cDNA Suppression Subtractive Hybridization(SSH) Library Construc-tion and Sequence Analysis of the Ovary of Chinese Merino Sheep(Ovis aries) in Different Period
Abstract：Seasonal oestrus is one of main restricted factors in the development of sheep industry. The ovaries are the important reproductive organs of sheep, and the periodicly expressed gene in ovary is likely to regulate the ewe seasonal breeding directly or undirectly. In order to discuss the molecular genetic mechanism of the sheep seasonal oestrus, the subtracted cDNA library of the Chinese Merino sheep(Ovis aries) ovary(estrus and anestrus) was conducted by the suppression subtractive hybridization(SSH). Random sampling of 386 positive clones were identified by PCR, and obtained about 306 effective clones and 126 non-redundant sequences after sequencing. The sequences were studied through BLAST on line with the GenBank dbase and GenBank EST dbase and the results showed that 21 of them were not matched to any of the databases which might represent new genes related to Chinese Merino sheep ovary tissue seasonal reproduction trait. Retinoic acid receptor response element1(Rarres), thymosin beta 4(TMSB4X), eukaryotic translation elongation factor 1 alpha 1(EEF1A1), prothymosin alpha(PTMA), and No.12 and No.89 genes were selected from the library, and tissue-specific detection of the 6 kind of genes screened was performed in heart, liver, spleen, lung, kidney, ovary, hypothalamus, pituitary, uterus and fallopian tubes tissues. The results showed that RARRES1 and No.89 gene over-expressed in ovary tissues, however, TMSB4X, EEF1A1, PTMA and No.12 gene expressed in all tissue. In order to further validate the obtained results, RARRES1, TMSB4X, EEF1A1, PTMA, No.12 and No.89 genes were selected for expression analysis by quantitative Real-time PCR. The results showd that their expression were all up-regulated in ovary of estrous Chinese Merino sheep. In this study, we got potential genes related with the seasonal reproduction trait of the Chinese Merino sheep, wihch provides a foundation to research the molecular mechanism of interaction between ovary function and seasonal reproduction trait of sheep in future.
