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作物学报 ›› 2013, Vol. 39 ›› Issue (04): 744-752.doi: 10.3724/SP.J.1006.2013.00744

• 研究简报 • 上一篇    下一篇

干旱胁迫下油菜消减文库的构建及分析

谢小玉,张兵,张霞,马仲炼,李加纳*   

  • 收稿日期:2012-08-04 修回日期:2012-12-08 出版日期:2013-04-12 网络出版日期:2013-01-28
  • 通讯作者: 李加纳, E-mail: ljn1950@swu.edu.cn
  • 基金资助:

    本研究由中央高校基本科研业务费专项资金(XDJK2009B020)和重庆市自然科学基金项目(CSTC2010BB1012)资助。

Construction and Analysis of SSH Library in Rapeseed (Brassica napus L.) under Drought Stress

XIE Xiao-Yu,ZHANG Bing,ZHANG Xia,MA Zhong-Lian,LI Jia-Na*   

  • Received:2012-08-04 Revised:2012-12-08 Published:2013-04-12 Published online:2013-01-28
  • Contact: 李加纳, E-mail: ljn1950@swu.edu.cn

摘要:

以抗旱能力强的油菜Holiday为材料,以干旱处理的样品为Tester,正常水分管理(对照)Driver构建干旱诱导的甘蓝型油菜叶片正向抑制性消减杂交 (SSH)文库随机挑选24个阳性克隆进行PCR验证,结果表明,其中23个含有插入片段,平均大小在750 bp左右。将96个阳性克隆测序,并拼接和去除冗余,获得重叠群4条,单拷贝序列82,平均长度为542 bp。经BlastX程序比对蛋白数据库发现,11EST没有找到同源性序列,75条有同源序列。KOBAS分析发现,28EST被定位到67条代谢途径中,根据P值推测,光合中的碳固定、有氧呼吸的电子供体、氮代谢、乙醛酸和二羧酸代谢在植物干旱胁迫中发挥着极为重要的作用。这些EST涉及到的功能中所占比例最大的分别是细胞器(58.82%)、结合(30.77%)和新陈代谢过程(43.72%),表明甘蓝型油菜面对干旱胁迫时,细胞器组件上的功能发挥了重要的作用,结合基因和蛋白被激活,加强了新陈代谢。

关键词: 油菜, 抑制差减杂交, 干旱胁迫, cDNA文库, 表达序列标签

Abstract:

The positive SSH library in leaves of rapeseed (Brassica napus L.) Holiday (with high drought resistance ability) was constructed using the sample under drought stress as Tester and the sample under normal water management as Driver. Twenty-four positive clones selected randomly were verified by using PCR. The result of electrophoresis showed that 23 clones contained inserts whose average length was 750 bp. Ninety-six positive clones were sequenced and analyzed by CAP3 Sequence Assembly Program, showing that four contigs and 82 singletons were contained and the average length of 86 ESTs was 542 bp. Blastx alignment with Nr database showed 11 ESTs without significant similarity and 75 ESTs with similarity in GenBank database. KOBAS suggested that 28 ESTs were mapped to 67 pathways. It could be predicted that the carbon fixation in photosynthetic organism, electron donor III of aerobic respiration, nitrogen metabolism, glyoxylate and dicarboxylate metabolism played a key role in drought tolerance ofrapeseed. The analysis of ESTs showed that a large share of function could be attributed to organelle (58.82%), binding (30.77%), and metabolic process (43.72%), which plays important roles in metabolism of rapeseed under drought stress.

Key words: Rapeseed (Brassica napus L.), Suppression subtractive hybridization (SSH), Drought stress, cDNA library, Express sequence tag

[1]Xiang J(项俊), Chen Z-B(陈兆波), Wang P(王沛), Yu L-J(余龙江), Li M-T(栗茂腾). The effect of CaCl2 treatment on the Chang of drought related physiological and biochemical indices of Brassica napus. J Huazhong Agric Univ (华中农业大学学报), 2007, 26(5): 607–611 (in Chinese with English abstract)



[2]Umezawa T, Yoshida R, Maruyama K, Yamaguchi-Shinozaki K, Shinozaki K. SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. Proc Natl Acad Sci USA, 2004, 101: 17306–17311



[3]Selvam J N, Kumaravadivel N, Gopikrishnan A, Kumar B K, Ravikesavan R, Boopathi M N. Identification of a novel drought tolerance gene in Gossypium hirsutum L. cv KC3. Commun Biometry Crop Sci, 2009, 4: 9–13



[4]Yan A H, Zhang L F, Zhang Y W, Wang D M. Early stage SSH library construction of wheat near isogenic line TcLr19 under the stress of Puccinia recondita f. sp. tritici. Front Agric China, 2009, 3: 146–151



[5]Islam M A, Du H, N J, Ye H Y, Xiong L Z. Characterization of Glossyl homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Mol Biol, 2009, 70: 443–456



[6]Poroyko V, Hejlek L G, Spollen W G, Springer G K, Nguryen H T, Sharp R E, Bohnert H J. The maize root transcriptome by serial analysis of gene expression. Plant Physiol, 2005, 138: 1700–1710



