| In the present study, a famous local breed rabbit-Harbin white rabbit and theworld-famous medium-sized meat rabbits-Californian rabbit were used to constructtwo ovary DGE libraries of Harbin white rabbits at different developmental periodsand one ovary DGE library of Californian Rabbit. By using the emerging RNA-sequencing technique, we have already finished sequencing, constructing andbioinformatics analysis. In addition, we used Real-time PCR to amplify sixdifferential expressed genes between different developmental state and differentbreeds randomly, and verified the experiment results by testing the expression level ofsix genes.The main results of the experiment above are:1. To construct three DGE libraries of low-yield Californian rabbit(poor),high-yieldHarbin white rabbit(prolific) and immature Harbin white rabbit(neanic), aftersequencing and analyzing sequence datum we got7,159,832,7,637,178and7,596,342raw tags, respectively. By wiping out impurity tags, we got7,079,460,7,469,924and7,388,202clean tags, and accounting for98.88%,97.81%and97.26%of the total, respectively.2. Analyzing the comparable group-neanic and poor, and serving poor as a control, wefound1,610differential expressed genes in neanic, which contain1284up-regulatedgenes and326down-regulated genes. Among those genes, the fold change of5genesbeyond10and4of them up-regulated, one down-regulated. The highest up-regulatedfold change arrived at12, and the lowest up-regulated fold change reach11.95,butmost between2and4.3. Analyzing the comparable group-neanic and prolific, and serving prolific as acontrol, we found882differential expressed genes in neanic, which contain727up-regulated genes and158down-regulated genes. Among those genes, the foldchange of24genes beyond5, and16of them up-regulated,8down-regulated. Thehighest up-regulated fold change arrived at11.88, and the lowest up-regulated foldchange reach9.442, but most between1and3.4. Analyzing the comparable group-prolific and poor, and serving prolific as a control,we found321differential expressed genes in poor, which contain180up-regulatedgenes and141down-regulated genes. Among those genes, the fold change of5genesbeyond10and4of them up-regulated, one down-regulated. The highest up-regulatedfold change arrived at12.04,and the lowest up-regulated fold change reach12.25,but most between2and3.5. After analyzing the GO functional annotations of differential expression genesbetween prolific and poor,we discovery that4significant enrichment items on cellularcomponent,10significant enrichment items on molecular function and15significantenrichment items on biology process. Analyzing the GO functional annotations ofdifferential expression genes between prolific and neanic, we discovery that23significant enrichment items on cellular component,17significant enrichment itemson molecular function and32significant enrichment items on biology process.6. The results of pathway analysis indicated that differential expression genes weresignificantly enriched in25pathways between prolific and neanic, and between poorand prolific, differential expression genes were significantly enriched in27pathways.7. The results of pathway analysis indicated that between comparison groupprolific/neanic,21,6,13,13differential expression genes were significantly enrichedin cell cycle, DNA replication, oocyte meiotic maturation and oocyte meioticsignaling pathways,respectively, and all of those were related to cell growth anddivision.12,8,7,10differential expression genes were enriched in NOD-likereceptor signaling pathway, the hedgehog signaling pathway, the sensing pathcytoplasmic DNA, P53signaling and TOLL-like receptor, respectively, and all thosepathway associated with signal transduction. Additionally,4,7,2differentialexpression genes were enriched in Steroid biosynthesis, Steroid hormone biosynthesisand Vitamin B6metabolism, respectively.8. The results of pathway analysis indicated that between comparison grouppoor/prolific,40,6,5,4,2,3,2,5,7differential expression genes were significantlyenriched in metabolism related pathways fructose metabolism of mannose, GSHmetabolism, pyruvate metabolism, fatty acid synthesis metabolism, galactosemetabolism, sulfur metabolism, mediated by cytochrome P450drug metabolism andmediated by cytochrome P450exogenous substance metabolism,respectively, and allof those were related to metabolism.4,6,3differential expression genes wereenriched in steroid biosynthesis,steroid hormone biosynthesis and renin-angiotensinsystem, respectively. Additionally, the number of differential expression genesinvolved in ABC transporters, PPAR signaling pathways and fat cytokine signalingpathways were4,7,5, respectively.9. By analyzing the321different expression genes obtained in the comparison groupprolific/neanic, we ultimately selected five genes closely associated with rabbit fecundity, they were FSHR, BMP6, BMP15, INHBB and HSD17B1.10. Selecting6differential expression genes randomly, and verifying the sequencingresults by using real-time PCR. The experiment results were consistent with thesequencing results, which further proved the accuracy and reliability of the presentDGE experiment. |
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