Construction Of The Full-length Cdna Library And Analysis In Part Of Ests In Zi Goose Ovary

In order to clone the full-length of gene related to egg laying performance and further study the molecular mechanisms for the process of egg laying in Zi geese, the experiment was conducted to constructed the full-length cDNA library of the ovary in Zi geese using TRIZOL method. SMART (Switching Mechanism at 5'end of RNA Transcript)technique and CDSШ/3' were used for first-strand cDNA synthesis.LD PCRs were then used to analyse the double-strand that were than digested by Sfi1 and fractionated by CHORMA SPIN-400 column.The longer 400bp cDNAs were collected and linked toλTriplEx2 vector.Thenλphage packaging reaction and library amplification were performed. Eleven plaques were random picked and tested using PCR with universal primers derived from the sequence flanking the vector.Then convertingλTriplEx2 to pTriplEx2,198 clones were random picked and tested using PCR with universal primers T7 and SP6, 120 clones were chosen at random for sequencing.The results were as follows:1. The full-length cDNA library of ovary 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 we constructed was 3.1×106 pfu/mL, the rate of recombination was 94 %, and the fragment length of inserted DNA ranged mainly from 0.5 to 2.5 kb. The library titer, recombination rate and average inserted size all match quality requirments for cDNA library and this indicated that cDNA library of ovary was successfully constructive.2. 120 clones were chosen at random for sequencing from the cDNA library, and 109 clones were sequenced successfully, 45 clones were favourable after abscise the redundancy. We analyzed the identity of these ESTs in the GenBank nr database and the EST database,and 34 clones were known ESTs, 11 clones were novel ESTs. and after BLASTn analysis of cDNAs,there possible function were predict. Through cross-species genenomic comparison,the one goose gene species that wasβ-actin mRNA; the one Anser anser gene species that was MHC.The results show that cDNA library we constructed can provide a base for further study on the full length cloning of important genes of egg laying.