李慧;阚家亮;王影;蔺经;杨青松;常有宏;

• 1:江苏省农业科学院果树研究所
• 2:江苏省高效园艺作物遗传改良重点实验室
• 3:南京农业大学园艺学院

摘要(Abstract)：

【目的】测定谷胱甘肽还原酶活性变化,结合其催化产物含量、编码基因表达特点分析,明确该酶是否参与豆梨应对镉胁迫的调控过程。【方法】采用紫外/可见分光光度法检测谷胱甘肽还原酶(glutathione reductase,GR)活性、谷胱甘肽池组成及过氧化氢(hydrogen peroxide,H_2O_2)含量,RT-PCR和PCR克隆PcGRchl和PcGRcyt的cDNA和DNA序列,利用生物信息学方法进行序列比较分析,荧光定量PCR检测它们在镉胁迫下转录水平变化。【结果】镉胁迫情况下,豆梨叶片GR活性上升,谷胱甘肽池中总谷胱甘肽(total glutathione,T-GSH)和还原型谷胱甘肽(reduced glutathione,GSH)减少、氧化型谷胱甘肽(oxidized glutathione,GSSG)上升,H_2O_2积累增加。豆梨叶绿体PcGRchl和胞质PcGRcyt的序列长度、基因结构及所编码蛋白特征各不相同,它们在豆梨叶片中的表达量上调以响应镉胁迫信号,以PcGRchl的转录占主导。外源GSH预处理有助于豆梨预先储存GSH,减缓镉胁迫下H_2O_2的积累,与Cd(2 mmol·L~(-1)CdCl2·2.5H_2O+Hoagland营养液)组相比较,GC(2 mmol·L~(-1),GSH预处理12 h转入2 mmol·L~(-1)CdCl2·2.5H_2O+Hoagland营养液)组PcGRchl和PcGRcyt的表达水平没有太大变化,但GR活性部分受抑制;外源BSO预处理抑制植株叶片GSH合成,镉胁迫下BC(2 mmol·L~(-1)丁硫氨酸-亚砜亚胺预处理12 h转入2 mmol·L~(-1)CdCl2·2.5H_2O+Hoagland营养液)组H_2O_2产生加剧,GR活性上升幅度变大,但PcGRchl和PcGRcyt的转录不受影响。上述结果表明,GSH预处理或BSO预处理,可改变豆梨叶片GSH池的组成,从而在蛋白质水平上反馈调节镉胁迫情况下GR活性。【结论】豆梨GR参与应对镉胁迫的调控过程。镉处理后,豆梨叶片中GSH减少,促使GR酶活性上升,以补充植株应对逆境所需GSH,这一过程主要通过叶绿体PcGRchl的转录上调来实现;GSH或BSO预处理,改变植株GSH含量,从而影响GR活性,减缓或加剧镉胁迫下H_2O_2的产生,这一过程主要是在GR基因翻译后的蛋白水平上进行反馈调节。

关键词(KeyWords)： 豆梨;镉胁迫;谷胱甘肽还原酶;活性调节;表达特点

Abstract:

Keywords:

基金项目(Foundation): 江苏省自然科学基金(BK20151361)

作者(Author): 李慧;阚家亮;王影;蔺经;杨青松;常有宏;

Email:

