西伯利亚蓼铜伴侣蛋白与铜锌超氧化物歧化酶基因共转化烟草的耐盐性分析

doi:10.7525/j.issn.1673-5102.2015.02.008

摘要/Abstract

摘要： 为验证西伯利亚蓼铜伴侣蛋白基因（PsATX）和铜锌超氧化物歧化酶基因（PsSOD）共转化的烟草是否具有耐盐功能，分别构建了植物单价表达载体pROKⅡ-PsATX、pROKⅡ-PsSOD和双价表达载体pROKⅡ-PsATX-PsSOD，通过农杆菌介导遗传转化烟草，PCR验证转基因植株后测定了盐胁迫条件下转基因烟草的丙二醛（MDA）、活性氧、脯氨酸含量及超氧化物歧化酶（SOD）活性，以此鉴定转基因植株的耐盐性。结果表明：（1）PsATX和PsSOD基因已成功整合到烟草基因组中；（2）在100 mmol·L^-1 NaCl处理条件下，转双价基因植株的MDA、活性氧、脯氨酸含量均比野生型和转单价基因植株低，而SOD活性则高于这两类植株。证明PsATX和PsSOD基因共转化的烟草植株比转单价基因植株具有更强的耐NaCl能力。本研究将为研究PsATX和PsSOD基因的抗逆机制奠定分子基础。

关键词: 铜伴侣蛋白基因, 铜锌超氧化物歧化酶基因, 双价表达载体, 耐盐性

Abstract: We analyzed the salt tolerance of tobacco plants which expressed both PsATX and PsSOD genes of Polygonum sibiricum Laxm., constructed univalent expression vector pROKⅡ-PsATX, pROKⅡ-PsSOD and dual-gene expression vector pROKⅡ-PsATX-PsSOD respectively, and then transferred them into tobacco mediated by Agrobacterium tumefaciens. The transgenic tobacco were identified by PCR method. We measured the content of malondialdehyde(MDA), reactive oxygen, proline(Pro), and the activity of superoxide dismutase(SOD) in the transgenic tobacco under salt stress condition to identify the salt tolerance of these plants. The transgenic tobacco containing PsATX, PsSOD and PsATX+PsSOD genes respectively were obtained successfully. The integration of the foreign PsATX and PsSOD genes were confirmed by molecular identification. Under the treatment of 100 mmol·L^-1 NaCl solution, the content of MDA, reactive oxygen and proline in the co-transformed plants was lower than that of wild type and univalent transgenic plants, but the SOD activity were higher than them. By the salt test, the transgenic tobacco contained PsATX+PsSOD genes were more tolerant to salt stress than the transgenic tobacco contained PsATX or PsSOD genes.

Key words: PsATX, PsSOD, dual-gene expression vector, salt tolerance

中图分类号:

马静1；曲春浦2；许志茹1,2*；杨传平2；刘关君2. 西伯利亚蓼铜伴侣蛋白与铜锌超氧化物歧化酶基因共转化烟草的耐盐性分析[J]. 植物研究, 2015, 35(2): 208-217.

MA Jing1；QU Chun-Pu2；XU Zhi-Ru1,2*；YANG Chuan-Ping2；LIU Guan-Jun2. Salt Tolerance of Transgenic Tobacco Containing PsATX and PsSOD Genes of Polygonum sibiricum Laxm.[J]. Bulletin of Botanical Research, 2015, 35(2): 208-217.

参考文献

1.Jin T C,Chang Q,Li W F,et al.Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa［J］.Plant Cell,Tissue Organ Culture,2010,100(2):219-227.

2.Wang W,Vinocur B,Altman A.Plant responses to drought,salinity and extreme temperatures:towards genetic engineering for stress tolerance［J］.Planta,2003,218(1):1-14.

3.Sairam R K,Tysgi A.Physiology and molecular biology of salinity stress tolerance in plants［J］.Current Science,2004,86(3):407-421.

4.Puig S,Andrés-Colás N,García-Molina A,et al.Copper and iron homeostasis in Arabidopsis:responses to metal deficiencies,interactions and biotechnological applications［J］.Plant Cell and Environment,2007,30(3):271-290.

5.Brewer G J.Copper toxicity in the general population［J］.Clinical Neurophysiology,2010,121(4):459-460.

6.Wang Q,Burger M,Doane T A,et al.Effects of inorganic v.organic copper on denitrification in agricultural soil［J］.Advances in Animal Biosciences,2013,4(1):42-49.

7.Lutsenko S,Kaplan J H.Organization of P-type ATPases:significance of structural diversity［J］.Biochemistry,1995,34(48):15607-15613.

8.Rosenzweig A C,Huffman D L,Hou M Y,et al.Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolutio［J］.Structure,1999,7(6):605-617.