[7]Yue G D, Zhang Y L, Li Z X, Sun L, Zhang J R. Differential gene expression analysis of maize leaf at heading stage in response to water-deficit stress. Bioscie Rep, 2008, 28: 125–134



[8]Zhang G Y, Chen M, Li L C, Xu Z, Chen X P, Guo J M, Ma Y Z. Overexpression of the soybean GmERF3 gene, and AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. J Exp Bot, 2009, 60: 3781–3796



[9]Buchanan C D, Lim S, Salzman R A, Kagiampakis I, Morishige D T, Weers B D, Klein R R, Pratt L H, Cordonnier-Pratt M M, Klein P E Mullet, J E. Sorghum bicolor’s transcriptome response to dehydration high salinity and ABA. Plant Mol Biol, 2005, 58: 699–720



[10]Kanneganti V, Gupta A K. Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice. Plant Mol Biol, 2008, 66: 445–462



[11]Yu Q, Hu Y, Li J, Wu Q, Lin Z. Sense and antisense expression of plasma membrane aquaporin BnPl from Brassica napus in tobacco and its effects on plant drought resistance. Plant Sci, 2005, 169: 647–656



[12]Rebrikov D V, Desai S M, Siebert P D, Lukyanov S A. Suppression subtractive hybridization. Methods Mol Biol, 2004, 258: 107–134



[13]Li H-Y(李惠勇), Huang S-H(黄素华), Shi Y-S(石云素), Song Y-C(宋燕春), Zhao J-R(赵久然), Wang F-G(王凤格), Wang T-Y(王天宇), Li Y(黎裕). Isolating soil drought-induced genes from maize seedling leaves through suppression subtractive hybridization. Sci Agric Sin (中国农业科学), 2007, 6(6): 647–651 (in Chinese with English abstract)



[14]Zhang H(张宏), Song G-Q(宋国琦), Ji W-Q(吉万全), Hu Y-G(胡银岗). Gene induction by drought stress in wheat variety Xiaoyan 22 and their expression analysis. J Agric Biotechnol (农业生物技术学报), 2009, 17(4): 670–676 (in Chinese with English abstract)



[15]Clement M, Lambert A, Herouart D, Boncompagni E. Identification of new up-regulated genes under drought stress in soybean nodules. Gene, 2008, 426: 15–22



[16]Wang D-L(王德龙), Ye W-W(叶武威), Wang J-J(王俊娟), Song L-Y(宋丽艳), Fan W-L(樊伟丽), Cui Y-P(崔宇鹏). Construction of SSH library and its analyses of cotton drought associated genes under drought stress. Acta Agron Sin (作物学报), 2010, 36(12): 2035−2044 (in Chinese with English abstract)



[17]Zhang L(张玲), Li F-G(李付广), Liu C-L(刘传亮), Zhang C-J(张朝军), Wu Z-X(武之霞). Isolation and analysis of drought-related gene from cotton (Gossypium arboreum L.) library. Cotton Sci (棉花学报), 2010, 22(2): 110–114 (in Chinese with English abstract)



[18]Liu W-R(刘文荣), Zhang J-S(张积森), Rao J(饶进), Cai Q-H(蔡秋华), Weng X-Y(翁笑艳), Ruan M-H(阮妙鸿), Que Y-X(阙友雄), Chen R-K(陈如凯), Zhang M-Q(张木清). Construction and analysis of suppression subtractive hybridization library for Saccharum arundinaceum Retz. leaves exposed to drought stress. Acta Agron Sin (作物学报), 2007, 33(6): 961–967 (in Chinese with English abstract)



[19]Tao S-H(陶士珩). Bioinformatics (生物信息学). Beijing: Science Press, 2007. pp 98–99 (in Chinese)



[20]Ashburner M, Ball C A, Blake J A, Botstein D, Butler H, Cherry J M, Davis A P, Dolinski K, Dwight S S, Eppig J T, Harris M A, Hill D P, Issel-Tarver L, Kasarskis A, Lewis S, Matese J C, Richardson J E, Ringwald M, Rubin G M, Sherlock G. Gene Ontology: tool for the unification of biology. Nat Genet, 2000, 25: 25–29



[21]Nguyen H T, Leipner J, Stamp P, Guerra-Peraza O. Low temperature stress in maize (Zea mays L.) induces genes involved in photosynthesis and signal transduction as studied by suppression subtractive hybridization. Plant Physiol Biochem, 2009, 47: 116–122



[22]Chaves M M, Flexas J, Pinheiro C. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot, 2009, 103: 551–560



[23]Turchetto-Zolet A C, Margis-Pinheiro M, Margis R. The evolution of pyrroline-5-carboxylate synthase in plants: a key enzyme in proline synthesis. Mol Genet Genom, 2009, 281: 87–97



[24]Ahmed N U, Park J I, Seo M S, Kumar T S, Lee I H, Park B S, Nou I S. Identification and expression analysis of chitinase genes related to biotic stress resistance in Brassica. Mol Biol Rep, 2012, 39: 3649–3657



[25]Raeini-Sarjanz M, Chalavi V. Effects of water stress and constitutive expression of a drought induced chitinase gene on water-use efficiency and carbon isotope composition of strawberry. J Appl Bot Food Qual, 2011, 84: 90–94

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