参考文献(References)：

• [1]GILL S S,ANJUM N A,HASANUZZAMAN M,GILL R,TRIVEDI D K,AHMAD I,PEREIRA E,TUTEJA N.Glutathione and glutathione reductase:A boon in disguise for plant abiotic stress defense operations[J].Plant Physiology and Biochemistry,2013,70(1):204-212.
• [2]NOCTOR G,MHAMDI A,CHAOUCH S,HAN Y,NEUKER-MANS J,MARQUEZ-GARCIA B,QUEVAL G,FOYER C H.Glutathione in plants:an integrated overview[J].Plant,Cell and Environment,2012,35(2):454-484.
• [3]GILL S S,TUTEJA N.Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J].Plant Physiology and Biochemistry,2010,48(12):909-930.
• [4]TAHMASEBI A,ARAM F,EBRAHIMI M,MOHAMMADI-DEHCHESHMEH M,EBRAHIMIE E.Genome-wide analysis of cytosolic and chloroplastic isoforms of glutathione reductase in plant cells[J].Plant Omics Journal,2012,5(2):94-102
• [5]GARNIK E Y,BELKOV V I,TARASENKO V I,KORZUN MA,KONSTANTINOV Y M.Glutathione reductase gene expression depends on chloroplast signals in Arabidopsis thaliana[J].Biochemistry(Moscow),2016,81(4):364-372.
• [6]HONG C Y,CHAO Y Y,YANG M Y,CHENG S Y,CHO S C,KAO C H.NaCl-induced expression of glutathione reductase in roots of rice(Oryza sativa L.)seedlings is mediated through hydrogen peroxide but not abscisic acid[J].Plant and Soil,2009,320(1/2):103-115.
• [7]WU T M,LIN W R,KAO Y T,HSU Y T,YEH C H,HONG CY,KAO C H.Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3involved in salt stress response in rice[J].Plant Molecular Biology,2013,83(4/5):379-390.
• [8]ACHARY V M M,REDDY C S,PANDEY P,ISLAM T,KAULT,REDDY M K.Glutathione reductase a unique enzyme:molecular cloning,expression and biochemical characterization from the stress adapted C4plant,Pennisetum glaucum(L.)R.Br[J].Molecular Biology Reports,2015,42(5):947-962.
• [9]邓治,刘辉,王岳坤,李德军.橡胶树胞质型谷胱甘肽还原酶基因的克隆与表达分析[J].植物生理学报,2014,50(11):1699-1706.DENG Zhi,LIU Hui,WANG Yuekun,LI Dejun.Molecular cloning and expression analysis of a cytosolic glutathione reductase gene from Hevea brasiliensis[J].Plant Physiology Journal,2014,50(11):1699-1706.
• [10]DENG Z,ZHAO M M,LIU H,WANG Y K,LI D J.Molecular cloning,expression profiles and characterization of a glutathione reductase in Hevea brasiliensis[J].Plant Physiology and Biochemistry,2015,96:53-63.
• [11]LEE H,WON S H,LEE B H,PARK H D,CHUNG W,JO J K.Genomic cloning and characterization of glutathione reductase gene from Brassica campestris var.Pekinensis[J].Molecules and Cells,2002,13(2):245-251.
• [12]朱守晶,余伟林,石朝燕,揭雨成.苎麻谷胱甘肽还原酶基因(BnGR1)的克隆和表达分析[J].农业生物技术学报,2015,23(10):1318-1326.ZHU Shoujing,YU Weilin,SHI Zhaoyan,JIE Yucheng.Cloning and expression analysis of glutathione reductase gene(BnGR1)from Ramie(Boehmeria nivea L.)[J].Journal of Agricultural Biotechnology,2015,23(10):1318-1326.
• [13]TORRES-FRANKLIN M L,CONTOUR-ANSEL D,ZUILY-FO-DIL Y,PHAM-THI A T.Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress[J].Journal of Plant Physiology,2008,165(5):514-521.
• [14]DIETZ K J,HELL R.Thiol switches in redox regulation of chloroplasts:balancing redox state,metabolism and oxidative stress[J].Biological Chemistry,2015,396(5):483-494.
• [15]ANJUM N A,AHMAD I,MOHMOOD I,PACHECO M,DU-ARTE A C,PEREIRA E,UMAR S,AHMAD A,KHAN N A,IQBAL M,PRASAD M N V.Modulation of glutathione and its related enzymes in plants’responses to toxic metals and metalloids-A review[J].Environmental and Experimental Botany,2012,75:307-324.
• [16]LI H,HAN J L,LIN J,YANG Q S,CHANG Y H.Aγ-glutamylcysteine synthetase gene from Pyrus calleryana is responsive to ions and osmotic stresses[J].Plant Molecular Biology Reporter,2015,33(4):1088-1097.
• [17]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1/2):248-254.
• [18]FOYER C H,HALLIWELL B.The presence of glutathione and glutathione reductase in chloroplasts:A proposed role in ascorbic acid metabolism[J].Planta,1976,133(1):21-25.
• [19]PATTERSON B D,MACRAE E A,FERGUSON I B.Estimation of hydrogen peroxide in plant extracts using titanium(Ⅳ)[J].Analytical Biochemistry,1984,139(2):487-492.
• [20]ANDERSON M E.Determination of glutathione and glutathione disulfide in biological samples[J].Methods in Enzymology,1985,113(4):548-554.
• [21]BAILEY T,ELKAN C.Fitting a mixture model by expectation maximization to discover motifs in biopolymers[C]//Second International Conference on Intelligent Systems for Molecular Biology,August 14-17,1994,Stanford,Menlo Park,California,AAAI Press Proceeding,1994,28-36.
• [22]LOU L L,KANG J Q,PANG H X,LI Q Y,DU X P,WU W,CHEN J X,LV J Y.Sulfur protects pakchoi(Brassica chinensis L.)seedlings against cadmium stress by regulating ascorbateglutathione[J].Metabolism International Journal of Molecular Sciences,2017,18(8):1628
• [23]KHAN M I R,NAZIR F,ASGHER M,PER T S,KHAN N A.Selenium and sulfur influence ethylene formation and alleviate cadmium-induced oxidative stress by improving proline and glutathione production in wheat[J].Journal of Plant Physiology,2015,173(3):9-18.
• [24]WANG Y,ZHAO W W,HAO J R,XU W T,LUO Y B,WU WH,YANG Z J,LIANG Z H,HUANG K L.Changes in biosynthesis and metabolism of glutathione upon ochratoxin A stress in Arabidopsis thaliana[J].Plant Physiology and Biochemistry,2014,79(3):10-18.
• [25]HAO J R,WANG Y,ZHAO W W,XU W T,LUO Y B,YANGZ J,WU W H,LIANG Z H,HUANG K L.Effect of ochratoxin A and buthionine sulfoximine on proteome and ascorbate-glutathione cycle enzymes in Arabidopsis thaliana[J].Biologia Plantarum,2015,59(2):331-340.
• [26]EBRAHIMI M,LAKIZADEH A,AGHA-GOLZADEH P,EBRAHIMIE E,EBRAHIMI M.Prediction of thermostability from amino acid attributes by combination of clustering with attribute weighting:a new vista in engineering enzymes[J].Plos One,2011,6(8):e23146.
• [27]KISA D.Expressions of glutathione-related genes and activities of their corresponding enzymes in leaves of tomato exposed to heavy metal[J].Russian Journal of Plant Physiology,2017,64(6):876-882.