacol oxidation;Proline was measured by the method of Sulosalicylic acid. Results:Chlorophyll content and photosynthetic O2 release were measured as indicators of cadmium toxicities.Results showed that,between 10 to 240μmol/L Cd2+ ,total chlorophyll content and photosynthesis O2 release decreased when the ion concentration increased.The lower concentration of Cd2+ ,10μmol/L induced higher chlorophyll-a,it was 130% of the control.Cadmium caused the ratio of chlorophyll-a/b rising. When Cd2+ was over 30μmol/L,the ratio of chla/b rose from 1.22 to 2.61 These showed Cd2+ in this concentration range could cause toxicities to the C.ellipsoidea. So this concentration range of Cd2+ was used as stress to induce radicals and observe the physical responses of the alga.Results showed Cd2+ produced more O2-. in algal cells.When Cd2+ was 90μmol/L,O2-. was 2.07 times as much as the control in 72hrs after incubated.Meanwhile,POD was induced strongly and reached the highest level.It’s content was 4.84 times concentration of the control.But for 120μmol/L and 240μmol/L Cd2+ ,POD content was 4.10 and 3.63 times respectively;There were four SOD isoenzyme bands in the electrophoresis gel.The least molecular band was Fe-SOD,and the other three were Mn-SOD.Highest SOD content was induced by 120μmol/L Cd2+ ,reached 1.3 times as much as the control.Two types of SOD showed different responses to cadmium stress.Mn-SOD obviously increased.That caused total amount of SOD increasing.But Fe-SOD decreased;Proline content and Cd2+ concentration in algal cells was correlated.When Cd2+ was 10μmol/L,proline content was 1 89 times of the control.Proline reached highest level,2.86 times of the control in 120μmol/L Cd2+ .These results suggested that increasing of free radical elimination enzymes and substances was helpful to the algae to resistant cadmium injuring.However,as one of the most important anti-oxidation enzymes,CAT showed an opposite response to the Cd2+ .Its content went down with Cd2+ going up.When Cd2+ was 90μmol/L,CAT activity was 23.36% of the control.This meant CAT was very sensitive to Cd2+ . Conclusion:Cd2+ could induce radicals which were toxic to algal cell.The high heavy metal resistant C.ellipsoidea strain could produce more SOD,POD and proline to decrease the injuries.
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[1] Hao Y T,LI J-H,Pan X et al.Tolerance of Chlorella ellipsoidea and its removal of 4 heavy metals[J].J Lake Sci.2001,13(2):158-162[浩云涛,李建宏,潘欣,等.椭圆小球藻对4种重金属的耐受性及富积.湖泊科学,2001,13(2):158-162][2] Stohs S J,Bagchi D.Oxidative mechanisms in the toxicity of metal ions[J].Free Radic Biol Med,1995,18:321-336[3] Noctor G,Arisi A-C M,Jouanin L,et al.Glutathione:biosynthesis,metabolism and relationship to stress tolerance explored in transformed plants[J].J Exp Bot,1998,49:623-647[4] Nagalakshmi N,Prasad M N.Responses of glutathione cycle enzymes and gulathione metabolism to copper stress in Scenedesmus bijugatus[J].Plant Sci,2001,160:291-299[5] PAN X,LI J-H,HAO Y-T et al.A improved method for chlorophylla extraction with DMF[J].Biotech.2001,11(1):39-41[潘欣,李建宏,浩云涛,等.DMF提取微藻叶绿素a方法的改进.