霜霉病菌侵染的黄瓜叶片cDNA文库的构建及抗病相关基因筛选  

Construction of cDNA Library from Cucumber Leaves Infection by Pseudoperonospora cubensis and Screening of Resistance-related Genes

作　　者：王丽娟[1] 牛德[1] 孙彩玉[1] 秦智伟[2] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]东北农业大学园艺学院,哈尔滨150030

出　　处：《园艺学报》2010年第11期1775-1782,共8页Acta Horticulturae Sinica

基　　金：国家‘863’计划项目(2002AA207013);黑龙江省博士后专项科研基金项目(LBH-Z07213)

摘　　要：以接种黄瓜霜霉病菌的抗病黄瓜品种‘649’的叶片为材料,使用改良SDS法提取总RNA,构建黄瓜叶片全长cDNA文库,得到的原始文库滴度为5.5×106pfu·mL-1,扩增后的文库滴度达6.5×109pfu·mL-1,重组率约为99%,插入片段在0.5~2.0kb之间,大多在1.0kb左右。随机选取3360个克隆进行测序,共拼接出2507个unigenes,其中包括211个重叠群（Contigs）和2296个单拷贝EST（Singlets）。生物信息学分析显示：这些unigenes中存在427条与植物防御/抗病相关的基因,其中包括两类抗病基因和细胞程序性死亡相关基因,这些基因的发现为下一步研究黄瓜抗病机理,克隆抗霜霉病相关基因提供了重要的参考。Total RNA was extracted from the leaves of the disease-resistant cucumber（Cucumis sativus L.）cultiva＇r649＇challenged by Pseudoperonospora cubensis using the modified SDS method.And then,a full-length cDNA library was constructed.The results showed that the primary titer of the constructed cDNA library was 5.5 × 106 pfu · mL-1,and titer of the amplified library was 6.5 × 109 pfu · mL-1.The recombination rate was about 99%.The size of inserted cDNA fragment ranged from 0.5 kb to 2.0 kb,majority at about 1.0 kb.Sequencing analysis showed that 2 507 unigenes,included 211 contigs and 2 296 singlets were identified in the 3 360 ESTs derived from the cDNA library.The result of the bioinformatics analysis indicated that there were 427 plant defense/resistance-related genes including two type resistance genes and programmed cell death related genes.The discovery of these genes provides an important reference for further studying cucumber disease resistance mechanism and cloning resistance related genes.

关 键 词：黄瓜 霜霉菌 RNA提取 cDNA文库 抗病相关基因 

分 类 号：S642.2[农业科学—蔬菜学]