欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2010, Vol. 36 ›› Issue (12): 2035-2044.doi: 10.3724/SP.J.1006.2010.02035

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

干旱胁迫下棉花SSH文库构建及其抗旱相关基因分析

王德龙,叶武威*,王俊娟,宋丽艳,樊伟丽,崔宇鹏   

  1. 中国农业科学院棉花研究所 / 农业部棉花遗传改良重点开放实验室，河南安阳 455000
  • 收稿日期:2010-05-18 修回日期:2010-08-01 出版日期:2010-12-12 网络出版日期:2010-10-09
  • 通讯作者: 叶武威，E-mail:yeww@cricaas.com.cn;Tel:0372-2562283
  • 基金资助:

    本研究由国家农作物转基因生物新品种培育重大专项(2008ZX08005-004)和中央级公益性科研院所基本科研业务专项。

Construction of SSH Library and Its Analyses of Cotton Drought Associated Genes under Drought Stress

WANG De-Long,YE Wu-Wei*,WANG Jun-Juan,SONG Li-Yan,FAN Wei-Li,CUI Yu-Peng   

  1. Cotton Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory for Cotton Genetic Improvement, Ministry of Agriculture, Anyang 455000, China
  • Received:2010-05-18 Revised:2010-08-01 Published:2010-12-12 Published online:2010-10-09
  • Contact: YE Wu-Wei，E-mail:yeww@cricaas.com.cn;Tel:0372-2562283

PDF

2209

被引次数

39

摘要： 以耐旱自交系邯郸177为材料，利用抑制性差减杂交技术(SSH)，构建棉花苗期叶片的正向差减文库。挑取300个阳性克隆进行PCR验证，并对验证后的单克隆进行测序和分析，共获得284个有效序列。聚类后得到202条uniESTs序列，其中174条singlets，28条contigs。经过BlastN分析，156个unigene可以在GenBank中找到同源序列，46个unigene未能找到同源匹配。经BlastX分析，40个unigene与未知功能蛋白或假定蛋白有较高相似性，116条unigene与已知功能蛋白有较高同源性。用KOBAS系统将33个unigene定位到55个Pathways中，其中P值小于0.5的Pathway有23条。初步分析发现, 丙酮酸盐代谢(pyruvate metabolism)途径、乙醛酸和二羧酸代谢(glyoxylate and dicarboxylate metabolism)途径与棉花抗旱相关性较大。这些unigene基因涉及信号传导、能量代谢、蛋白质代谢、核酸代谢、光合作用及膜运输等代谢过程。发现了苹果酸合成酶基因(Ms1, 001_B03; Ms2, 003_E04)、苹果酸脱氢酶基因(Md1, 001_C12; Md2, 002_F01)；NAC(001_C08)、锌指蛋白(zfp, 003_C06)、BZR1/BES1(003_G04)等转录调节因子，以及翻译控制肿瘤蛋白基因(TCTP，002_C04)等耐旱相关基因。

关键词: 干旱胁迫, SSH, unigene, 耐旱

Abstract: A forward cDNA-SSH library was established by suppression subtractive hybridization using seedling leaf of Handan 177, a drought-tolerant cotton (Gossypium hirsuutm L.) inbred line, among which 300 positive clones were selected for sequencing. After detection by PCR for each clone, each single clone was sequenced. Totally 284 available sequences and 202 uniESTs which 28 were contigs and 174 were singlets were obtained by cluster analyses of the ESTs sequencing. The results of BlastN showed that 156 uniESTs had homologous sequences in GenBank database while the other 46 had no protein homologous. The BlastX results indicated that 116 uniESTs had significant protein homology and 40 uniESTs were unknown proteins and putative proteins. KOBAS mapped 33 ESTs of the 202 uniESTs to 55 KEGG pathways, in which there were 23 pathways at P-value<0.05. This study suggested that there were closely relationships with cotton drought tolerance among pyruvate metabolism, glyoxylate and dicarboxylate metabolism. A large group of drought stress-induced genes were found in the cDNA library, which involved in many metabolism pathways such as signal transduction, energy metabolism, protein metabolism, nucleic acid metabolism, photosynthesis, transmembran. And some genes related to drought tolerance were found, such as malate synthase genes (MS1,0001_C12; MS2, 0002_F01) and malate dehydrogenase genes(Md1, 001_C12; Md2, 002_F01), some transcription factors like NAC(001_C08), BZR1/BES1(003_G04) , zinc finger protein genes (zfp, 003_C06), and the translationally controlled tumor protein gene(TCTP，002_C04).

