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【目的】构建甘蔗根系全长cDNA文库,并进行测序,为分离克隆甘蔗抗旱候选基因奠定基础。【方法】在干旱胁迫条件下,以新台糖22号甘蔗根系为材料,采用SMART技术构建cDNA文库,并进行EST序列分析。【结果】文库构建质量鉴定结果显示,文库库容量为1.0×106PFU/mL,重组率约95%,文库插入片段长度在500～2000bp,平均长度约1102.1bp。挑选526个阳性克隆进行单向测序后,共获得523个ESTs序列,去除载体、接头序列以及长度低于100bp的序列后,进行聚类分析并组装,共得到468个Uni-ESTs,其中congtig29个,singlets439个,分别占总数的6.5%和93.5%。通过与NCBI非冗余蛋白库进行BLASTX比对,发现444个Uni-ESTs在GenBank有同源序列,占94.87%;获得24个未知蛋白功能基因,占5.13%。【结论】构建了干旱胁迫下甘蔗苗期根系全长cDNA文库,获得523个ESTs序列和468个Uni-ESTs,并获得一批新的未知蛋白功能基因。
【Objective】 The full-length cDNA library of sugarcane root was constructed and sequenced, which laid the foundation for the isolation and cloning of candidate genes for drought resistance of sugarcane. 【Method】 Under drought stress conditions, a new cDNA library was constructed from sugarcane root of Xintai Sugarcane 22 using SMART technique and analyzed by EST sequence. 【Result】 The results of library construction showed that the capacity of the library was 1.0 × 106 PFU / mL and the recombination rate was about 95%. The inserted length of the library was 500-2000 bp with an average length of 1102.1 bp. A total of 528 ESTs were obtained after 526 positive clones were sequenced. After removing the vector, linker sequence and the length of less than 100 bp, 468 Uni-ESTs were obtained, of which congtig29 , singlets439, accounting for 6.5% and 93.5% of the total respectively. By BLASTX alignment with NCBI non-redundant protein library, we found 444 Uni-ESTs have homologous sequences in GenBank, accounting for 94.87%; 24 unknown protein function genes were obtained, accounting for 5.13%. 【Conclusion】 The full-length cDNA library of sugarcane seedlings under drought stress was constructed. 523 ESTs and 468 Uni-ESTs were obtained and a number of new unknown protein functional genes were obtained.