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利用DSN(duplex-specific nuclease)均一化与SMART(switching mechanism at5′end of RNA transcript)技术构建玉米弯孢霉叶斑病菌菌丝的均一化全长cDNA文库。经检测原始文库的滴度为1.4×106pfu/mL,重组率为96.9%,插入片段平均长度大于1.0 kb。从文库中随机挑取192个cDNA克隆测序,获得173个高质量的EST序列,其中有5个跨叠群(conting),168个独立克隆,全长cDNA克隆占57%。BLASTx分析表明,有18.5%EST与GenBank中无任何同源性,有81.5%EST与GenBank中报道的功能已知或未知蛋白具有相似性。
A homogeneous full-length cDNA library was constructed by using duplex-specific nuclease (DSN) and switching mechanism of SMART (switch mechanism at5’end of RNA transcript). The titer of the original library tested was 1.4 × 106pfu / mL, the recombination rate was 96.9%, and the average length of the inserted fragment was greater than 1.0 kb. A total of 192 cDNA clones were randomly selected from the library and sequenced. A total of 173 high quality ESTs were obtained, including 5 contings and 168 independent clones. The full-length cDNA clones accounted for 57%. BLASTx analysis showed that 18.5% of ESTs have no homology with GenBank, and 81.5% of ESTs have similarities with known or unknown proteins reported in GenBank.