条锈菌诱导的小麦叶片cDNA文库构建及表达序列标签分析

 以小麦品种水源11和条锈菌CY31号小种为材料,用SMARTTMcDNA Library Construction Kit构建条锈菌诱导的小麦叶片cDNA文库。得到原始文库的滴度为6×10⁶pfu/mL,扩增文库滴度9×10⁹pfu/mL,重组率98%,插入片段在0.5~2.2kb。随机挑取600个克隆,测序获得594条高质量ESTs,与GenBank序列进行BLASTx分析,已知功能ESTs与植物同源比例最高,占44%;其次为真菌,占32%;有18%ESTs在GenBank中没有匹配的同源产物。蛋白功能分析表明,PIG28编码蛋白、ABC转运子、金属硫因、泛素、质膜H⁺-ATPase和氨基酸透酶等可能参与了寄主与病原菌互作过程。

Construction of cDNA library from wheat leaves chanllenged by Puccinia striiformis and analysis of expressed sequence tags

A cDNA library was constructed with wheat leaves challenged by Puccinia striiformis race CY31 by using SMART cDNA Library Construction Kit. The primary library had a high titer of 6×10⁶ pfu/mL, of which 98% clones were recombinant and insert cDNAs were from 0.5 to 2.2 kb. The amplified library had a titer of 9×10⁹ pfu/mL. 594 ESTs were acquired, and ESTs similarity analysis based on BLASTx software was finished by comparing sequences in non-redundance database of GenBank. 44% ESTs had higher homology with plant, which was the largest category; 32% ESTs had higher homology with fungi, and no-hits found ESTs were 18%. The results showed that PIG28 encoding protein,ABC transporter,metallothioneine, polyubiquitin 2, plasma membrane H⁺-ATPase, amino acid permease etc were supposed to involve in the process of compatible interaction.