Abstract: 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.

Key words: Cycas debaoensis, coralloid root, normalized, cDNA library

摘要： 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。

关键词: 德保苏铁, 珊瑚根, 均一化, cDNA文库