| Sheep lambing performance is the most important economic trait and is regarded as a critic factor affecting the productivity in sheep industry, in which the twinning trait is regarded as the ideal lambing performance favoring by sheep industry. However, the litter size affected by many factors such as genetic and environmental factors, the mechanisms of molecular genetics and regulation for controlling sheep twinning traits are still unknown, and their breeding techniques are scare. Ovary and gonadal axis are the main functional organs involved in reproduction and its regulation. During each estrous cycle, the follicle has experienced development, pregnancy and implantation process, and these process more or less directly affect and/or determine the ovulation,the rate of fertilization and the litter size of ewes. This research takes Mongolia sheep(ewes with singleton and twin lambs), Poll Dorset(ewes with singleton and twin lambs) and Small Tail Han sheep(ewes with three lambs) as the test materials, based on the reproductive endocrinology, genomics, proteomics and bioinformatics, aimed at : 1) clarify the differences between reproductive hormones in peripheral blood of three breeds; 2) state the spatio temporal pattern of three candidate genes expression distributions, variations; 3) screen the differenced expressed proteins; and 4) screen nucleic acid difference of each candidate genes mutation and polymorphism. And make the correlation analysis between those differences and lambing traits. The main results were as follows:1. All the seven hormones concentration of the Poll Dorset ewes was higher than that of the Mongolian ewes in the three periods. And the hormones secretion shown different patterns in three breeds. The hormones relationships between lamb performances showed that there were significant differences(P<0.05) in PRL, LH,FSH concentrations in both breeds, and MT concentrations in Mongolian ewes, and PROG in Poll Dorset ewes during post-partum anoestrus. While in the estrous period of the first day, there were significant differences(P<0.05) in PROG concentrations in both breeds, PRL, LH, FSH, MT concentrations in Mongolian ewes, T concentrations in Poll Dorset ewes. But the MT and T concentrations of ewes with single lamb ewes were higher than that of ewes with twin lambs. In the estrous periodof the 11 th day, only PROG concentrations was significant differences(P<0.05) in both breeds. There were significant differences(P<0.05) in LH, FSH and T concentrations in Mongolian ewes, PRL concentrations in Poll Dorset ewes, there were no differences in other hormones.2. Real-time reverse transcriptase-polymerase chain reaction was used to analyse the changes of m RNA expression levels of 3 genes in ovary and gonadal axis organs. The results showed that different expression patterns: Mongolia sheep and Dorset GDF9 and RXRG gene expression have similar pattrern, but differ from the BMPR-IB gene.All the three genes have the same pattern in Small Tail Han sheep. Aggression analysis was performed to investigate the correlation between gene expression levels and litter sizes. BMPR-IB gene expression levels in anestrus of Mongolia sheep and Dorset sheep ovarian pituitary, hypothalamus and Mongolia in Dorset, estrus period of 1st day of Mongolia sheep and Dorset pituitary significantly positively correlated with litter size. GDF9 gene expression levels in pituitary and hypothalamus significantly positively correlated with litter size. Mongolia sheep RXRG gene in anestrus ovarian, Dorset RXRG gene in oestrus 1st day, 11 th day of ovarian and pituitary ovarian oestrus were also significantly positively correlated with litter size.3. Based on separation technology of differential protein electrophoresis, combined with mass spectrometry, the correlation between protein expression levels and litter sizes were analyzed. The results showed that the expression of BMPR-IB protein of ewes with twin lambs were greater than that of ewes with single lamb. The expression of that of Small Tail Han was the highest. The expression of GDF9 protein shared theh same pattern with BMPR-IB protein. RXRG protein was missed, only the retinoic acid receptor RXR-beta were detected in anestrus stage, while the negative correlation with litter size detected.4. Sequence homology, amino acid structure, the potential functional site prediction,protein secondary and tertiary structure of BMPR-IB, GDF9 and RXRG genes were compared among goat, sheep, bovine, human, mouse and pig using bioinformatics softwares. We found the amino acid constitutions of each of the 3 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. Based on the sequencing, the mutation of G4(G721A), G549 C, T640 A and C694 T 4 in GDF9 in Mongolia sheep, Fec B genotype in Small Tai Han, and C1113 A in Mongolia and Dorset were detected. The base difference of 597 locus was detected: total A in SmallTai Han ewes, total G in Poll Dorset, both A and G in Mongolia ewes. And the mutation T864 C in Mongolia and Poll Dorset ewes with single lamb. Those SNPs provided the novel markers in breeding, but need to check in population level. |
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