夜香树均一化全长cDNA文库的构建及EST分析

Construction of Normalized Full-length c DNA Library and EST Analysis from Cestrum nocturnum L.

为构建高质量的夜香树(Cestrum nocturnum)均一化全长c DNA文库,以其花朵为材料,采用DSN均一化技术与改进的SMART技术相结合,将连接产物进行脱盐浓缩后电转化进行构建。结果表明,未脱盐连接产物的转化效率为1μL连接产物有7000个菌落,理论重组率为96%;脱盐后,提升为4.1×106个菌落,理论重组率为98%;蓝斑也有插入片段,实际重组率应为100%;插入片段大小平均为1.6 kb。随机挑取500个单克隆(含50个蓝斑)测序,共获得464条EST,单一序列(unigene)为426条,占91.8%,其中片段重叠群26个,单基因400个,冗余率仅为8.1%,表明构建文库的均一化效果较好,可满足后续功能基因的筛选和基因信息的研究。

In order to construct high-quality normalized full-length c DNA library of Cestrum nocturnum L., the library from its flowers was established successfully by combined modified SMART technology, DSN digestion with desalting of ligation product. The results showed that transformation efficiency of the ligation product without desalting was 7000 colonies per microliter, and theoretical recombination rate was 96%, whereas, the transformation efficiency increased up to 4.1×106 colonies per microliter, and theoretical recombination rate was 98% after the ligation product desalting. Actually, blue colonies also contained the insert fragments, and, the recombination rate was to 100%. The average size of inserts in the library was about 1.6 kb. Five hundred colonies including 50 blue colonies were randomly picked for sequencing from the library, 464 effective sequences were obtained including 426 unigenes(accounting for 91.8%), which were composed of 26 contigs and 400 single genes, redundant rate was only 8.1%. So, it was suggested that the quality of the normalized c DNA library was better than that of ordinary c DNA libraries, which could be used for further research of screening of functional genes and their related information.