Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2015, Vol. 41 ›› Issue (04): 565-573.doi: 10.3724/SP.J.1006.2015.00565


Cloning of BnADH3 Gene from Brassica napus L. and Submergence Tolerance of BnADH3 Transgenic Arabidopsis

LÜ Yan-Yan,FU San-Xiong,CHEN Song,ZHANG Wei,QI Cun-Kou*   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing 210014, China
  • Received:2014-07-11 Revised:2015-02-05 Online:2015-04-12 Published:2015-03-02
  • Contact: 戚存扣, E-mail: qck9898@sina.com


BnADH3 gene was highly homologous to BoADH3 from Brassica oleracea and AtADH3 from Arabidopsis, with the BnADH3  expression was induced by submergence and the up-regulation occurred since 6-hour post treatment. The BnADH3 transgenic Arabidopsis was obtained and the transgenic seedBnADH3 gene was cloned from submergence-tolerant line WR-4 of Brassica napus L. using RT-PCR technique. The full-length open reading frame is 1137 bp, encoding 379 amino acids. Homology analysis showed that BnADH3 gene was highly homologous to BoADH3 from Brassica oleracea and AtADH3 from Arabidopsis, with the 96% and 91% similarity, respectively. Quantitative RT-PCR assay was carried out to compare BnADH3 expression between the submergence-tolerant line WR-4 and the submergence-susceptible line WR-24. The result showed that the BnADH3  expression was induced by submergence and the up-regulation occurred since 6-hour post treatment. The BnADH3 transgenic Arabidopsis was obtained and the transgenic seedlings were exposed to three-day submergence stress. Overexpression of BnADH3 resulted in higher ADH activity in leaf and root of transgenic Arabidopsis compared to that of the wild type. The four- and six-week seedlings of T2 generation showed higher tolerance to submergence stress after three-day submergence treatment and most T2 seedlings were recovered with normal growth when the stress was relieved for three days. However, the wild-type seedlings withered until death. After five-day submergence, the survival ratios were 26.7% for the wild type, 80.0% for transgenic line ADH33, and 66.7% for transgenic line ADH44.

Key words: Brassica napus L., Gene BnADH3, Transgenic Arabidopsis, Flood tress, Flood tolerance

[1]谭筱玉, 程勇, 郑普英, 张学昆, 周广生. 油菜湿害及耐湿性机理研究进展. 中国油料作物学报, 2011, 33: 306–310

Tan X Y, Cheng Y, Zheng P Y, Zhang X K, Zhou G S. Research progress on waterlogging resistance in rapeseed (Brassica napus L.). Chin J Oil Crop Sci, 2011, 33: 306–310 (in Chinese with English abstract)

[2]Kato-Noguchi H. Pyruvate metabolism in rice coleoptiles under anaerobiosis. Plant Growth Regul, 2006, 50: 41–46

[3]Tadege M, Dupuis I, Kuhlemeier C. Ethanolic fermentation: new functions for an old pathway. Trends Plant Sci, 1999, 4: 320–325

[4]Dennis E S, Dolferus R, Ellis M, Rahman M, Wu Y, Hoeren F U, Grover A, Ismond K P, Good A G, Peacock W J. Molecular strategies for improving waterlogging tolerance in plants. J Exp Bot, 2000, 51: 89–97

[5]姜华武, 张祖新. 淹水对玉米根系几种酶活性的影响. 湖北农学院学报, 1999, 19: 209–211

Jiang H W, Zhang Z X. Effect of flooding on activities of several enzymes in maize roots. J Hubei Agric Coll, 1999, 19: 209–211 (in Chinese with English abstract)

[6]刘登望, 李林. 湿涝对幼苗期花生根系ADH活性与生长发育的影响及相互关系. 花生学报, 2007, 36(4): 12–17

Liu D W, Li L. The response of alcohol dehydroganase activity and development of Peanut roots to waterlogging and their relationships. J Peanut Sci, 2007, 36(4): 12–17 (in Chinese with English abstract)

