Screening, Identifying And Function Analysis Of Differentially Expressed Genes Of Prolific Trait In Dairy Goat

In the study, we took Xinong saanen goats as our main research object, and collected ovary samples of multiparous goat (three lambs) and monotocous goat (one lamb) at 10th day after estrus to construct forward and reverse subtracted cDNA libraries of ovarian tissues, further identify and clone the differentially expressed genes, and predict their structure and function by bioinformatics, and analyze their expression pattern by real-time reverse transcriptase-polymerase chain reaction, identify the new genes controlling prolific traits. The main results were showed as following:1. Suppression subtractive hybridization technology is used to construct forward and reverse subtracted cDNA libraries of ovarian tissues at 10th day after estrus called H-L (ovary of multiparous goat as tester and ovary of monotocous goat as driver) and L-H (Ovary of monotocous goat as tester and ovary of multiparous goat as driver) subtracted cDNA libraries. The subtraction efficiency was estimated by a housekeeping gene named GAPDH, and the results showed that GAPDH was subtracted efficiently at 25 folds for H-L and L-H subtracted cDNA library which demonstrated that differentially expressed genes were enriched in ovarian tissues of multiparous goat and monotocous goat.2. 86 and 91 clones were isolated from H-L and L-H subtracted cDNA library, H-L subtracted cDNA library, 72 clones were a single band by PCR detection, which accounted for 83.7% of total number of clones. In L-H subtracted cDNA library, 84 clones were a single band by PCR detection, which accounted for 92.3% of total number of clones. 40 clones selected randomly from each library which had different insert fragments were sequenced. The results showed that there are 7 goat ESTs in H-L subtracted cDNA library (Nucleobindin 1, Ornithine Decarboxylase Antizyme 1, Metallopeptidase Inhibitor 1 (TIMP1), CXXC-type zinc finger protein 5, Collagen type I alpha 1, GTPase activating protein 21, Solute Carrier Family 29 Member 1-SLC29A1), which are unknown in goat but have more than 90% homology with bovine and sheep. Some of these genes associate with follicular growth and development. In H-L subtracted cDNA library there are 9 goat ESTs and 1 of them is known in goat (Cytochrome C Oxidase Subunit II), 5 are unknown in goat (Calpain II Small Subunit, Transmembrane Emp24 Protein Transport Domain Containing 4, Ribosomal Protein L5, Hydroxyacyl-Coenzyme A Dehydrogenase, , Eukaryotic Translation Initiation Factor 1) but have more than 95% homology with bovine and sheep. These genes involved in egg activation, energy metabolism and regulation of gene transcription. In addition, three gene fragments did not match all the sequences of NCBI database.3. Real-time reverse transcriptase-polymerase chain reaction was used to analyse the change of mRNA expression level of TIMP-1, CXXC5, OAZ1 and Calpain II in H-L subtracted cDNA library. The results showed that TIMP-1, CXXC5 and OAZ1 had 3.32, 38.34 and 1.41 folds higher in ovary of multiparous goat than that in ovary of monotocous goat.