9.Glerum D M,Shtanko A,Tzagoloff A.Characterization of COX17,a yeast gene involved in copper metabolism and assembly of cytochrome oxidas［J］.Journal of Biological Chemistry,1996,271(24):14504-14509.

10.Balandin T,Castresana C.AtCOX17,an Arabidopsis homolog of the yeast copper chaperone COX17［J］.Plant Physiology,2002,129(4):1852-1857.

11.Gamonet F,Lauquin G J.The Saccharomyces cerevisiae LYS7 gene is involved in oxidative stress protection［J］.European Journal of Biochemistry,1998,251(3):716-723.

12.Shin L J,Lo J C,Yeh K C.Copper chaperone antioxidant protein1 is essential for copper homeostasis［J］.Plant Physiology,2012,159(3):1099-1110.

13.Lin S J,Culotta V C.The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity［J］.Proceedings of the National Academy of Sciences,1995,92(9):3784-3788.

14.Jithesh M N,Prashanth S R,Sivaprakash K R,et al.Antioxidative response mechanisms in halophytes:their role in stress defense［J］.Jouranl of Genetics,2006,85(3):237-254.

15.Abreu I A,Cabelli D E.Superoxide dismutases-a review of the metal-associated mechanistic variations［J］.Biochimica et Biophysica Acta-biomembranes,2010,1804(2):263-274.

16.曾秀存,孙万仓,孙佳,等.白菜型冬油菜铁超氧化物歧化酶（Fe-SOD）基因的克隆及表达［J］.中国农业科学,2013,46(21):4603-4611.

17.Ogawa K,Kanematsu S,Asada K.Intra-and-extra-cellular localization of “cytosolic” Cu/Zn-superoxide dismutase in spinach leaf and hypocotyl［J］.Plant and cell physiology,1996,37(6):790-799.

18.Nath K,Kumar S,Poudyal R S,et al.Developmental stage-dependent differential gene expression of superoxide dismutase isoenzymes and their localization and physical interaction network in rice(Oryza sativa L.)［J］.Genes & Genomics,2014,36(1):45-55.

19.Tseng M J,Liu C W,Yiu J C.Enhanced tolerance to sulfur dioxide and salt stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts［J］.Plant Physiology Biochemistry,2007,45(10-11):822-833.

20.赵淑婷,曲春浦,许志茹,等.西伯利亚蓼谷氨酰胺合成酶基因的克隆及碱性盐胁迫下的表达［J］.植物研究,2014,34(2):252-257.

21.刘关君,曲春浦,刘明坤,等.西伯利亚蓼铜伴侣蛋白基因在盐胁迫条件下的表达分析［J］.中国生物化学与分子生物学报,2008,24(11):1034-1039.

22.吕艳芳,王大海,刘关君,等.盐碱胁迫下西伯利亚蓼体内无机离子的变化［J］.广西植物,2006,26(3):304-307.

23.张海娜,李小娟,李存东,等.过量表达小麦超氧化物歧化酶(SOD)基因对烟草耐盐能力的影响［J］.作物学报,2008,34(8):1403-1408.

24.汪本勤,陈曦,向成斌.SOD活性增高的拟南芥晚花突变体具有增强的非生物胁迫耐受性.植物学通报,2007,24(5):572-580.

25.Faize M,Faize L,Petri C,et al.Cu/Zn superoxidedismutase and ascorbate peroxidase enhance in vitro shoot multiplication in transgenic plum［J］.Journal of Plant Physiology,2013,170(7):625-632.

26.Diaz-Vivancos P,Faize M,Barba-Espin G,et al.Ectopic expression of cytosolic superoxide dismutase and ascorbate peroxidase leads to salt stress tolerance intransgenic plums［J］.Plant Biotechnology Journal,2013,11(8):976-985.

27.伍小兵,成雨洁,邓西平,等.叶片喷施H₂O₂以及转入Cu/Zn-SOD和APX基因对甘薯幼苗冷后恢复的作用［J］.中国农业科学,2010,43(7):1379-1388.

28.尤佳,张菁,王文瑞,等.NaCl处理下黄花补血草幼苗生理特性的变化［J］.植物研究,2013,33(1):45-50.

29.刘建新,王鑫,王瑞娟,等.碱胁迫对黑麦草幼苗根系活性氧代谢和渗透溶质积累的影响［J］.植物研究,2011,31(6):674-679.

30.Puig S,Mira H,Dorcey E,et al.Higher plants possess two different types of ATX1-like copper chaperones［J］.Biochemical and Biophysical Research Communications,2007,354(2):385-390.

31.Itoh S,Ozumi K,Kim H W,et al.Novel mechanism for regulation of extracellular SOD transcription and activity by copper:role of antioxidant-1［J］.Free Radical Biology and Medicine,2009,46(1):95-104.

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