甘薯茎线虫诱导抑制差减杂交cDNA文库的构建及表达序列标签分析  

Construction of SSH cDNA Library Induced by Ditylenchus destructor in Sweetpotato and Analysis of its Expressed Sequence Tags

作　　者：翟红[1] 商丽丽[1] 刘庆昌[1] 

机构地区：[1]中国农业大学农业部作物基因组学与遗传改良重点开放实验室,北京市作物遗传改良重点实验室,教育部作物杂种优势研究与利用重点实验室,北京100193

出　　处：《农业生物技术学报》2010年第1期141-148,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(30871570);国家科技支撑计划(2006BAD01A06);农业部948项目(2006-G21-01)共同资助

摘　　要：以甘薯(Ipomoea batatas(L.)Lam.)抗茎线虫病品种鲁薯3号为材料,利用抑制差减杂交(suppression subtractive hybridization,SSH)技术,构建了甘薯茎线虫诱导的甘薯块根cDNA文库。从中筛选出1379个阳性克隆进行测序,用CAP3软件聚类拼接,得到185个unique EST。Blast2Go比对ESTs,并进行GO功能分类,结果发现151条非重复序列与已知基因同源性很高,占全部非重复序列的81.6%。已知功能的EST涉及植物的能量代谢、信号转导、抗病防卫、蛋白质合成等多方面。通过对已知功能的基因进行分析,推测促分裂原活化蛋白激酶、丝氨酸/苏氨酸蛋白激酶、过氧化氢酶、几丁质酶、葡聚糖酶等可能参与甘薯接种茎线虫后相关的抗性反应。实时荧光定量PCR分析结果表明,与抗茎线虫病相关的4个基因在甘薯接种线虫后不同时间点具有不同的表达模式。A cDNA library was constructed based on suppression subtractive hybridization（SSH） from the storage roots of stem-nematode resistant sweetpotato varity（Ipomoea batatas（L.） Lam.） cv.Lushu 3.A total of 1 379 positive clones were screened and sequenced,and then clustered by CAP3 software.As a result,185 unique expressed sequence tags（ESTs） were obtained.The ESTs were blasted in GenBank,innotated GO and matched metabolism pathway in KEGG by Blast2Go software,151 of the 185 ESTs,accounting for 81.6%,showed high homology with the function-known genes or ESTs,These function-known ESTs are involved in energy metabolism,signal transduction,defense response and protein synthesis metabolism.Analysis of the function-known genes reveals that mitogen-activated protein kinase,serine/threonine protein kinase,catalase,chitinase and glucanase might be involved in the resistance reaction to sweetpotato stem nematode.The results of real-time quantitative reverse-transcriptase PCR indicated that the four disease-related genes showed different expression patterns at different times after inoculated with stem nematodes.

关 键 词：甘薯 茎线虫 抑制差减杂交 表达序列标签 实时荧光定量PCR 

分 类 号：S435.315.9[农业科学—农业昆虫与害虫防治]