东方山羊豆Cu/ZnSOD基因的克隆及表达分析

doi:10.3724/SP.J.1005.2012.00095

遗传 ›› 2012, Vol. 34 ›› Issue (1): 95-101.doi: 10.3724/SP.J.1005.2012.00095

东方山羊豆Cu/ZnSOD基因的克隆及表达分析

李玉坤, 王学敏, 高洪文, 仁爱琴, 王赞, 孙桂枝

中国农业科学院北京畜牧兽医研究所, 北京 100193

收稿日期:2011-03-29 修回日期:2011-10-15 出版日期:2012-01-20 发布日期:2012-01-25
通讯作者: 王学敏 E-mail:wangxm@iascaas.net.cn
基金资助:
十二五科技支撑项目(编号：2011BAD17B01)和国际科技合作与交流专项(编号: 2010DFR30620-2)资助

Cloning and expression analysis of Cu/ZnSOD gene from Galega orientalis L

LI Yu-Kun, WANG Xue-Min, GAO Hong-Wen, REN AI-Qin, WANG Zan, SUN Gui-Zhi

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2011-03-29 Revised:2011-10-15 Online:2012-01-20 Published:2012-01-25

摘要/Abstract

摘要： 超氧化物歧化酶是一种广泛存在于真核生物中的金属酶类, 在植物的抗逆性中起到重要的作用。文章采用RACE方法, 从东方山羊豆中克隆了Cu/ZnSOD基因, 并对其进行了初步分析。该基因cDNA序列全长935 bp, 开放阅读框600 bp, 编码199个氨基酸, 蛋白质分子量为20.35 kDa。通过实时荧光定量PCR结果分析, 该基因在东方山羊豆叶中表达量最多, 茎中次之, 根中最少。在NaCl和PEG诱导下, Cu/ZnSOD基因表达量先上调后下降。NaCl诱导24 h后, 该基因的表达量显著低于对照。ABA胁迫抑制了该基因的表达。亚细胞定位结果表明, Cu/ZnSOD蛋白定位于叶绿体中。实验结果证明, Cu/ZnSOD基因主要在东方山羊豆的绿色组织中表达, 在抵抗渗透性胁迫方面起到一定作用。

关键词: 东方山羊豆, Cu/ZnSOD基因, 基因克隆, 实时荧光定量PCR, 亚细胞定位

Abstract: SOD is an important enzyme which exists in eukaryote extensively and plays an essential role in stress-tolerance of higher plants. A cDNA of Cu/ZnSOD gene was cloned from Galega orientalis L. using rapid amplification of cDNA ends (RACE) method. The full-length of cDNA sequence is 935 bp, included a 600 bp open reading frame which encoded a 199-amino-acid polypeptide. The molecular weight of this protein was 20.35 kDa. The results of Real-Time PCR indicated that the expression level of Cu/ZnSOD gene was the highest in leaves, moderate in stems, and the least in roots. The expression of Cu/ZnSOD gene under stress of NaCl and PEG was up-regulated firstly and then declined. The expression level was significantly lower than the control after 24 h treated with NaCl. Abscisic acid downregulated the expression of Cu/ZnSOD gene. The result of subcellular localization indicated that Cu/ZnSOD was located in chloroplast. Gene Cu/ZnSOD mainly expressed in the green organs of G. orientalis and played a certain role in resisting osmotic stress.

Key words: Galega orientalis L, Cu/ZnSOD gene, cloning, real-time PCR, subcellular localization

李玉坤，王学敏，高洪文，仁爱琴，王赞，孙桂枝. 东方山羊豆Cu/ZnSOD基因的克隆及表达分析[J]. 遗传, 2012, 34(1): 95-101.

LI Yu-Kun, WANG Xue-Min, GAO Hong-Wen, REN AI-Qin, WANG Zan, SUN Gui-Zhi. Cloning and expression analysis of Cu/ZnSOD gene from Galega orientalis L[J]. HEREDITAS, 2012, 34(1): 95-101.

