| 大豆花叶病毒侵染初期的抗病性表达序列标签分析 |
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| 引用本文： | 刘春燕,陈庆山,辛大伟,邱红梅,单大鹏.大豆花叶病毒侵染初期的抗病性表达序列标签分析[J].作物学报,2005,31(11):1394-1399. |
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| 作者姓名： | 刘春燕 陈庆山 辛大伟 邱红梅 单大鹏 |
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| 作者单位： | 东北农业大学大豆研究所，黑龙江哈尔滨150030 |
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| 基金项目： | 新材料领域项目，黑龙江省哈尔滨市青年科学基金，科技部专项基金 |
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| 摘 要： | 以抗花叶病毒病品系东农8143为材料，利用抑制消减杂交技术构建了一个大豆花叶病毒接种初期的cDNA文库，共获得2000个阳性克隆。随机挑取64个阳性克隆测序，与GenBank进行BLASTx和BLASTn同源比较，其中49个有比较明确的比对结果，占测序EST的76.6%。测序的ESTs片段中最短的为136 bp，最长的691 bp，平均长度为456 bp，有41条序列带有Poly(A)尾。功能已知基因的EST涉及大豆的细胞自身保护、信号传导、抑制病原菌生长、系统获得性抗性以及持家基因等许多方面，其中SAR（Systemic aquired resistance）系统基因在表达谱中的种类和数量最多。未知功能ESTs与GenBank的dbEST库中序列比较表明，其中许多与生物、非生物胁迫cDNA文库来源的ESTs同源。
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| 关 键 词： | 大豆 花叶病毒病 抑制消减杂交 表达序列标签 |
| 收稿时间： | 2004-09-17 |
| 修稿时间： | 2005-01-16 |
ESTs Analysis of Resistance to Soybean Mosaic Virus (SMV) in Soybean at Primary Infected Stage |
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| LIU Chun-Yan,CHEN Qing-Shan,XIN Da-Wei,QIU Hong-Mei,SHAN Da-Peng.ESTs Analysis of Resistance to Soybean Mosaic Virus (SMV) in Soybean at Primary Infected Stage[J].Acta Agronomica Sinica,2005,31(11):1394-1399. |
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| Authors: | LIU Chun-Yan CHEN Qing-Shan XIN Da-Wei QIU Hong-Mei SHAN Da-Peng |
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| Institution: | Soybean Research Institute of Northeast Agricultural University, Harbin 150030，Heilongjiang, China |
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| Abstract: | Soybean is a major source of edible oil and high-quality protein in the world, but the disease make the yield of soybean lower, even have no harvests. To study the resistance mechanism to soybean mosaic virus(SMV), a soybean line, Dongnong 8143, with resistance to SMV, was used, and a subtractive cDNA library was constructed from soybean leaves infected by SMV No.1 at primary stage with SSH. DongNong 8143 was planted in culture pans. When the plants were at V1 stage, the fully-developed unifoliate leaves were inoculated with SMV No.1, then the sampling from inoculated plants and non-inoculated plants was made once every 24 hours and 8 times totally. Total RNA was extracted with TRIZOL Reagent according to the kit manual. To perform SSH, total RNA was pooled for both uninfected and infected samples. Poly(A)+RNA was purified with the PolyATract® mRNA Isolation Kit. SSH was performed using the PCR-select cDNA Subtraction Kit. SMV infected leaves RNA as the tester and uninfected leaves RNA was used as the driver. The cDNA was then digested with RsaⅠ. The tester cDNA then was subdivided into 2 portions, and each was ligated with a different cDNA adaptor. Two rounds of hybridization were performed. After filling in the ends by DNA polymerase, 2 rounds of PCR amplification were performed using the primers that matched the different adaptors to the 5′ and 3′ ends. cDNA dominantly or specifically expressed in infected leaves was purified using PCR Purification Kit and cloned into pGEM-T easy vector. Colonies were grown on LB-agar plates containing ampicillin and IPTG. As a result, A subtractive plasmid library was constructed by SSH. Two thousands positive clones were acquired. 64 clones were picked up randomly and sequenced, and the results showed that the shortest length of ESTs was 136 bp, the longest length of ESTs was 691 bp, and the average length was 456 bp. 41 ESTs of them had poly(A)+ sequence. All ESTs were compared with sequences in unigene database of GenBank with BLASTn and BLASTx algorithm, and 49 ESTs of them had comparatively clear results. ESTs with known function involved in cellular protect of soybean, signal transduction, restrict pathogen growth, system acquired resistance, and housekeeping genes related with photosynthesis, respiration, and protein synthesis, etc. SAR genes were the richest not only in varieties but also in quantity. In the function unknown ESTs, many homologous ESTs are found from other biotic and abiotic-stresses selected cDNA libraries after compared with dbEST in GenBank. |
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| Keywords: | Soybean Soybean mosaic virus SSH Expressed sequence tags |
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