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作物学报 ›› 2008, Vol. 34 ›› Issue (12): 2121-2125.doi: 10.3724/SP.J.1006.2008.02121

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦抗白粉病SSH-cDNA文库中差异基因的表达模式

吴金华1,2;胡银岗1;王新茹3;张宏1;王长有1;王秋英1;吉万全1,*   

  1. 1 西北农林科技大学农学院 / 陕西省农业分子生物学重点实验室 / 国家小麦改良中心杨凌分中心，陕西杨凌712100；2 河北农业大学农学院，河北保定071001；3 陕西省农药管理检定所，陕西西安710003
  • 收稿日期:2008-03-07 修回日期:2008-06-05 出版日期:2008-12-12 网络出版日期:2008-09-06
  • 通讯作者: 吉万全
  • 作者简介:吴金华(1978-),女,山西朔州人,博士,主要从事小麦抗病育种及其分子生物学研究.
  • 基金资助:

    国家重点基础研究发展计划(973计划)前期项目(2006CB708208);国家科技支撑计划项目(2006BAD01A02)

Expression Pattern of Special Genes Resistant to Powdery Mildew (Blumeria graminis f. sp. tritici) in SSH-cDNA Library of Wheat

WU Jin-Hua1,HU Yin-Gang1,WANG Xin-Ru2,ZHANG Hong1,WANG Chang-You1,WANG Qiu-Ying1,JI Wan-Quan1*   

  1. 1 College of Agronomy, Northwest A＆F University / Yangling Branch of China Wheat Improvement Center / Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, Shaanxi; 2 College of Agronomy, Agricultural University of Hebei, Baoding 071001, Hebei; 3 Institute for the Control of Agrochemicals of Shaanxi Province, Xi’an 710003, Shaanxi, China
  • Received:2008-03-07 Revised:2008-06-05 Published:2008-12-12 Published online:2008-09-06
  • Contact: JI Wan-Quan

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2247

被引次数

20

摘要：

为了解白粉菌诱导下抗白粉病小麦的抗病机制，构建了白粉菌接种初期的抑制性消减杂交cDNA(SSH-cDNA)文库。从中随机挑取140个阳性克隆进行测序，去除冗余序列和重复序列后，得到94条EST，利用NCBI的BLAST在线序列比对工具对GenBank的蛋白质数据库进行同源性比对及功能分类。结果表明，49个EST与已知功能蛋白同源性较高，主要涉及初级代谢(2%)、能量代谢(24%)、细胞结构(2%)、转录(2%)、蛋白质合成加工与储藏(16%)、转运(4%)、信号转导(4%)和抗病与防御(30%)。经文库比较，筛选出与病程相关蛋白基因同源的EST(Z25-1)和与谷胱甘肽硫转移酶同源的EST(Z440-1)，GenBank登录号为EX567369和EX567360。以其EST序列为依据设计引物，通过RT-PCR分析它们在白粉菌诱导下的表达模式，结果该2基因表达存在明显差异。其中，Z25-1在未接种白粉菌时具有一定的表达量，白粉菌侵染后表达量开始上升，侵染72 h时表达量最高，然后下降；Z440-1在未接种时也具有一定的表达量，但在接种初期表达微弱，甚至不表达，24 h后表达开始上升，到72 h时达最高，然后下降。本研究表明，病程相关蛋白和谷胱甘肽硫转移酶属于诱导型表达基因，且参与白粉病抗病反应，在白粉菌诱导72 h时表达量最高。

关键词: 小麦, 白粉病, 抑制性消减杂交(SSH), 反转录聚合酶链式反应(RT-PCR)

Abstract:

