以德保苏铁珊瑚根为材料,利用DSN(duplex-specific nuclease)均一化与SMART(switching mechanism at 5′end of RNA transcript)技术,构建均一化全长cDNA文库。经检测原始文库滴度为1.5×106 cfu·mL-1,文库重组率达97%,插入片段平均长度为1 kb。从文库中随机挑取192个cDNA克隆测序,获得175条高质量EST序列,装备成165条unique序列,其中包括7条contigs和158条singlets,EST冗余度为5.71%。将获得的174条干净的高质量EST序列与NCBI非冗余核酸库(NT)和非冗余蛋白库(NR)数据库进行Blast比对(分值≥200,一致性≥60),其中66.1%(115)的EST序列发现了与其同源的序列,而33.9%(59)的EST序列未发现与其显著同源的序列。构建德保苏铁珊瑚根均一化全长cDNA文库为功能性标记开发和功能基因研究提供了丰富的信息平台。%A normalized full-length cDNA library was constructed with the coralloid roots of Cycas debaoensis by DSN (duplex-specific nuclease) normalization method combined with SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique. The titer of original cDNA library was about 1.5×106 cfu·mL-1 and the average insertion size was about 1 kb with high recombination rate (97%). The 175 high quality expressed sequence tags (ESTs) were obtained from 192 randomly picked cDNA clones. Clusting and assembly of ESTs resulted in 165 unique sequences, consisting of 7 contigs and 158 singlets. Using a generic similarity threshold (score≥200, identity≥60), approximately 115 (66.1%) of all 174 clean ESTs matched corresponding homologous sequences according to BLAST analysis of the nr-nt (non-redundant protein and non-redundant nucleotide sequence) database in GenBank. The 33.9% (59) ESTs have no significant match. These results showed preliminarily that we successfully constructed a normalized full-length cDNA library of coralloid roots in C. debaoensis, which could serve as an abundant information platform for functional marker development and functional gene study.
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