甘蓝型油菜BnADH3基因的克隆及转BnADH3拟南芥的耐淹性

doi:10.3724/SP.J.1006.2015.00565

摘要/Abstract

摘要：

利用RT-PCR技术从甘蓝型油菜耐淹品系WR-4中克隆获得BnADH3基因，其完整开放阅读框为1137 bp。该基因编码379个氨基酸，与甘蓝BoADH3基因和拟南芥AtADH3基因高度同源，同源性分别达到96%和91%。利用定量RT-PCR检测BnADH3基因在油菜耐淹系WR-4和不耐淹系WR-24中的表达表明，在淹水处理下该基因表达受到一定的诱导，淹水处理6 h后表达开始上调，说明BnADH3基因在油菜耐淹机制中发挥作用；将其转化到模式生物拟南芥中，采用幼苗淹水3 d后去水处理，测定表明转基因株系叶片和根系中乙醇脱氢酶活性均高于野生型；生长4周和6周的拟南芥植株淹水3 d后的表型显示，BnADH3的表达可增强拟南芥对淹水胁迫的耐性，处理的T₂代转基因幼苗大部分恢复，但野生型幼苗枯死；调查表明，淹水5 d后野生型植株的存活率为26.7%，转基因株系ADH33和ADH44的存活率分别为80.0%和66.7%。

关键词: 甘蓝型油菜, 基因BnADH3, 转基因拟南芥, 淹水胁迫, 耐淹性

Abstract:

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

吕艳艳,付三雄,陈松,张维,戚存扣*. 甘蓝型油菜BnADH3基因的克隆及转BnADH3拟南芥的耐淹性[J]. 作物学报, 2015, 41(04): 565-573.

Lü Yan-Yan,FU San-Xiong,CHEN Song,ZHANG Wei,QI Cun-Kou*. Cloning of BnADH3 Gene from Brassica napus L. and Submergence Tolerance of BnADH3 Transgenic Arabidopsis[J]. Acta Agron Sin, 2015, 41(04): 565-573.

参考文献

[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

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