参考文献

[1] Alscher RG, Erturk N, Heath LS. Role of superoxide dis-mutases (SODs) in controlling oxidative stress in plants. J Exp Bot, 2002, 53(372): 1331-1341.
[2] Sen Raychaudhuri S, Deng XW. The role of superoxide dismutase in combating oxidative stress in higher plants. Bot Rev, 2000, 66(1): 89-98.
[3] Slooten L, van Montagu M, Inzé D. Manipulation of oxi-dative stress tolerance in transgenic plants. In: Lindsey K, ed. Transgenic Plant Research. Amsterdam: Harwood Academic Publishers, 1998: 241-262.
[4] Bannister JV, Bannister WH, Rotilio G. Aspects of the structure, function, and applications of superoxide dismutas. Cri Rev Biochem Mol Biol, 1987, 22(2): 111-180.
[5] 杜金芳, 曾勇庆, 陈伟, 崔景香, 陈其美, 杨伦, 胡艳霞. 猪CuZnSOD基因的克隆、表达及功能分析. 遗传, 2010, 32(10): 1037-1042.
[6] Song FN, Yang CP, Liu XM, Li GB. Effect of salt stress on activity of superoxide dismutase (SOD) in Ulmus pumila L. J Forestry Res, 2006, 17(1): 13-16.
[7] 郭丽红, 吴晓岚, 龚明. 谷胱甘肽还原酶和超氧化物歧化酶在玉米幼苗热激诱导的交叉适应中的作用. 植物生理学通讯, 2005, 41(4): 429-432.
[8] 冯昌军, 罗新义, 沙伟, 王凤国. 低温胁迫对苜蓿品种幼苗SOD、POD活性和脯氨酸含量的影响. 草业科学, 2005, 22(6): 29-32.
[9] Ronald E, Joseph A, John G. Cloning of cDNA for maize superoxide dismutase2 (SOD2). Proc Nat Acad Sci USA, 1987, 84(1): 179-183.
[10] Bowler C, Alliotte T, De Loose M, van Montagu M, Inzé D. The induction of manganese superoxide dismutase in response to stress in Nicotiana plumbaginifolia. EMBO J, 1989, 8(1): 31-38.
[11] Cannon RE, Scandalios JG. Two cDNAs encode two nearly identical Cu/Zn superoxide dismutase proteins in maize. Mol Gen Genet, 1989, 219(1): 1-8.
[12] Kaminaka H, Morita S, Yokoi H, Masumura T, Tanaka K. Molecular cloning and characterization of a cDNA for plastidic copper/zinc-superoxide dismutase in rice (Oryza sativa L.). Plant Cell Physiol, 1997, 38(1): 65-69.
[13] Perl-Treves R, Nacmias B, Aviv D, Zeelon EP, Galun E. Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences. Plant Mol Biol, 1988, 11(5): 609-623.
[14] Sakamoto A, Ohsuga H, Tanaka K. Nucleotide sequences of two cDNA clones encoding different Cu/Zn-superoxide dismutases expressed in developing rice seed (Oryza sativa L.). Plant Mol Biol, 1992, 19(2): 323-327.
[15] Bueno P, Piqueras A, Kurepa J, Savouré A, Verbruggen N, van Montagu M, Inzé D. Expression of antioxidant en-zymes in response to abscisic acid and high osmoticum in tobacco BY-2 cell cultures. Plant Sci, 1998, 138(1): 27-34.
[16] Hernández-Nistal J, Dopico B, Labrador E. Cold and salt stress regulates the expression and activity of a chickpea cytosolic Cu/Zn superoxide dismutase. Plant Sci, 2002, 163(3): 507-514.
[17] McKersie BD, Chen Y, De Beus M, Bowley SR, Bowler C, Inze D, D'Halluin K, Botterman J. Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiol, 1993, 103(4): 1155-1163.
[18] McKersie BD, Murnaghan J, Jones KS, Bowley SR. Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance. Plant Physiol, 2000, 122(4): 1427-1438.
[19] van Camp W, Capiau K, van Montagu M, Inze D, Slooten L. Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts. Plant Physiol, 1996, 112(4): 1703-1714.
[20] Dorofejuk MT, Dorofejuk VF, Addrusewitsch VT. Cultivation of eastern goat's rue (Galega orientalis Lam) as a perennial fodder plant on turf-podzol soils in the Republic of Belarus. M Archives of Agron Soil Sci, 1999, 44(2): 131-148.
[21] 刘法涛, 杨志忠. 优良豆科牧草——东方山羊豆. 草食家畜, 2000, (2): 42.
[22] 张自和, 于应文. 东方山羊豆在俄罗斯的研究和应用. 草业科学, 2006, 23(1): 26-33.
[2