[7]赵森, 陈永华, 陈昊和, 肖国樱. 荧光定量PCR检测淹涝胁迫下水稻Adh2基因的表达量变化. 中国生态农业学报, 2008, 16: 455–458

Zhao S, Chen Y H, Chen H H, Xiao G Y. Dynamic analysis of Adh2 gene of rice (Oryza sativa L.) under submergence stress using real-time quantitative PCR. Chin J Eco-Agric, 2008, 16: 455–458 (in Chinese with English abstract)

[8]Xie Y, Wu R. Rice alcohol dehydrogenase genes: anaerobic induction, organ specific expression and characterization of cDNA clones. Plant Mol Biol, 1989, 13: 53–68

[9]Andrews D L, Drew M C, Johnson J R, Cobb B G. The response of Maize seedlings of different ages to hypoxic and anoxic stress. Plant Physiol, 1994, 105: 53–60

[10]Clough S J, Bent A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16: 735–743

[11]Jefferson R A, Kayanagh T A, Bevan M W. GUS fusion: β-glucuronidase as a sensitive and versative gene fusion marker in higher plants. EMBO J, 1987, 6: 3901–3907

[12]Zhang J, Van Toai T, Huynh L, Preiszner J. Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production. Mol Breed, 2000, 6: 135–144

[13]Huynh L N, VanToai T, Streeter J, Banowetz G. Regulation of flooding tolerance of SAG12:ipt Arabidopsis plants by cytokinin. J Exp Bot, 2005, 56: 1397–1407

[14]Grichko V P, Glick B R. Flooding tolerance of transgenic tomato plants expressing the bacterial enzyme ACC deaminase controlled by the 35S, rolD or PBR-1b promoter. Plant Physiol Biochem, 2001, 39: 19–25

[15]Farwell A J, Vesely S, Nero V, Rodriguez H, McCormack K, Shsh S, Dixon D G, Glick B R. Tolerance of transgenic canola plants (Brassica napus) amended with plant growth-promoting bacteria to flooding stress at a metal-contaminated field site. Environ Pollut, 2007, 147: 540–545

[16]Xu K, Xu X, Fukao T, Canlas P, Maghirang-Rodriguez R, Heuer S, Ismail A M, Bailey-Serres J, Ronald P C, MacKill D J. Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature, 2006, 442: 705–708

[17]Fukao T, Xu K, Ronald P C, Bailey-Serres J. A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice. Plant Cell, 2006, 18: 2021–2034

[18]Fukao T, Bailey-Serres J. Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice. Proc Natl Acad Sci USA, 2008, 105: 16814–16819

[19]Licausi F, van Dongen J T, Giuntoli B, Novi G, Santaniello A, Geigenberger P, Perata P. HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana. Plant J, 2010, 62: 302–315

[20]Kürsteiner O, Dupuis I, Kuhlemeier C. The Pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses. Plant Physiol, 2003, 132: 968–978

[21]Lasanthi-Kudahettige R, Magneschi L, Loreti E, Gonzali S, Licausi F, Novi G, Beretta O, Vitulli F, Alpi A, Perata F. Transcript profiling of the anoxic rice coleoptiles. Plant Physiol, 2007, 144: 218–231

[22]Komatsu S, Thibaut D, Hiraga S, Kato M, Chiba M, Hashiguchi A, Tougou M, Shimamura S, Yasue H. Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots. Plant Mol Biol, 2011, 77: 309–322

[23]Christie P J, Hahn M, Walbot V. Low-temperature accumulation of alcohol dehydrogenase1 mRNA and protein activity in maize and rice seedlings. Plant Physiol, 1991, 95: 699–706

[24]Kato-Noguchi H, Yasuda Y. Effect of low temperature on ethanolic fermentation in rice seedlings. J Plant Physiol, 2007, 164: 1013–1018

[25]Dolferus R, Jacobs M, Peacock W J, Dennis E S. Differential interaction of promoter elements in stress responses of the Arabidopsis adh gene. Plant Physiol, 1994, 105: 1075–1087

