| Reproductive trait is one of the important traits of rabbits,and its performance not only affects the fertility of female rabbits,but also directly affects economic benefits of rabbit production.There is a certain gap between the annual number of commercial rabbits in China,s breeding and developed European countries.Based on the universality of postpartum estrus in female rabbits,New Zealand white rabbits were selected as the research object for this experiment,containing frequent breeding and half-frequency breeding aspairing groups to explore effective methods for breeding commodity rabbits from modern large-scale rabbit farm.In addition,two comparative groups of frequency-tolerant and frequency-intolerant breeding were selected from the intensive frequency breeding group of New Zealand female rabbits.The ovarian tissues of female rabbits were taken to analyseaccording to transcriptome and proteomics in order to filter differential genes and differential proteins which are related to New Zealand white rabbits’tolerance in reproduction.The experimental results are as follows:1.In order to analyze the effects of different breeding methods on the reproduction and production performance of rabbits,frequent breeding and half frequency breeding groups were set,and each group contains 30 New Zealand female rabbits.The frequent breeding group(Group A)mated within 1 day after delivery,while the half-frequency breeding group(Group B)mated 18 days later after delivery,twice consecutively,collecting statistics of female rabbits’ weight,litter size,little weight and other indicators.The results showed that there was no significant difference in the number of litters,live births,rabbit litter weight and newborn weight between the frequent breeding group and the half-frequency breeding group(P>0.05).Female rabbits in the frequent breeding group differed significantly from the ones in the half-frequent breeding group in aspect of weight(P<0.05).The weight of the frequent breeding group showed a downward trend after parturition and the weight of the other group remained unchanged.Therefore,although frequent reproduction can obtain a larger number of offspring,the high-intensity reproduction method is not suitable for the long-term reproduction.which causes greater damage to mother rabbits2.The RNA-Seq technology was further used to analyze the ovarian tissues of the tolerant group and the intolerant group in the frequent-produced group,using |log2fold change|≥1&P<0.05 as the differential gene to filter threshold.The results showed that the tolerant group and the intolerant group contained a total of 1648 differentially expressed genes,including 563 up-regulated genes and 1085 down-regulated genes The results of differentially expressed genes GO and KEGG analysis showed that the differentially expressed genes GO were enriched in 99 genes related to reproduction,including 16 up-regulated genes,83down-regulated genes,and genes related to differentiation and development of reproductive organs such as IHH and OVGP1.KEGG mass spectrometry showed that the signaling pathways related to the significant enrichment of differentially expressed genes in reproduction include steroid biosynthesis,ovarian steroid hormone synthesis,and folic acid biosynthesis.Through qPCR technology,detected ZP1、CYP19A1、OVGPland BMP6,which is consistent with RNA-Seq sequencing results.Through the analysis of RNA-Seq data,IHH,OVGP1,BMP-6,CYP19A1,etc.may be related to New Zealand white rabbit reproductive tolerance3.Using 4D-LFQ technology,proteomics analysis was performed on the ovarian tissues of the frequency-tolerant breeding group and the frequency-tolerant breeding group.FC1.5 times&P<0.05 were used as the screening criteria for differentially expressed proteins and collected 427 differential proteins in total.Among them,253 were up-regulated differential proteins and 174 were down-regulated differential proteins,includingCYP51 A1、CYP17A1、CYP11A1 and LDLR reproduction-related proteins.Differentially expressed protein GO analysis is mainly enriched in cholesterol biosynthesis,glycol biosynthesis,steroid metabolism regulation,cholesterol metabolism regulation,negative regulation of peptidase activity,cholesterol biosynthesis regulation,bicarbonate transport and other processes.KEGG enrichment mass spectrometry showed that differential proteins are involved in steroid biosynthesis pathways,ovarian steroid production and other reproduction-related pathways.Further transcriptome and proteome joint analysis found that CD68,GPNMB,MCM4 and other proteins closely related to ovarian cancer,suggesting that these three genes may be negatively correlated with New Zealand female rabbit reproductive tolerance.4.According to the transcriptome and proteome sequencing results,it was found that there was no significant difference in the CYP11A1 gene mRNA expression level between the tolerant group and the intolerant group,but the protein level was significantly different.The CDS full-length sequence of CYP11A1 was further cloned,which was 1557bp.Using bioinformatics method to forecast analysis of the structure and function of CYP11A1,found CYP11A1 protein of unstable protein,isoelectric point 4.91,protein secondary structure is given priority to with a spiral and random coil,tertiary structure is curved spiral,a total of 40 potential phosphorylation sites.Ovarian granulosa cells were further isolated and identified,and CYP11A1 gene overexpression and interference experiments were conducted.It was found that the changes in the expression of CYP11A1 gene could significantly affect the expression of HSD17B1,BMP 15 and FSHR genes(P<0.05),thus affecting the reproductive function of female rabbits.These results provide a theoretical basis for further study on the regulatory role of CYP11A1,a key protein,in the reproductive performance of female rabbits. |
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