低温环境对球等鞭金藻3011抗氧化系统和二十二碳六烯酸产量的影响
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摘要
以球等鞭金藻(Isochrysis galbana)3011为对象,通过研究降低培养温度后其超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)和过氧化氢酶(catalase,CAT)活性以及还原型谷胱甘肽(glutathione,GSH)、脂质过氧化产物丙二醛(malondialdehyde,MDA)、活性氧(reactive oxygen species,ROS)和二十二碳六烯酸(docosahexaenoic acid,DHA)含量的变化,以阐明低温环境对球等鞭金藻细胞抗氧化系统和DHA含量的影响。用流式细胞术结合荧光染色法测定低温环境对球等鞭金藻细胞内ROS水平的影响;并采用气相色谱法检测球等鞭金藻细胞内DHA含量。结果表明:在21、18、15℃低温环境处理下,球等鞭金藻细胞SOD、CAT和POD活性均随培养时间延长而呈现先升高后降低的趋势;温度越低,峰值出现越早,且峰值越大,峰后酶活性下降越快;GSH含量的变化趋势与上述酶活性的变化相似,MDA含量则持续增加;ROS水平随着温度的降低而呈现出较为复杂的变化,15℃和18℃诱导16 h出现ROS水平爆发,20 h时达到峰值,分别为(14.11±0.11)%和(14.74±0.58)%(P<0.05);经18℃低温诱导24 h后,球等鞭金藻细胞内DHA含量为0.105 mg/g,比对照组高0.06 mg/g。因此,低温环境可以作为提高代谢物产量的诱导子,使球等鞭金藻细胞产生主动防御反应,引起清除活性氧相关的酶活性的升高,同时也提高了DHA产量。
Effects of low environmental temperature on the activities of superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT), and the contents of glutathione peroxidase(GSH), malondialdehyde(MDA), docosahexaenoic acid(DHA) and reactive oxygen species(ROS) in Isochrysis galbana 3011 were investigated for exploring the relationship among antioxidant enzyme activities, ROS levels and DHA contents at different temperatures. The enzyme activities were determined by corresponding kits. ROS levels were detected by flow cytometry and inverted fluorescence microscope, and DHA contents were determined by gas chromatography(GC). The results showed that activities of SOD, CAT and POD increased firstly and then decreased with increasing culture time at 15, 18 and 21 ℃. Lower temperature could result in earlier and higher enzyme activity peaks followed by a faster decline. The profile of GSH was similar to that of antioxidant enzyme activities, while content of MDA increased continuously. ROS levels showed a complex change with declining temperature, and ROS burst was appeared after induction for 16 h at 15 and 18 ℃, with maximum values of(14.11 ± 0.11)%and(14.74 ± 0.58)% at 20 h, respectively(P < 0.05). DHA was 0.105 mg/g at 18 ℃ for 24 h, which was higher than that at 24 ℃ for 24 h by 0.06 mg/g. Therefore, low environmental temperature can improve the levels of metabolic products by activating microalgae defense responses, increasing ROS-related enzyme activities, ROS levels and DHA contents.
Effects of low environmental temperature on the activities of superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT), and the contents of glutathione peroxidase(GSH), malondialdehyde(MDA), docosahexaenoic acid(DHA) and reactive oxygen species(ROS) in Isochrysis galbana 3011 were investigated for exploring the relationship among antioxidant enzyme activities, ROS levels and DHA contents at different temperatures. The enzyme activities were determined by corresponding kits. ROS levels were detected by flow cytometry and inverted fluorescence microscope, and DHA contents were determined by gas chromatography(GC). The results showed that activities of SOD, CAT and POD increased firstly and then decreased with increasing culture time at 15, 18 and 21 ℃. Lower temperature could result in earlier and higher enzyme activity peaks followed by a faster decline. The profile of GSH was similar to that of antioxidant enzyme activities, while content of MDA increased continuously. ROS levels showed a complex change with declining temperature, and ROS burst was appeared after induction for 16 h at 15 and 18 ℃, with maximum values of(14.11 ± 0.11)%and(14.74 ± 0.58)% at 20 h, respectively(P < 0.05). DHA was 0.105 mg/g at 18 ℃ for 24 h, which was higher than that at 24 ℃ for 24 h by 0.06 mg/g. Therefore, low environmental temperature can improve the levels of metabolic products by activating microalgae defense responses, increasing ROS-related enzyme activities, ROS levels and DHA contents.
引文
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[2]宰学明,吴国荣,陆长梅,等.低温预处理对大豆萌芽活力及其活性氧代谢的影响[J].大豆科学,2001,20(3):163-166.DOI:10.3969/j.issn.1000-9841.2001.03.002.
[3]朱素琴.高盐/低温胁迫下水稻叶细胞内ASC的氧化还原状态和外源ABA的作用研究[D].扬州:扬州大学,2011:12.
[4]WINSTON S W,DIGIULIO R T.Prooxidant and antioxidant mechanisms in aquatic organisms[J].Aquatic Toxicology,1991,19(2):137-161.DOI:10.1016/0166-445X(91)90033-6.
[5]YOSHIOKA H,ASAI S,YOSHIOKA M,et al.Molecular mechanisms of generation for nitric oxide and reactive oxygen species,and role of the radical burst in plant immunity[J].Molecules and Cells,2009,28(4):321-329.DOI:10.1007/s10059-009-0156-2.
