小麦条锈菌cDNA文库构建和表达序列标签(ESTs)分析(英文)

Construction and Characterization of a cDNA Library from Germinated Urediospores of Puccinia striiformis f. sp. tritici

小麦条锈菌(Puccinia striiformisf.sp.tritici)是全世界范围内小麦生产上的重要病原真菌,但是对小麦条锈菌的基因组和基因功能却了解甚少。为了促进小麦条锈菌基因组学的发展和大规模基因发现,我们以噬菌体λTrip1Ex2为载体,采用SMART技术构建了小麦条锈菌萌发夏孢子的cDNA文库。原始文库的滴度为1.1×106pfu/mL,平均插入片段长度为750 bp。从文库中随机挑取279个cDNA克隆测序,分析发现这些ESTs的平均GC含量为45.08%。通过聚类分析,279个ESTs拼接成31个contigs和80 singletons。BLASTx分析表明,47%的ESTs与GenBank中报道的功能已知或未知蛋白具有相似性。tBLASTx分析表明12个uniseqs与EST数据库中的序列具有相似性,其中9个是来自担子菌的cDNA文库。几个EST与已知的真菌致病相关基因具有高度相似性。RT-PCR分析了几个基因在小麦条锈菌侵染过程中的表达水平。这些结果为小麦条锈菌夏孢子萌发以及侵染寄主过程中的基因表达研究奠定了很好的分子生物学基础。

Puccinia striiformis f. sp. tritici is one of the most economically important fungal pathogen of wheat worldwide. However, very little is known about its genome and gene functions, which can be critical for developing the disease management strategies. To provide new impetus for the studies of its functional genomics and discovery of large-scale gene, we generated a cDNA library from germinated urediospores of P. striiformis f. sp. tritici. The cDNA library was constructed from total RNA with bacteriophage λTrip1Ex2 vector by using SMART technology. Analysis of the cDNA library suggested that the titer of unamplified library was 1.1 × 10^6 pfu/mL, and the average size of inserts was about 750 bp. A total of 279 randomly selected cDNA clones has been sequenced and analyzed. The average of GC content for the sequenced ESTs was 45.08%. The ESTs were clustered into 31 contigs and 80 singletons to give a set of 111 unisequences. BLASTX analysis revealed that 47% of the ESTs displayed the significant homology to functionally known or unknown genes from GenBank database, tBLASTx analysis suggested that 12 unisequences showed significant similarity to dbEST entries, of which 9 unisequences were from cDNA libraries of other biotrophic basidiomycetes. Several sequenced cDNA clones showed a high homology to known pathogenicity-related genes of plants. Three identified genes were selected for RT-PCR experiments. Results of the analysis displayed that the level of expression were different during the infection process of P. striiformis on host. It is the indication that the cDNA library from germinated urediospores can provide a useful resource for mining new genes expressed during rust infection.