小麦叶锈菌与TcLr19非亲和互作cDNA文库的EST分析

Analysis of Expression Sequence Tags During Incompatible Interaction Between Leaf Rust (Puccinia triticina) and TcLr19 Using a cDNA Library

【目的】建立小麦叶锈菌与小麦非亲和互作的基因表达数据库,为从分子水平阐明小麦抗叶锈病机制奠定基础。【方法】对叶锈菌诱导的TcLr19小麦叶片cDNA文库随机测序,利用BLASTx对所得序列进行功能注释,按照Bevan的植物基因功能分类标准进行分类。采用RT-PCR技术,以Actin为参照,对2条信号转导相关基因和3条抗病与防御相关基因进行表达分析。【结果】随机对cDNA文库中756个阳性克隆测序,获得649条高质量EST。对EST序列聚类拼接获得472条非重复序列(Unisequence)。功能分类结果表明,25.2%为未知功能蛋白,21.0%与能量代谢基因相关,17.8%与抗病防御基因及信号转导基因相关,其余EST分别与转录、转运、蛋白代谢等功能基因相关。RT-PCR分析结果表明同一基因在叶锈菌侵染后不同时间点的小麦叶片中表达量不一致,且各基因有不同的表达模式。【结论】推测钙转运ATP酶,谷胱甘肽-S-转移酶,蛋白激酶Pti1,NBS-LRR抗病蛋白和分子伴侣DnaJ等参与了小麦与叶锈菌的非亲和互作过程。该cDNA文库可用于了解小麦与叶锈菌非亲和互作过程中的功能基因,为建立研究病原菌基因及小麦抗病基因数据库奠定基础。

【Objective】 The objective of this study is to develop an EST database of wheat infected by avirulent Puccinia triticina isolate and provide a base for understanding the wheat resistant mechanism to the pathogen at the molecular level.【Method】The positive clones of TcLr19 cDNA library from wheat leaves induced by P.triticina were picked up randomly for sequencing,and the qualified expressed sequence tags （ESTs） were annotated.The functional classification of all ESTs was established based on the database entry giving the best E-value using the Bevan＇s classification categories.The expression profiles of two signal transduction genes and three disease/defence genes were analyzed by normalization with Actin.【Result】 Seven hundred and fifty-six clones of the cDNA library were sequenced randomly,and 649 qualified ESTs were acquired and clusterted into 472 unisequences.All ESTs were classified,25.2% of these genes were similar to genes encoding proteins with unknown functions,and 21.0% genes shared high homology with genes involved in energy metabolism and photosynthesis.And 17.8% of the genes with functions related to disease defence and signal transduction,and those in the remaining groups to genes involved in transcription,transport processes,protein metabolism,etc,respectively.The results of RT-PCR showed that the transcripts of the selected genes were different at different time points of wheat leaves after inoculated with the pathogen,and the expression patterns of each gene were inconsistent in wheat leaves.【Conclusion】The putative calcium-transporting ATPase,glutathione-S-transferase,putative protein kinase Pti1,NBS-LRR disease resistance protein homologue,Chaperone protein DnaJ,etc.,were supposed to involve in the process of the incompatible interaction between wheat TcLr19 and the P.triticina race.The cDNA library is useful for identifying the functional genes involved in the wheat-leaf rust incompatible interaction,and thus laying a foundation for establiment of a new database for studying leaf rust pathogenesis genes and wheat defense genes.