Construction Of The Full-length CDNA Library And Analysis In Part Of ESTs In Zi Goose Pituitary Gland

In order to further study the molecular mechanism of reproductive physiology controlled by pituitary of Zi-goose. The experiment was conducted to constructe the full-length cDNA library from the pituitary gland of the Zi-goose (Anser anser) by SMART (switching mechanism at 5′end of RNA Transcript) technique, which usedλTriplEx2 as a vector. Thenλphage packaging reaction and library amplification were performed. Eleven plaques were randomly picked and tested using PCR method with universal primers derived from the sequence flanking the vector. Then convertingλTriplEx2 to pTriplEx2, 217 clones were randomly picked and tested using PCR with universal primers T7 and SP6, 143 clones were chosen at randomly for sequencing.The results were as follows:1. The full-length cDNA library of pituitary gland from Zi-goose was constructive withλTriplEx2 vector and the qualities of original cDNA library were strictly checked by conventional titer ditermination. The results showed that the titration of the cDNA library constructed was 6×105 pfu/mL, the rate of recombination was 94 %, and the fragment length of inserted DNA ranged mainly from 0.5 to 2.0kb. The library titer, recombination rate and average inserted size all match quality requirments for cDNA library and this indicated that cDNA library of pituitary gland was successfully constructed.2. 143 clones were chosen at randomly for sequencing from the cDNA library, and 140 clones were sequenced successfully, 108 unigene weregained after unigene clusters. The ratio of unigene obtained was up to 87%. The BLAST sequence analysis indicated that 73 clones were known ESTs, and 35 clones were novel ESTs, and after BLASTn analysis of cDNAs, there possible function were predict. Through cross-species genenomic comparison, one of these are similar to the prolaction(PRL) gene of Anser anser ,homology was up to 99%; one of these are similar to the growth hormone(GH) of Anser anser, homology was up to 100%. one of these are similar to the ferritin heavy chain(FTH)of Gallus gallus, homology was up to 90%. This stady laid the foundation for further research on the mechanism of reproductive physiology controlled by pituitary of Zi-goose.