Key words: Drought-stress, Suppression subtractive hybridization (SSH), unigene, Drought tolerance

[1]Zhao K-F(赵可夫), Li Z-F(李法曾). China Halophyte (中国盐生植物). Beijing: Science Press, 1999. pp 28-29 (in Chinese)
[2]Zhang-T(张彤), Qi-L(齐麟). Progress of the research on plant drought-resistant mechanism. Hubei Agric Sci (湖北农业科学), 2005, (4): 107-110 (in Chinese with English abstract)
[3]Li Z-N(李智念), Wang G-M(王光明), Zeng Z-W(曾之文). The study on ABA in plants under drought stressed. Agric Res Arid Areas (干旱地区农业研究), 2003, 21(2): 99-104 (in Chinese with English abstract)
[4]Yang R-L(杨瑞丽), The study on plant drought resistance. Inner Mongolia Sci Technol Econ (内蒙古科技与经济), 2003, 4: 107-108 (in Chinese with English abstract)
[5]Liu J-D(刘金定), Ye W-W(叶武威). Cotton resistant research and utilization in our country. China Cotton (中国棉花), 1998, 25(3): 5-6 (in Chinese with English abstract)
[6]Wang W, Vinocur B, Altman A. Plant responses to drought, salinity and extreme temperatures: Towards genetic engineering for stress tolerance. Planta, 2003, 218: 1-14
[7]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
[8]Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to dehydration and low temperature: Differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol, 2000, 3: 217-223
[9]Bray E. Classification of genes differentially expressed during water-deficit stress in Arabidopsis thaliana: An analysis using microarray and differential expression data. Ann Bot, 2002, 89: 803-811
[10]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
[11]Hao X-Y(郝晓燕), Chen M(陈明), Xu H-J(徐慧君), Gao S-Q(高世庆), Cheng X-G(程宪国), Li L-C(李连成), Du L-P(杜丽璞), Ye X-G(叶兴国), Ma Y-Z(马有志). Obtaining of transgenic wheat with GH-DREB gene and their physiological index analysis on drought tolerance. Southwest China J Agric Sci (西南农业学报), 2005, 18: 616-620 (in Chinese with English abstract)
[12]Islam M A, Du H, N J, Ye H Y, Xiong L Z. Characterization of Glossy1-homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Mol Biol, 2009, 70: 443-456
[13]Rabello A R, Guimaraes C M, Rangel P H, Silva F R, Seixas D, Souza E, Brasileiro A C, Spehar C R, Ferreira M E, Mehta A. Identification of drought-responsive genes in roots of upland rice (Oryza sativa L.). BMC Genomics, 2008, 9: 485
[14]Agalou A, Purwantomo S, Overnas E, Johannesson H, Zhu X Y, Estiati A, Kam R J, Engstrom P, Slamet-Loedin I H, Zhu Z, Wang M, Xiong L Z, Meijer A H, Ouwerkerk P B. Genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members. Plant Mol Biol, 2008, 66: 87-103
[15]Jeanneau M, Gerentes D, Foueillassar X, Zivy M, Vidal J, Toppan A, Perez P. Improvement of drought tolerance in maize: towards the functional validation of the Zm-Asr1 gene and increase of water use efficiency by over-expressing C4-PEPC. Biochimie, 2002, 84: 1127-1135
[16]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
[17]Zinselmeier C, Sun Y, Helentjaris T, Beatty M,Yang S, Smith H, Habben J. The use of gene expression profiling to dissect the stress sensitively of reproductive development in maize. Field Crops Res, 2002, 75: 111-121
[18]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
[19]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
[20]Schafleitner R, Gaudin A, Rosales R O G, Aliaga C A A, Bonierbale M. Proline accumulation and real time PCR expression analysis of genes encoding enzymes of proline metabolism in relation to drought tolerance in Andean potato. Acta Physiol Plant, 2007, 29: 19-26
[21]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
[22]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
[23]Kosmas S A, Argyrokastritis A, Loukas M G, Eliopoulos E, Tsakas S, Kaltsikes P J. Isolation and characterization of drought-related trehalose 6-phosphate-synthase gene from cultivated cotton (Gossypium hirsutum L.). Planta, 2006, 223: 329-339
