叶锈菌诱导的小麦叶片抑制差减杂交文库构建及其分析

Construction and Analysis of a Wheat Leaf Suppression Subtractive Hybridization Library Induced by Puccinia triticina

【目的】研究叶锈菌诱导小麦叶片的基因表达情况,从分子水平阐明小麦的抗病机制。【方法】以小麦抗叶锈病近等基因系TcLr41为材料,利用抑制差减杂交技术,构建叶锈菌诱导的小麦叶片cDNA文库,随机挑取文库中的阳性克隆测序,并对测序结果进行功能注释和分类。【结果】成功构建了叶锈菌诱导的小麦叶片抑制差减杂交文库,共获得3 456个阳性克隆。挑取165个阳性克隆进行测序,获得160条高质量EST。对EST序列进行聚类后,共获得107条非重复序列(Unigene),与GenBank进行BLASTx和BLASTn同源比对,其中70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65.4%。已知功能的EST涉及植物的能量代谢、信号转导、转录调控及抗病防卫等多方面。【结论】通过对功能已知的基因分析,推测促分裂原活化蛋白激酶(mitogen activated protein kinase)、丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase)、GTP结合蛋白(GTP-binding protein)、钙网蛋白(calreticulin)、过氧化氢酶(catalase)、谷胱甘肽-S-转移酶(glutathione-S-transferase)、多聚泛素基因(polyubiquitin)、锌指蛋白(zinc-finger protein)、肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase)、转脂蛋白(lipid transfer protein)、类甜蛋白(thaumatin-like)、几丁质酶(chitinase)等可能参与了小麦与叶锈菌的非亲和互作过程。

【Objective】The objective of this paper is to study the gene expression profile of wheat leaves induced by Puccinia triticina and the wheat resistant mechanism to the pathogen at the molecular level.【Method】TcLr41,a near isogenic line resistant to wheat leaf rust,was used to construct a suppression subtractive hybridization（SSH） library from wheat leaves induced by Puccinia triticina.After sequencing the positive clones picked up randomly,the qualified expressed sequence tags（ESTs） were annotated and classified.【Result】A high quality SSH cDNA library was constructed successfully and 3456 positive clones were obtained in the SSH library.One hundred and sixty five clones of the library were picked out randomly and sequenced,and then 160 qualified ESTs were obtained.The ESTs clustering was analyzed and 107 unigenes were acquired.ESTs similarity analysis of these unigenes was performed with the Blastx and Blastn by comparing sequences in non-redundance database from GenBank.As a result,seventy unigenes accounted for 65.4% of all the unigenes showed high homology with the function known genes or ESTs.The function-known ESTs related to energy metabolism,signal transduction,transcription regulation and defense response.【Conclusion】Through analysing the genes with known function,mitogen-activated protein kinase,serine/threonine protein kinase,GTP-binding protein,calreticulin,catalase,glutathione-S-transferase,polyubiquitin,zinc-finger protein,cinnamyl alcohol dehydrogenase,lipid transfer protein,thaumatin-like protein and chitinase,etc.,were supposed to involve in the process of the incompatible interaction between wheat TcLr41 and the Puccinia triticina race.