[26]Sánchez F J, Manzanares M, de Andres E F, Tenorio J L, Ayerbe L. Turgor maintenance, osmotic adjustment and soluble sugar and proline accumulation in 49 pea cultivars in response to water stress. Field Crops Res, 1998, 59: 225–235

[27]张学昆, 范其新, 陈洁, 李加纳, 王汉中. 不同耐湿基因型甘蓝型油菜苗期对缺氧胁迫的生理差异响应. 中国农业科学, 2007, 40: 485–491

Zhang X K, Fan Q X, Chen J, Li J N, Wang H Z. Physiological reaction differences of different waterlogging-tolerant genotype rapeseed (Brassica napus L.) to anoxia. Sci Agric Sin, 2007, 40: 485–491 (in Chinese with English abstract)

[28]Jacobs M, Dolferus R, van den Bossche D. Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants of Arabidopsis thaliana (L.), Heynh. Biochem Genet, 1988, 26: 105–122

[29]Johnson J R, Cobb B G, Drew M C. Hypoxic induction of anoxia tolerance in roots of Adh1 null Zea mays L. Plant Physiol, 1994, 105: 61–67

[30]Matsumura H, Takano T, Takeda G, Uchimiya H. Adh1 is transcriptionally active but its translational product is reduced in a rad mutant of rice (Oryza sativa L.), which is vulnerable to submergence stress. Theor Appl Genet, 1998, 97: 1197–1203

[31]Shiao T L, Ellis M H, Dolferus R, Dennis E S, Doran P M. Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations. Biotechnol Bioeng, 2002, 77: 455–461

[32]Ismond K P, Dolferus R, Pauw M D, Dennis E S, Good A G. Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway. Plant Physiol, 2003, 132: 1292–302

[33]Ellis M H, Millar A A, Llewellyn D J, Peacock W J, Dennis E S. Transgenic cotton (Gossypium hirsutum) over-expressing alcohol dehydrogenase shows increased ethanol fermentation but no increase in tolerance to oxygen deficiency. Aust J Plant Physiol, 2000, 27: 1041–1050