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

东方山羊豆Cu/ZnSOD基因的克隆及表达分析

Cloning and expression analysis of Cu/ZnSOD gene from Galega orientalis L

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	汪德州,莫晓婷,张霞,徐妙云,赵军,王磊. 玉米逆境响应相关转录因子ZmC2H2-1基因克隆及功能验证[J]. 遗传, 2018, 40(9): 767-778.
[2]	徐宗昌,孔英珍. 普通烟草CESA基因家族成员的鉴定、亚细胞定位及表达分析[J]. 遗传, 2017, 39(6): 512-524.
[3]	李月, 刘晓东, 董永梅, 谢宗铭, 陈受宜. 棉花Trihelix转录因子GhGT29基因的克隆及功能分析[J]. 遗传, 2015, 37(12): 1218-1227.
[4]	何风华朱碧岩高峰李韶山李娘辉. 孟德尔豌豆基因克隆的研究进展及其在遗传学教学中的应用[J]. 遗传, 2013, 35(7): 931-938.
[5]	杨剑波，甘尚权，杨永林，张红琳，宋天增，冯静，杨井泉，高磊，石国庆，沈敏. 绵羊ILK基因的克隆及其在毛囊生长期的表达[J]. 遗传, 2012, 34(6): 719-726.
[6]	宋冰，王丕武，付永平，范旭红，夏海丰，高玮，洪洋，王贺，张卓，马建. 大豆C₂H₂型锌指蛋白基因SCTF-1的克隆及功能分析[J]. 遗传, 2012, 34(6): 749-756.
[7]	林佳丽，沈良才，潘登科，张瑾. 猪Gli1基因的克隆、表达谱分析及脂肪组织特异性表达载体的构建[J]. 遗传, 2012, 34(10): 1291-1297.
[8]	王伟伟，朱长青，刘小花，陈昆松，徐昌杰. 番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用[J]. 遗传, 2011, 33(9): 1017-1022.
[9]	刘伍限，贾斌，石国庆，任建功，刘卡，马润林. 绵羊FGF5基因的克隆、表达及RNA干扰[J]. 遗传, 2011, 33(9): 982-988.
[10]	苏建民，许文兵，李艳艳，王丽君，王勇胜，张涌. 转基因克隆牛胎盘中印迹基因PEG10的DNA甲基化水平[J]. 遗传, 2011, 33(5): 533-538.
[11]	帅建军，熊飞，彭日荷，姚泉洪，熊爱生. 多氯联苯的生物修复[J]. 遗传, 2011, 33(3): 219-227.
[12]	徐丽华，常玉梅，刘春雷，梁利群，刘金亮，池炳杰. 鲤脑组织低温差异表达候选基因的筛选[J]. 遗传, 2011, 33(3): 262-269.
[13]	尹佟明. 林木基因组及功能基因克隆研究概述[J]. 遗传, 2010, 32(7): 677-684.
[14]	林荆，付凤玲，蒋伟，牟禹，雍太明，李晚忱. 垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析[J]. 遗传, 2010, 32(5): 498-504.
[15]	杜金芳，曾勇庆，陈伟，崔景香，陈其美，杨伦，胡艳霞. 猪CuZnSOD基因的克隆、表达及功能分析[J]. 遗传, 2010, 32(10): 1037-1042.