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 SMARTTM 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(+) Sfi Ⅰ vector and transferred into the competent cells of Escherichia coli TOP10 strain. The obtained N. coibor cDNA library contains 1.0105 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.