[1] WANG Wen-Xiang,HU Qiong,MEI De-Sheng,LI Yun-Chang,ZHOU Ri-Jin,WANG Hui,CHENG Hong-Tao,FU Li,LIU Jia*. Genetic Effects of Branch Angle Using Mixture Model of Major Gene Plus Polygene in Brassica napus L. [J]. Acta Agron Sin, 2016, 42(08): 1103-1111.
[2] LU Kun,QU Cun-Min,LI Sha,ZHAO Hui-Yan,WANG Rui,XU Xin-Fu,LIANG Ying,LI Jia-Na. Expression Analysis and eQTL Mapping of BnTT3 Gene in Brassica napus L. [J]. Acta Agron Sin, 2015, 41(11): 1758-1766.
[3] JIAO Cong-Cong,HUANG Ji-Xiang,WANG Yi-Long,ZHANG Xiao-Yu,XIONG Hua-Xin,NI Xi-Yuan,ZHAO Jian-Yi. Genetic Analysis of Yield-Associated Traits by Unconditional and Conditional QTL in Brassica napus [J]. Acta Agron Sin, 2015, 41(10): 1481-1489.
[4] TANG Min-Qiang,CHENG Xiao-Hui,TONG Chao-Bo,LIU Yue-Ying,ZHAO Chuan-Ji,DONG Cai-Hua,YU Jing-Yin,MA Xiao-Gen,HUANG Jun-Yan,LIU Sheng-Yi. Genome-wide Association Analysis of Plant Height in Rapeseed (Brassica napus) [J]. Acta Agron Sin, 2015, 41(07): 1121-1126.
[5] ZHANG Ya-Jie,LI Jing,PENG Hong-Kun,CHEN Xiu-Bin,ZHENG Hong-Yu,CHEN Sheng-Bei,LIU An-Guo,HU Li-Yong. Dynamic Simulation Model for Growth Duration of Rapeseed (Brassica napus) [J]. Acta Agron Sin, 2015, 41(05): 766-777.
[6] ZHANG Wei-Xin,CAO Hong-Xin,ZHU Yan,LIU Yan,ZHANG Wen-Yu,CHEN Yu-Li,FU Kun-Ya. Morphological Structure Model of Leaf Space Based on Biomass at Pre-Overwintering Stage in Rapeseed (Brassica napus L.) Plant [J]. Acta Agron Sin, 2015, 41(02): 318-328.
[7] WEN Juan,XU Jian-Feng,LONG Yan,XU Hai-Ming,MENG Jin-Ling,WU Jian-Guo,SHI Chun-Hai. QTL Mapping and Analysis Based on Embryo and Maternal Genetic Systems for Semi-Essential Amino Acid Contents in Rapeseed (Brassica napus L.) [J]. Acta Agron Sin, 2015, 41(01): 57-65.
[8] JIN Yan,Lü Yan-Yan,FU San-Xiong,QI Cun-Kou. Inheritance of Major Gene Plus Polygene of Water-logging Tolerance in Brassica napus L. [J]. Acta Agron Sin, 2014, 40(11): 1964-1972.
[9] QU Cun-Min,LU Kun,LIU Shui-Yan,BU Hai-Dong,FU Fu-You,WANG Rui,XU Xin-Fu,LI Jia-Na. SNP Detection and Analysis of Genes for Flavonoid Pathway in Yellow- and Black-Seeded Brassica napus L. [J]. Acta Agron Sin, 2014, 40(11): 1914-1924.
[10] ZHOU Qing-Yuan,CUI Cui,YIN Tao,CHEN Dong-Liang,ZHANG Zheng-Sheng,LI Jia-Na. Genetic Analysis of Silique Length Using Mixture Model of Major Gene Plus Polygene in Brassica napus L. [J]. Acta Agron Sin, 2014, 40(08): 1493-1500.
[11] ZUO Qing-Song,YANG Hai-Yan,LENG Suo-Hu,CAO Shi,ZENG Jiang-Xue,WU Jiang-Sheng,ZHOU Guang-Sheng. Effects of Nitrogen Fertilizer on Nitrogen Accumulation, Translocation and Nitrogen Use Efficiency in Rapeseed (Brassica napus L.) [J]. Acta Agron Sin, 2014, 40(03): 511-518.
[12] HU Mao-Long,LONG Wei-Hua,GAO Jian-Qin,FU San-Xiong,CHEN Feng,ZHOU Xiao-Yin,PENG Qi,ZHANG Wei,PU Hui-Ming*,QI Cun-Kou,ZHANG Jie-Fu,CHEN Song. Development and Application of Allele-Specific PCR Markers for Imidazolinone-Resistant Gene BnALS1R in Brassica napus [J]. Acta Agron Sin, 2013, 39(10): 1711-1719.
[13] HUANG Hai-Tao,RONG Xiang-Min,SONG Hai-Xing,LIU Qiang,LIAO Qiong,LUO Ji-Peng,GU Ji-Dong,GUAN Chun-Yun,ZHANG Zhen-Hua. Effect of Nitrate Reductase (NR) Inhibitor on NR Activity in Oilseed Rape (Brassica napus L.) and Its Relation to Nitrate Content [J]. Acta Agron Sin, 2013, 39(09): 1668-1673.
[14] MA Zhen-Zhen,LI Jia-Na,Benjiamin WITTKOP,Martin FRAUEN,YAN Xing-Ying,LIU Lie-Zhao,XIAO Yang. Analysis of QTLs for Oil, Protein, Cellulose and Hemicellulose Contents of Seeds in Brassica napus L. [J]. Acta Agron Sin, 2013, 39(07): 1214-1222.
[15] XIE Xiao-Yu,ZHANG Bing,ZHANG Xia,MA Zhong-Lian,LI Jia-Na. Construction and Analysis of SSH Library in Rapeseed (Brassica napus L.) under Drought Stress [J]. Acta Agron Sin, 2013, 39(04): 744-752.
Full text



No Suggested Reading articles found!