Screening, Identifying And Analysis Of Differently Expressed Genes In Ovary Of Multiparous And Uniparous Dairy Goat During The Estrous

Reproductive rate is an important economic trait in goat reproduction. To improve the fecundity, also known as litter size, is of special meaning in the selection of animals with high reproductivity. But the litter size is a result of interaction between genetic and environmental factors, how precisely the litter size is controlled remains a critical and important question in reproductive biology. Ovary is the main functional organ in reproduction. During each estrous cycle, ovaries experience the changes of proliferation, invasion, differentiation and cell apoptosis, and these normal physiological changes of ovaries more or less directly affect and/or determine the ovulation, the rate of fertilization and the litter size of goats. But Studies of gene expressions using ovaries during the estrous cycle of goats are limited. Thus we collected ovary samples of multiparous and uniparous Guanzhong dairy goats during the estrous cycle to construct forward and reverse subtracted cDNA libraries of ovaries, and identified differentially expressed genes enriched in multiparous and uniparous Guanzhong dairy goats. The main results were as follows:1. Forward and reverse subtracted cDNA libraries of ovaries at estrous stage were constructed using suppression subtractive hybridization technology, called M-U (ovary of multiparous Guanzhong dairy goat as Tester, and ovary of uniparous Guanzhong dairy goats as Driver) and U-M (ovary of uniparous Guanzhong dairy goat as Tester, and ovary of multiparous Guanzhong dairy goat as Driver) subtracted cDNA library respectively. The subtraction efficiency was estimated by a housekeeping gene GAPDH, and the result showed that GAPDH was subtracted efficiently by 211 and 28 folds for M-U and U-M subtracted cDNA library respectively, which indicated that differentially expressed genes were also enriched by the same corresponding folds.2. Screening and identification of the constructed M-U and U-M libraries. Dot blot hybridization was used for the screening of the differentially expressed clones. From the sequenced clones we identified 77 completely different genes in M-U subtracted cDNA library, of which 4 were known in goats, 53 were unknown in goats but have high homology with Ovis aries, Bos taurus, Sus scrofa or other species, and the rest 20 were unknown genes. Whereas we identified 50 completely different sequences from the sequenced clones in the U-M library, of which no one of them was known in goats, 30 were unknown in goats but have high homology with Ovis aries, Bos taurus, Sus scrofa or other species, and 20 were completely unknown genes. The 77 goat ESTs in M-U subtracted cDNA library involved in 139 biological processes based on the GO annotation for biological process; whereas, there were only 22 biological processes involved for the sequences identified in the U-M library. Enrichment analysis of the biological processes involved by the 77 sequences was carried out, and 17 different processes were overrepresented, which included biological regulation, biological adhesion, death, growth, locomotion, metabolic process, cell proliferation, signaling, localization, cellular component biogenesis, cellular component organization, immune system process, reproduction, multicellular organismal process, developmental process, response to stimulus and cellular process. The same analysis was done to the sequences from the U-M subtracted cDNA library, but only 4 biological processes were enriched, which comprised response to stimulus, cellular process, biological regulation and metabolic process.3. Real-time reverse transcriptase-polymerase chain reaction was used to analyse the changes of mRNA expression levels of 12 genes selected from M-U subtracted cDNA library in ovaries of multiparous and uniparous Guanzhong dairy goats. The results showed that they increased 16.03 (DCN), 11.87 (OAZ1), 5.28 (EPHX1), 5.68 (FSHR), 5.36 (TIMP1), 9.67 (RUNX1), 3.90 (ADAMTS1), 7.97 (BMP6), 2.91 (RPL5), 6.01 (VIM), 2.23 (ZNF207) and 4.91 (SF3B5) folds in ovaries of multiparous than that of uniparous Guanzhong dairy goats. Aggression analysis was performed to investigate the correlation between gene expression levels and litter size in multiparous goats. Of the 12 genes, only DCN, OAZ1, EPHX1 and FSHR were significantly positively correlated with litter size, with R2= 0.9783, 0.9747, 0.9669 and 0.902 respectively. We then cloned the complete coding region of DCN, OAZ1, EPHX1 and FSHR from goats, namely, Capra hircus Decorin (ORF 1083bp, Accession No:HQ326237), Capra hircus Epoxide Hydrolase 1 (ORF 1356bp, Accession No:HQ326238), Capra hircus Follicle Stimulating Hormone Receptor (ORF 2055bp, Accessoin No:HQ326239) and Capra hircus Ornithine Decarboxylase Antizyme 1 (ORF 577bp, Accession No:HQ326240), which laied a foundation for further functional research.4. Sequence homology, amino acid structure, the potential functional site prediction, protein secondary and tertiary structure of DCN, OAZ1, EPHX1 and FSHR were compared among goat, sheep, bovine, human, mouse and pig using bioinformatics softwares. We found the ORFs and amino acid constitutions of each of the 4 genes in different species had high homology; the protein structures and potential functional domains of each gene among species were similar. The signal peptides were predicted for DCN and FSHR of goat, sheep, bovine, human, mouse and pig. No signal peptides and curls of EPHX1 and OAZ1 were found, so there were no complex spatial structures. These four genes have N-glycosylation sites. Only OAZ1 does not have transmembrane helix.