生物技术,2001,11(1):39-41][6] LI D Y,Ye J Y.Preparation of membrane oxygen electrode and measurements of respiration and photosynthesis[J].Plant Physiol Com 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erythrocuprein(hemocuprein[J]).J.Biol.Chem.,1969,244:6049-6055[14] Zhou C F,Wu G R,Shi G X et al,The role of antioxidant system in Cu2+ stress resistance in Alternanthera philoxeroides[J].Acta Bot Sin 2001,43(4):389-394.[周长芳,吴国荣,施国新等.水花生抗氧化系统在抵御Cu2+胁迫中的作用.植物学报,2001,43(4):389-394][15] Buonaurio R,Torre G D,Montalbini P.Soluble superoxide dismutase(SOD)in susceptible and resistant hostparasite complex of Paseolus vulgaris and Uromyces phaseoli[J].Physiol.Mol.Plant Pathol.,1987,31:173[16] Mehta S K,Gaur J P.Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris[J].New Phytol.,1999,143:253-259[17] Alia,Mohanty P,Matysik J.Effect of proline on the production of single oxygen[J].Amino Acids,2001,21:195-200 Hao Y T,LI J-H,Pan X et al.Tolerance of Chlorella ellipsoidea and its removal of 4 heavy metals[J].J Lake Sci.2001,13(2):158-162[浩云涛,李建宏,潘欣,等.椭圆小球藻对4种重金属的耐受性及富积.湖泊科学,2001,13(2):158-162][2] Stohs S J,Bagchi D.Oxidative mechanisms in the toxicity of metal ions[J].Free Radic Biol Med,1995,18:321-336[3] Noctor G,Arisi A-C M,Jouanin L,et al.Glutathione:biosynthesis,metabolism and relationship to stress tolerance explored in transformed plants[J].J Exp Bot,1998,49:623-647[4] Nagalakshmi N,Prasad M N.Responses of glutathione cycle enzymes and gulathione metabolism to copper stress in Scenedesmus bijugatus[J].Plant Sci,2001,160:291-299[5] PAN X,LI J-H,HAO Y-T et al.A improved method for chlorophylla extraction with DMF[J].Biotech.2001,11(1):39-41[潘欣,李建宏,浩云涛,等.DMF提取微藻叶绿素a方法的改进.生物技术,2001,11(1):39-41][6] LI D Y,Ye J Y.Preparation of membrane oxygen electrode and measurements of respiration and photosynthesis[J].Plant Physiol Com 1980,(1):35-40[李德耀,叶济宇.薄膜氧电极的制作与呼吸或光合控制的测定.植物生理学通讯,1980,(1):35-40][7] Bates LS,Waldren RP,Teare ID.Rapid determination of free proline for water stress studies[J].Plant and Soil,1973,39:205-207[8] Wang A G,Luo G H.Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants[J].Plant Physiol Com 1990,(6):55-57[王爱国,罗广华.植物的超氧自由基与羟胺反应的定量关系.植物生理学通讯,1990,(6):55-57][9] Wang A G,Luo G H.Electrophoreses and activity expression of plant SOD[J].Plant Physiol Com 1983,(6):44-45[罗广华,王爱国.植物SOD的凝胶电泳及活性的显示.植物生理学通讯,1983,(6):44-45][10] Maehly A C.Plant Peroxidase.Methods in Enzymology,[M]Vol II,San Diego:Academic Press,1955,801-813[11] Stobart A K,Griffiths W T,et al.The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley[J].Physiol.Plant,1985,63:293-298[12] Wang H X,Basic Pollution Ecology,[M]Kunming:Yun Nan University Press,1990,71-148[王焕校.污染生态学基础.昆明:云南大学出版社,1990:71-148][13] McCord J M,Fridovich I.Superoxide dismutase:An enzymic function for erythrocuprein(hemocuprein[J]).J.Biol.Chem.,1969,244:6049-6055[14] Zhou C F,Wu G R,Shi G X et al,The role of antioxidant system in Cu2+ stress resistance in Alternanthera philoxeroides[J].Acta Bot Sin 2001,43(4):389-394.[周长芳,吴国荣,施国新等.水花生抗氧化系统在抵御Cu2+胁迫中的作用.植物学报,2001,43(4):389-394][15] Buonaurio R,Torre G D,Montalbini P.Soluble superoxide dismutase(SOD)in susceptible and resistant hostparasite complex of Paseolus vulgaris and Uromyces phaseoli[J].Physiol.Mol.Plant Pathol.,1987,31:173[16] Mehta S K,Gaur J P.Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris[J].New Phytol.,1999,143:253-259[17] Alia,Mohanty P,Matysik J.Effect of proline on the production of single oxygen[J].Amino Acids,2001,21:195-200
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