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important fungal diseases of common wheat (Triticum aestivum L.) worldwide and causes severe yield losses. Wheat germplasm N9436, developed by our research group, is a resistant material to powdery mildew. In the present study, a suppression subtraction hybridization (SSH) cDNA library was constructed with cDNA from N9436 leaf inoculated by Blumeria graminis as the tester and cDNA from N9436 healthy leaf as the driver. A total of 140 positive clones were randomly chosen from the SSH-cDNA library and were amplified with sp6 and t7 primers to examine the insert size, which ranged from 200 to 1 000 bp with an average of 238 bp. After screening repeat and redundancy sequences, 94 expressed sequence tags (ESTs) were acquired. Protein homology search in nr-protein database revealed that 49 ESTs were highly homologous with known proteins functioning in primary metabolism (2%), energy metabolism (24%), cell structure (2%), transcription (2%), protein synthesis and processing (16%), transport (4%), signal transduction (4%), and disease resistance and defenses (30%). Compared with the EST sequences among the SSH-cDNA library, 2 ESTs (GenBank accession number: EX567369 and EX567360) were highly similar to pathogenesis-related protein (Z25-1) and glutathione-S-transferase (Z440-1), respectively. On the basis of the EST sequences, 2 pairs of primers were designed and used to detect the expression differences of the 2 genes when inoculating with powdery mildew by Reverse Transcriptase PCR (RT-PCR). Z25-1 and Z440-1 were both expressed under inoculation. Their expression amounts were the largest in 72 h after inoculation with powdery mildew. But their expression trends were different. At early stage of inoculation (24 h after inoculation), the expression amount of Z440-1 was very a little, then increased in 72 h, and finally decreased in 96 h. The expression amount of Z25-1 increased from 24 h to 72 h after inoculation and decreased after 96 h. It suggests that pathogenesis-related protein and glutathione-S-transferase belong to inducible expression genes and are involved in the resistance to powdery mildew.

Key words: Wheat, Powdery mildew, Suppression subtraction hybridization (SSH), Reverse transcriptase PCR

