Construction Of CDNA Subtractive Library Of Ovarian Tissues In Sexual Maturity Zi Geese

Suppression subtractive hybridization (SSH) is an effective technique to isolate and clone differentially expressed genes. The study was conducted by using technique of SSH to construct cDNA subtractive library of ovarian tissues in sexual maturity Zi geese, and to screen differentially expressed genes that were highly expressed in the ovarian tissues of laying geese compared with prelaying geese. The aim of the present study is to lay theoretical foundations for clarifying the molecular mechanism that the differentially expressed genes affected the egg-laying and reproductive performance, and to improve the egg-laying performance of Zi geese fundamentally.Take the prelaying and laying ovarian tissues in sexual maturity Zi geese as study object. Isolate the total RNA from two tissues to construct RNA pools and purify into mRNA, then became the cDNA with reverse transcription. Take the laying ovarian tissues cDNA as the Tester cDNA and the prelaying ovarian tissues cDNA as the Driver cDNA. After digested with the RsaⅠ, the digested cDNA ligated with adaptors, then two steps of hybridization and two round of PCR were followed to normalize and enrich differentially expressed genes. Ligate the production of Nested PCR with pGEM-T vector, then transformed into the E.coli competent cell TG1 to construct cDNA subtractive library of ovarian tissues in sexual maturity Zi geese. The monoclonal coaenobium were screened through the blue-white screening system. White coaenobium were randomly picked up shaking in LB medium at 37℃for 12 h, then identify the positive clones by PCR. Those clones were screened and identified by Northern dot blot technique which the probes were labeled with DIG. Positive clones were sequenced and compared with known sequences in the public databases of GenBank for homology analysis.Total RNA and mRNA were respectively exacted and purified from prelaying and laying ovarian tissues in good quality and quantity. Double-strand cDNA were reverse transcripted integrally, and cut by RsaⅠinto even length short segments. Ligation was effective. After two steps of hybridization and Nested PCR, the differentially expressed genes were obtained and enrich. Subtractive hybridization was effective. Subtractive products were inserted into the pGEM-T clone vector; then transformed into the E.coli competent cell TG1 successfully. After constructing subtracted library of ovarian tissues in sexual maturity Zi geese, total 467 positive clones were confirmed by PCR from 500 white clones and the results showed that the positive rate was 93.4%. After screening 69 clones were confirmed to be positive differentially expressed genes fragments. These clones were selected for sequencing and analyzed with BLAST program in GenBank, and 24 differentially expressed genes were obtain that were highly expressed in the ovarian tissues of laying geese compared with prelaying geese. The 16 differentially expressed genes were homologous with known genes, and 8 differentially expressed genes were unknown gene fragments.