Mining And Functional Validation Of Key Genes For Ovarian Development In The Reproduction Cycle Of Yili Goose

Xinjiang Yili goose is the only herbivorous special local poultry species from the greylag in China.As a high-quality local special poultry,its meat quality is excellent,rich in nutritional value,and has the characteristics of strong adaptability,rough feeding resistance,disease resistance and strong resistance to adversity,and is an irreplaceable breed resource for the development of local special goose breeding industry.However,as a local goose species,the annual egg production of Yili goose is low and shows obvious individual differences,with the highest annual egg production reaching 27,the lowest annual egg production being only 1,and the average annual egg production being 11,which seriously restricts the rapid development of the Yili goose industry.The development of ovaries and follicles is an important factor affecting the egg production performance of poultry.For egg-laying poultry,there are three different physiological stages of ovarian development: the development and growth stage,the egg-laying and reproduction stage,and the resting stage.During ovarian development and growth,the primordial follicle is recruited and remains quiescent.During egg production,follicles are activated and ovulation becomes the main activity of the ovaries,regulated by the secretion of sex hormones.In contrast to the egg-laying period,most reproductive activity in female birds ceases during the resting or cessation phase.The dramatic differences in the performance of the poultry ovary during the three physiological stages are largely influenced by differential gene expression.Therefore,studying the expression characteristics of ovarian tissues at the transcriptional level at various stages of egg laying in geese can provide an important basis for screening and identifying key genes regulating ovarian development in geese,and thus for improving egg laying performance.The Yili goose is one of the typical seasonal breeding animals in China.It enters its peak egg-laying period at the age of 3 and has only 1 egg-laying cycle per year,a feature that also makes the Ili goose an ideal animal model for studying the gene expression patterns of geese in each egg-laying cycle.Therefore,in this study,mRNA and non-codingRNAs of ovarian tissues were identified at the whole transcriptome level in 3-year-old female Yili geese before and after egg laying(n=4/period).Combining bioinformatics analysis,RT-q PCR,dual luciferase assay,Western blot,CCK-8 and flow-through apoptosis assay,we deeply explored and identified key genes and non-codingRNAs affecting goose ovarian development,and provided a theoretical basis for exploring the molecular regulatory mechanisms of genes and non-codingRNAs mediating goose ovarian development.The main results of this study are as follows.(1)Using whole transcriptome sequencing technology,we revealed the differences in the expression of mRNAs and lncRNAs in ovarian tissues of Yili goose before and after egg laying.258 differential mRNAs and 466 differential lncRNAs were found in the laying group compared with the pre-laying group;compared with the ceased-laying group,1136 differential mRNAs and 925 differential lncRNAs were found in the laying group,and 525 differential mRNAs and 742 differential lncRNAs were found in the pre-laying group.There were four and six differential mRNAs and differential lncRNAs shared by the three periods,respectively.GO enrichment analysis revealed that differentially expressed mRNAs and lncRNAs target genes were mainly enriched in bioregulatory,cellular process and metabolic process;KEGG enrichment analysis revealed that differentially expressed mRNAs and lncRNAs target genes were significantly enriched in neuroactive ligand-receptor interaction,ECM-receptor interaction and steroid biosynthesis pathways related to ovarian development.In addition,the protein interaction network of differential mRNAs was constructed to screen for BTK,PDGFRA and ITGB3,which are potential regulatory genes associated with goose ovarian development.(2)In this study,a total of 2700 known-miRNAs and 2983 novel-miRNAs were identified from 12 Yili goose ovary tissues.Compared with the pre-laying group,there were 258 differential miRNAs in the laying group;compared with the ceased-laying group,there were 1131 differential miRNAs in the laying group and 909 differential miRNAs in the pre-laying group.There were 44 differential miRNAs common to the three periods.GO enrichment analysis showed that the differential miRNAs target genes were mainly related to catalytic activity and cellular processes;KEGG enrichment analysis showed that the differential miRNAs target genes were mainly involved in ECM-receptor interactions,calcium signaling pathway and Notch signaling pathway related to egg production process.Based on the above data,a differential lncRNA-miRNA-mRNA regulatory network involved in follicular development and associated with cell proliferation,differentiation and apoptosis was established and initially validated in this study,and the expression trends of key nodes in the lncRNA-miRNA-mRNA regulatory network were verified by real-time fluorescence quantitative PCR.(3)In this study,a total of 4483 circRNAs were identified in the ovarian tissues of Yili goose,and there were 159 differential circRNAs in the laying group compared with the pre-laying group,455 differential circRNAs in the laying group compared with the ceased-laying group,and 383 differential circRNAs in the pre-laying group.Functional analysis showed that the source genes of differential circRNAs in ovarian tissues at all stages were mostly enriched in biological processes such as bioregulation,catalytic activity and related pathways such as signaling and metabolism.Screening to the core regulator circRNA NW＿013186107.1:36835|52574 with gga-miR-34b-5p based on differential circRNA-miRNA regulatory network.(4)The expression trends of MSTRG.5970.28,miR-133a-3p and ANOS1 in ceased-laying and laying stages were screened and validated based on the differential lncRNA-miRNA-mRNA regulatory network in ovarian tissues of Yili goose at each stage.After treatment of goose granulosa cells with h CG(5 IU/m L)or FSH(10 IU/m L),the expression of MSTRG.5970.28 was found to be extremely significantly reduced in both granulosa cells,suggesting that MSTRG.5970.28 may be involved in the regulation of the follicular development process in geese.MSTRG.5970.28 was found to have a target binding site for miR-133a-3p with ANOS1 by a dual luciferase activity assay.In addition,this study found that overexpression of miR-133a-3p both highly significantly inhibited the expression of MSTRG.5970.28 and ANOS1 in goose granulosa cells,and the expression of both was highly significantly increased after interference with miR-133a-3p.Overexpression of MSTRG.5970.28 was found to inhibit proliferation and promote apoptosis of granulocytes significantly by CCK-8 cell proliferation and flow apoptosis assay.In contrast,when MSTRG.5970.28 was interfered,the proliferative capacity of granulosa cells was highly significantly increased and apoptosis was highly significantly decreased.miR-133a-3p had the opposite effect on proliferation and apoptosis of granulosa cells as that of MSTRG.5970.28.Meanwhile,in this study,the overexpression of MSTRG.5970.28,miR-133a-3p plasmid was cotransfected into granulocytes,and it was found that the proliferative and apoptotic effects of granulocytes caused by MSTRG.5970.28 overexpression were attenuated by miR-133a-3p.By RT-q PCR and WB assay,MSTRG.5970.28 was found to competitively bind miR-133a-3p as a ceRNA of miR-133a-3p,thus attenuating the inhibitory effect of miR-133a-3p on ANOS1.In summary,this study constructed whole transcriptome gene expression profiles of ovarian tissues of Yili goose at different periods,revealed the expression differences of mRNAs,lncRNAs,miRNAs and circRNAs in ovarian tissues of Yili goose at each period,initially explored the involvement of differentially expressed mRNAs,lncRNAs,miRNAs and circRNAs in ovarian development of Yili goose.We constructed a ceRNA regulatory network,and found that MSTRG.5970.28,a key node of the network,could act as a ceRNA for miR-133a-3p to regulate the expression of ANOS1 and affect the proliferation and apoptosis of goose granulosa cells.In this study,through the mining of key genes of ovarian development in Yili goose before and after egg laying and the functional validation of MSTRG.5970.28-miR-133a-3p-ANOS1,we laid the theoretical foundation to reveal the molecular mechanism of ovarian development in goose,and then to improve the egg laying performance of goose.