The construction of pituitary gland cDNA library of Nibea coibor

A cDNA expression library from the pituitary gland of Nibea coibor was constructed into pcDNA3.1 (+) eukarytical expression plasmid using SMART^TM cDNA library construction protocol. The anchor first strand cDNA containing asymmetrical SfiⅠ restriction enzyme sites (A & B) was synthesized by transcription of total RNA with the SMART technique. The long-distance(LD PCR) was performed using a modified oligo(dT) prime and anchor prime as the prime set, and anchor fist-strand cDNA as the template to enrich the cDNA population for full-length sequences. After digestion with SfiⅠ and size fractionation using CHROM SPIN-400 column, SMART cDNA was ligated into the SfiⅠ-digested pcDNA3.1 (+) -SfiⅠ vector and transferred into the competent cells of Escherichia coli TOP10 strain. The obtained N.coibor cDNA library contains 1.0×10⁵ recombinants, the percentage of vectors with inserts was 90% and the average insert size was between 0.4~3.0 kb. The N.coibor cDNA library gives an ideal base for further study structure and characterization of the gene in relation to growth and development of N.coibor.