[24]Gu K Y, Zhai H Q. Advances in the study on the suppression subtractive hybridization. Biotechnol lnform, 1999, 2: 13-16
[25]Diatchenko L, Lau Y F, Campbell A P, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov E D, Siebert P D. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025-6030
[26]Wan J, Mattehew B W, Li C. Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics, 2002, 3: 12
[27]Rebrikov D V, Desai S M, Siebert P D, Lukyanov S A. Suppression subtractive hybridization. Methods Mol Biol, 2004, 258: 107-134
[28]Shen G-S(沈国顺), Liu L-X(刘丽霞). SSH method and its application. Chin J Vet Med (中国兽医学报), 2004, 24(5): 511-514 (in Chinese with English abstract)
[29]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).
[30]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)
[31]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
[32]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.) SSH library. Cotton Sci (棉花学报), 2010, 22(2): 110-114 (in Chinese with English abstract)
[33]Yu S-X(喻树迅). Short-season Cotton Breeding in China (中国短季棉育种学). Beijing: Science Press, 2007. p 560 (in Chinese)
[34]Mao X, Cai T, Olyarchuk J G, Wei L. Automated genome annotation and pathway identification using the KEGG orthology (KO) as a controlled vocabulary. Bioinformatics, 2005, 21(19): 3787-3793
[35]Zhang M-Q(张木清), Chen R-K(陈如凯). Molecular Physiology and Genetic Improvement for Drought Resistance in Crop (作物抗旱分子生理与遗传改良). Beijing: Science Press, 2005, pp 67-506 (in Chinese)
[36]Guo X-H(郭新红), Jiang X-C(姜孝成), Pan X-L(潘晓玲). SSH method and its application in gene cloning. Acta Laser Bid Sin (激光生物学报), 2001, 10(3): 236-239 (in Chinese with English abstract)
[37]Olsen A N, Ernst H A, Leggio L L. NAC transcription factors: Structurally distinct functionally diverse. Trends Plant Sci, 2005, 10: 79-87
[38]He J X, Gendron J M, Sun Y. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth response. Science, 2005, 307: 1634-1638
[39]Li F, Zhang D, Fujise K. Characterization of fortilin, a novel antiapoptotic protein. Biol Chem, 2001, 276: 47542-47549
[40]Berkowitz O, Jost R, Pollmann S, Masle J. Characterization of TCTP, the translationally controlled tumor protein, from Arabidopsis thaliana. Plant Cell, 2008, 20: 3430-3447
[1] 王霞, 尹晓雨, 于晓明, 刘晓丹. 干旱锻炼对B73自交后代当代干旱胁迫记忆基因表达及其启动子区DNA甲基化的影响[J]. 作物学报, 2022, 48(5): 1191-1198.
[2] 丁红, 徐扬, 张冠初, 秦斐斐, 戴良香, 张智猛. 不同生育期干旱与氮肥施用对花生氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 695-703.
[3] 张明聪, 何松榆, 秦彬, 王孟雪, 金喜军, 任春元, 吴耀坤, 张玉先. 外源褪黑素对干旱胁迫下春大豆品种绥农26形态、光合生理及产量的影响[J]. 作物学报, 2021, 47(9): 1791-1805.
[4] 李洁, 付惠, 姚晓华, 吴昆仑. 不同耐旱性青稞叶片差异蛋白分析[J]. 作物学报, 2021, 47(7): 1248-1258.
[5] 李鹏程, 毕真真, 孙超, 秦天元, 梁文君, 王一好, 许德蓉, 刘玉汇, 张俊莲, 白江平. DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘[J]. 作物学报, 2021, 47(4): 599-612.
[6] 秦天元, 刘玉汇, 孙超, 毕真真, 李安一, 许德蓉, 王一好, 张俊莲, 白江平. 马铃薯StIgt基因家族的鉴定及其对干旱胁迫的响应分析[J]. 作物学报, 2021, 47(4): 780-786.
[7] 蒙姜宇, 梁光伟, 贺亚军, 钱伟. 甘蓝型油菜耐盐和耐旱相关性状的QTL分析[J]. 作物学报, 2021, 47(3): 462-471.
[8] 周练, 刘朝显, 熊雨涵, 周京, 蔡一林. 质膜内在蛋白ZmPIP1;1参与玉米耐旱性和光合作用的功能分析[J]. 作物学报, 2021, 47(3): 472-480.
[9] 刘亚文, 张红燕, 曹丹, 李兰芝. 基于多平台基因表达数据的水稻干旱和盐胁迫相关基因预测[J]. 作物学报, 2021, 47(12): 2423-2439.
[10] 秦天元, 孙超, 毕真真, 梁文君, 李鹏程, 张俊莲, 白江平. 基于WGCNA的马铃薯根系抗旱相关共表达模块鉴定和核心基因发掘[J]. 作物学报, 2020, 46(7): 1033-1051.
[11] 张海燕, 汪宝卿, 冯向阳, 李广亮, 解备涛, 董顺旭, 段文学, 张立明. 不同时期干旱胁迫对甘薯生长和渗透调节能力的影响[J]. 作物学报, 2020, 46(11): 1760-1770.
[12] 李旭凯,李任建,张宝俊. 利用WGCNA鉴定非生物胁迫相关基因共表达网络[J]. 作物学报, 2019, 45(9): 1349-1364.
[13] 袁溢,朱双,方婷婷,蒋金金,王幼平. 人工合成甘蓝型油菜抗旱性及DNA甲基化水平分析[J]. 作物学报, 2019, 45(5): 693-704.
[14] 李萍,侯万伟,刘玉皎. 青海高原耐旱蚕豆品种青海13号响应干旱胁迫蛋白质组学分析[J]. 作物学报, 2019, 45(2): 267-275.
[15] 李鹏程,毕真真,梁文君,孙超,张俊莲,白江平. DNA甲基化参与调控马铃薯干旱胁迫响应[J]. 作物学报, 2019, 45(10): 1595-1603.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2