Cloning, Sequence Analysis And Quantification Of Egg-laying-related Genes In Ovarian Tissue Of Zi-goose

It is obtained 5 differentially expressed genes related to egg production by suppression subtractive hybridization of Zi-goose ovarian tissue. To further verify the results of subtractive hybridization, and to identify these genes and the correlation of egg production. 5 were selected from 26 ESTs as study subjects.The test materials were pre-laying period of 5-month-old and 8-month-old Zi-goose ovarian tissue.Total RNA in ovarian was obtained two months of age with Trizol,than was reversed transcription to cDNA. Using RT-PCR, 5 gene fragments of expressed sequence tags were amplified. SYBR Greenâ… Real Time PCR was performed to analyze mRNA expression levels of 5 egg production related genes in Zi-goose ovary tissue during pre-laying and laying periods. Primers about EST4, EST5, EST7, EST8 were designed by the comparison with other species to identify conserved regions.PCR method were used in the expansion of these sequence length. A novel EST named Syndecan 2 (SDC2) which comes from the differences expressed EST sequences of the same subtracted cDNA library was amplified to full length cDNA using 5' -RACE methord .It's Accession No.is GE616652. After 5 ' end were sequenced, the result contains the complete CDS.Primers were designed for PCR to get the full-length CDS .The result are:(1) Results indicated the mRNA expression levels of the genes EST4, EST5, EST6, and EST8 during laying period were significantly higher than that during pre-laying period (P<0.05), and the mRNA expression level of the gene EST7 was extremely significantly higher than that during pre-laying period (P<0.01). EST4 relative mRNA gene expression early egg laying period is 10.36 times than that in egg laying period. EST5 is 3.54 times, EST6 is 4.35 times, EST7 is 3.06 times, and EST8 is 4.64 times.(2)EST4 were amplified from 288bp to 537bp, EST5 were amplified from 433bp to 866bp, EST7 were amplified from 398bp to818bp, and EST8 were amplified from 322bp to 915bp. It obtained with the EST corresponding goose ovary RPL10A, ATP5A1, RNF130 part of the coding sequence, complete CDS area by OAZ1.(3) It is obtained complete CDS of goose SDC2, ORF length of it is 606bp, encoding 201 nucleotides, isoelectric point of 4.53, molecular weight 22kDa. Phylogenetic tree was constructed with the multiple sequence comparison to determine goose SDC2 polymer proteoglycans are members of the family is highly conserved in species evolution and embryonic development and related growth factors relevant to study the protein's specific function and regulation mechanism of the production eggs of great significance to improve the performance.This test verified the test results of pre-dot blot hybridization using the SSH technique an.The mRNA expression of the five genes in Zi-goose ovary of egg laying period was higher than that of early egg production period. The four novel genes long sequence molecular cloning and fragment analysis in order to carry out the full length of egg-laying related genes cloning and function studies and molecular mechanism of goose laying the foundation for further study. The nature and function of the amino acids and Zi-goose SDC2 genes were conducted to do the preliminary inference.