[6]TAMAS L,MISTRIK I,HUTTOVA J,et al.Role of reactive oxygen species generating enzymes and hydrogen peroxide during cadmium mercury and osmotic stresses in barley root tip[J].Planta,2010,231(2):221-231.DOI:10.1007/s00425-009-1042-z.
[7]PU G B,MA D M,CHEN J L,et al.Salicylic acid activates artemisinin biosynthesis in Artemisia annua L.[J].Plant Cell Reports,2009,28(7):1127-1135.DOI:10.1007/s00299-009-0713-3.
[8]ZHAO J,HU Q,GUO Y Q,et al.Elicitor-induced indole alkaloid biosynthesis in Catharanthus roseus cell cultures is related to Ca2+influx and the oxidative burst[J].Plant Science,2001,161(3):423-431.DOI:10.1016/S0168-9452(01)00422-8.
[9]林武杰,吴云辉.海洋微藻多不饱和脂肪酸研究进展[J].福建水产,2013,35(1):78-82.DOI:10.3969/j.issn.1006-5601.2013.01.014.
[10]赵培,王雪青,陈庆森,等.Zn2+对球等鞭金藻3011细胞膜电位和膜通透性的影响[J].食品科学,2012,33(5):66-70.
[11]赵培,王雪青,彭博丽,等.荧光分析法检测金藻对Z n2+的利用[J].食品研究与开发,2011,32(8):87-90.DOI:10.3969/j.issn.1005-6521.2011.08.027.
[12]赵培,王雪青,魏丹,等.赤霉素对球等鞭金藻3011生物量、细胞形态与不同生长时期群体细胞活性的影响[J].环境与健康杂志,2011,28(5):383-386.
[13]王海波,黄雪梅,张昭其.植物逆境胁迫中活性氧和钙信号的关系[J].北方园艺,2010(22):189-194.
[14]张怡,路铁钢.植物中的活性氧研究概述[J].生物技术进展,2011,1(4):242-248.
[15]ZHOU D,SHAO L,SPITZ D R.Reactive oxygen species in normal and tumor stem cells[J].Advances in Cancer Research,2014,122:1-67.DOI:10.1016/B978-0-12-420117-0.00001-3.
[16]毛永强.低温下胁迫螺旋藻(节旋藻)几种氧化还原酶的研究[D].呼和浩特:内蒙古师范大学,2007:3-17.
[17]刘娟妮,王雪青,庞广昌.温度和光照对极大螺旋藻多糖含量和SOD酶活力的影响[J].食品工业科技,2008,29(9):132-134.
[18]RADY A A,EI-SHEEKH M M,MATKOVICS B.Temperature shift induced changes in the antioxidant enzyme system of Cyanobacterium ynechocystis PCC 6803[J].International Journal of Biochemistry,1994,26(3):433-435.DOI:10.1016/0020-711X(94)90064-7.
[19]阚光锋.南极冰藻Chlamydomona sp.L4的逆境适应性及其抗逆蛋白质组学的研究[D].青岛:中国海洋大学,2005:26-33.
[20]丁燏,缪锦来,王全富,等.温度对南极衣藻ICE-L(Chlamydomonassp.ICE-L)谷胱甘肽含量及其相关酶活性的影响[J].海洋与湖沼,2006,37(2):154-161.DOI:10.3321/j.issn:0029-814X.2006.02.009.
[21]夏利花,何剑锋,高岩,等.三种北极微藻对不同温度的适应性研究[J].极地研究,2009,21(4):279-287.
[22]郭兵,龚阳,万霞,等.光强和温度对球等鞭金藻(Isochrysis sphaerica)生长及其脂肪酸的影响[J].中国油料作物学报,2011,33(3):295-301.
[23]CONVERTI A,CASAZZA A A,ORTIZ E Y,et al.Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production[J].Chemical Engineering and Processing:Process Intensification,2009,48(6):1146-1151.DOI:10.1016/j.cep.2009.03.006.
[24]CAI Fang,DUPERTUIS Y M,PICHARD C.Role of polyunsaturated fatty acids and lipid peroxidation on colorectal cancer risk and treatments[J].Current Opinion in Clinical Nutrition&Metabolic Care,2012,15(2):99-106.DOI:10.1097/MCO.0b013e32834feab4.
[25]蒋汉明,翟静,张媛英,等.温度对海洋微藻生长及脂肪酸组成的影响[J].食品研究与开发,2005,26(6):9-12.DOI:10.3969/j.issn.1005-6521.2005.06.003.
[26]FISARAKIS I,CHARTZOULAKIS K,STAVRAKAS D.Response of Sultana vines(V.vinifera L.)on six rootstocks to Na Cl salinity exposure and recovery[J].Agricultural Water Management,2001,51(1):13-27.DOI:10.1016/S0378-3774(01)00115-9.
[27]范博.低温胁迫下ABA诱导冬小麦抗氧化防护系统的研究[D].哈尔滨:东北农业大学,2012:6.
[28]程艳丽,宋纯鹏.植物细胞H2O2的信号转导途径[J].中国科学:生命科学,2005,35(6):480-489.DOI:10.3321/j.issn:1006-9259.2005.06.001.
[29]GECHEV T,WILLEKENS H,van MONTAGU M,et al.Different responses of tobacco antioxidant enzymes to light and chilling stress[J].Journal of Plant Physiology,2003,160(5):509-515.DOI:10.1078/0176-1617-00753.
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