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

Abstract:

In order to construct high-quality normalized full-length cDNA 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 cDNA library was better than that of ordinary cDNA libraries, which could be used for further research of screening of functional genes and their related information.

Key words:Cestrum nocturnum;Normalized cDNA library;EST