[1]Liao Y(廖勇), Zhang Z-Y(张增艳), Du L-P(杜丽璞), Xu H-J(徐惠君), Yao W-L(姚乌兰), Shi J-Y(石建业), Ren Z-L(任正隆), Xin Z-Y(辛志勇). Isolation of RAR1 gene from Thinopyrum in-termedium and analysis of its function in wheat background. Sci Agric Sin (中国农业科学), 2007, 40(8): 1667–1674 (in Chinese with English abstract)
[2]Luo M(骆蒙), Kong X-Y(孔秀英), Huo N-X(霍纳新), Zhou R-H(周荣华), Jia J-Z(贾继增). ESTs analysis of resistance to powdery mildew in wheat at primary infected stage. Acta Genet Sin (遗传学报), 2002, 29(6): 525–530 (in Chinese with English abstract)
[3]Wang Z Y, Wang Y C, Chen X R, Shen G, Zhang Z G, Zheng X B. Differential screening reveals genes differentially expressed in low- and high-virulence near-isogenic Phytophthora sojae lines. Fung Genet Biol, 2006, 43: 826–839
[4]Chen Y, Wang H, Wang X, Cao A, Chen P. Cloning and expres-sion of peroxisomal ascorbate peroxidase gene from wheat. Mol Biol Rep, 2006, 33: 207–213
[5]Wong Y Y, Ho C L, Nguyen P D, Teo S S, Harikrishna J A, Ra-him R A, Wong M C V L. Isolation of salinity tolerant genes from the mangrove plant, Bruguiera cylindrica by using suppres-sion subtractive hybridization (SSH) and bacterial functional screening. Aquat Bot, 2007, 86: 117–122
[6]Ouyang B, Yang T, Li H, Zhang L, Zhang Y, Zhang J, Fei J, Ye Z. Identification of early salt stress response genes in tomato root by suppression subtractive hybridization and microarray analysis. J Exp Bot, 2007, 58: 507–520
[7]Lin C S, Lai Y H, Sun C W, Liu N T, Tsay H S, Chang W C, Chen J J W. Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppres-sive subtractive hybridization (SSH) and microarray analysis. Plant Cell Tissue Organ Cult, 2006, 86: 169–175
[8]Chen Y P, Wang H Z, Cao A Z, Wang C M, Chen P D. Cloning of a resistance gene analog from wheat and development of a codominant PCR marker for Pm21. J Integr Plant Biol, 2006, 48: 715–721
[9]Wang Y-Z (汪以真), Lin W-X(林文学), Xu Z-R(许梓荣). Anti-bacterial peptide pr-39 gene-expression in different growth stages of pig evaluated by semi-quantitative RT-PCR method. J Agric Biotechnol (农业生物技术学报), 2004, 12(1): 52–55(in Chinese with English abstract)
[10]Dong H-P(董宏平), Peng J-L(彭建令), Yu X-J(余晓江), Zhao J(赵静), Wang Y(王颖), Dong H-S(董汉松). Studies on methods in cloning and expression quantification of genes that regulate plant disease resistance signal transduction. J Nanjing Agric Univ (南京农业大学学报), 2003, 26(4): 30–35(in Chinese with Eng-lish abstract)
[11]Sun X-H(孙晓红), Feng A-P(冯爱萍), Chen M-J(陈明杰), Pan Y-J(潘迎捷). Cloning and sequence analysis of cold induced genes in Chinese straw mushroom, Volvariella volvacea. Myco-systema (菌物学报), 2006, 25(1): 88–93(in Chinese with English abstract)
[12]Zhang L-N(张丽娜), Niu J-S(牛吉山), Yu L(于玲). Expression analysis of wheat TaMlo-A1c gene by semi-QRT-PCR. Acta Bot Boreal-Occident Sin (西北植物学报), 2005, 25(7): 1368–1371(in Chinese with English abstract)
[13]Li R, Rimmer R, Buchwaldt L, Sharpe A G, Séguin-Swartz G, Coutu C, Hegedus D D. Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: Expressed sequence tag analysis identifies genes associated with fungal pathogenesis. Fung Genet Biol, 2004, 41: 735–753
[14]Bevans M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, Dirkse W, Van Staveren M, Stiekema W, Drost L, Ridley P, Hudson S A, Patel K, Murphy G, Piffanelli P, Wedler H, Wedler E, Wambutt R, Weitzenegger T, Pohl T M, Terryn N, Gielen J, Villarroel R, De Clerck R, Van Montagu M, Lecharny A, Auborg S, Gy I, Kreis M, Lao N, Kavanagh T, Hempel S, Kotter P, Entian K D, Rieger M, Schaeffer M, Funk B, Mueller-Auer S, Silvey M, James R, Montfort A, Pons A, Puigdomenech P, Douka A, Voukelatou E, Milioni D, Hatzopoulos P, Piravandi E, Obermaier B, Hilbert H, Düsterh?ft A, Moores T, Jones J D G, Eneva T, Palme K, Benes V, Rechman S, Ansorge W, Cooke R, Berger C, Delseny M, Voet M, Volckaert G, Mewes H W, Klosterman S, Schueller C, Chalwatzis N. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature, 1998, 391: 485–488
[15]Cao A-Z(曹爱忠), Li Q(李巧), Chen Y-P(陈雅平), Zou X-W(邹晓文), Wang X-E(王秀娥), Chen P-D(陈佩度). Screening resis-tance-related gene to powdery mildew in Haynaldia villosa using barly genechip and studying its mechanism of resistance. Acta Agron Sin (作物学报), 2006, 32(10): 1444–1452 (in Chinese with English abstract)
[16]Luo M(骆蒙), Kong X-Y(孔秀英), Huo N-X(霍纳新), Zhou R-H(周荣华), Jia J-Z(贾继增). Analysis of gene resistant to powdery mildew by suppression subtractive hybridization. Chin Sci Bull (科学通报), 2002, 47(16): 1237–1241 (in Chinese)
[17]He D-Y(何道一), Wang H-G(王洪刚). Cloning of a new phos-phoserine aminotransferase in the substitution resistant to pow-dery mildew of wheat—Agropyron elongatum. Chin Sci Bull (科学通报), 2005, 50(6): 535–539 (in Chinese)
[18]Ma J-B(马金彪), Wang X-J(王晓杰), Yu X-M(于秀梅), Xu L-S(徐亮胜), Han Q-M(韩青梅), Huang L-L(黄丽丽), Kang Z-S(康振生). Construction of cDNA library from wheat leaves challenged by Puccinia striiformis and analysis of expressed se-quence tags. Acta Phytopathol Sin (植物病理学报), 2007, 37(5): 487–499 (in Chinese with English abstract)
[19]Yang P(杨平), Hu J(胡军), Wang Y-C(王玉成), Yin W-B(尹维波), Chen Y-H(陈宇红), Jiang J(姜静), Li J-L(李集临), Hu Z-M(胡赞民). Cloning and function characterization of glu-tathione-S-transferase gene from Tamarix and rossowii. J Agric Biotechnol (农业生物技术学报), 2007, 15(1): 76–80 (in Chinese with English abstract)
[20]Enayati A A, Ranson H, Hemingway J. Insect glutathione trans-ferases and insecticide resistance. Insect Mol Biol, 2005, 14: 3–8
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