| The egg production traits of chicken are an important economic trait,which mainly depends on the degree of follicle development,the development of follicles is regulated by a complex and highly coordinated comprehensive system.This complex system is not only a product of environmental factors but a product regulated by hormones secreted by the hypothalamic-pituitary-gonadal(HPG)axis and other local ovarian factors.The aim of this study was to identify key candidate genes closely related to egg-laying traits by use of mRNA transcriptome analysis of the ovarian follicles at different developmental stages.The results of this research not only provide an omics basis for analyzing the molecular mechanism of egg-laying traits,but also provide important references for molecular breeding and genetic improvement of local chicken with egg-laying traits as the target,which has very important academic value and application prospect.In this study,to identify genes associated with egg production,high-yielding,and low-yielding chickens were used.We selected 21 weeks old Roman brown laying hens,with high yield and Jilin black chicken,an indigenous low yield breed as the experimental material.The ovarian follicle tissues were collected at different developmental stages with diameters of3.5-5.5 mm(LWF),6.0-8.0 mm(SYF),and 8.5-10.5 mm(LYF).Transcriptome sequencing technology was used to screen and analyze the differentially expressed genes(DEGs)in different follicular tissues between high and low laying hens.The results showed that 236,544,and 386 DEGs were identified in LWF,SYF,and LYF follicles,respectively.Among them,47 were DEGs co-expressed in LWF and SYF follicles,and 68 were DEGs co-expressed in SYF and LYF follicles.There were 54 DEGs co-expressed in LWF and LYF follicles,and 13 DEGs were co-expressed in LWF,SYF,and LYF follicles.22 differentially expressed genes were randomly selected for verification by RT-qPCR,and later focusing the molecular biological function analysis of two key differentially expressed genes from LWF,SYF,and LYF co-expression contained NDUFAB1 and GABRA1.To reveal the molecular role of NDUFAB1 and GABRA1 genes in regulating follicular development.The main research contents and experimental results are as follows:1.Screening and validation of key genes for egg production traits in chickens based on follicular mRNA transcriptome analysis(1)Analysis of follicular mRNA transcriptome sequencing data at three different developmental stagesIn this experiment,transcriptome sequencing was performed on the follicles(LWF,SYF,and LYF)at three different developmental stages for the high-yield LB and low-yield JB hens.A total of 18 c DNA libraries were constructed and the raw reads of each library were 41.61 million.The quality scores of Q20 and Q30 were greater than 97.35% and 93.87%,respectively.The clean reads obtained after filtration were greater than 41.41 million.Total mapped sequences(reads)possessed 85.11 to 86.49% of the clean reads.The total reads mapped to genes(MGs)shared 81.69 to 83.29%.(2)Screening of Differentially Expressed Genes in Follicles with Different Egg Production LevelsScreening of differentially expressed genes from different follicular tissues of LB and JB hens showed that there were 236 differentially expressed genes in LWF follicles,including 137up-regulated genes and 99 down-regulated genes.There were 544 differentially expressed genes in SYF follicles including 447 up-regulated genes and 97 down-regulated genes.386 differentially expressed genes were revealed in LYF follicles,including 229 up-regulated genes and 157 down-regulated genes.(3)The GO and KEGG functional enrichment analysis of differentially expressed genes related to egg production traitsTo enrich the differentially expressed genes closely related to egg production traits,GO functional annotation and KEGG signaling pathway analysis were conducted.Results showed that in LWF follicles,203 DEGs have GO annotations,which were significantly enriched in 162 biological process(BP)term,179 cell component(CC)term and 159 molecular function(MF)term.In SYF follicles,486 DEGs obtained GO functional annotations,which were significantly enriched in 404 BP term,444 CC term and 375 MF term.In LYF follicles,386 DEGs obtained GO functional annotations and were significantly enriched in 246 BP term,301 CC term and241 MF term.In the KEGG enrichment analysis,10 signal pathways were enriched and 44 DEGSs were detected in LWF follicles.7 signal pathways were enriched and 87 DEGSs were detected in SYF follicles.11 signal pathways were enriched and 71 DEGSs were detected in LYF follicles.(4)Screening of key candidate genes related to egg production traits between follicles at different levels of hensThe screening results of key candidate genes related to egg production traits among different follicles showed that 47 DEGs were co-expressed in the LWF and SYF follicular,68 DEGs were co-expressed in the SYF and LYF follicular,54 DEGs were co-expressed in the LWF and LYF,and 13 DEGs were co-expressed in the three follicular groups of LWF,SYF and LYF.(5)Validation of key candidate genes for egg production traits in hensUsing RT-qPCR technology,22 DEGs co-expressed in each group were randomly selected.These included P2RX1,CAB39 L,BLK,CSMD3,GPR65,ADRB2,CSMD1,PLPP4,ATF3,PRLL,STMN3,RORB,PIK3R1,PERP1,ACSBG1,MRTO4,CDKN1 A,EDA2R,GABRA1,NDUFAB1,NCAM2,and LOC424014.The results showed that 17 of the 22 genes(P2RX1,CAB39 L,CSMD1,CSMD3,PRLL,NDUFAB1,NCAM2,etc.)had different mRNA expression levels from theRNA-Seq sequencing results,but their expression trends were consistent,which proved the reliability and accuracy ofRNA-Seq sequencing results.The differentially expressed genes co-expressed in the follicles of LWF,SYF,LYF,NDUFAB1,and GABRA1,were further verified by functional analysis.2.Molecular role of NDUFAB1 gene in promoting the development of chicken follicles(1)NDUFAB1 gene stimulates the synthesis and secretion of estradiol(E2)and progesterone(P4)hormonesAfter using shRNA interference to silence the NDUFAB1 gene,RT-qPCR and Western Blot were used to detect St AR and CYP11A1 gene expression levels in granulosa cells of ovarian follicles.The results showed that the mRNA and protein expression levels of St AR and CYP11A1 genes were significantly down-regulated(P<0.05),which indicated that the NDUFAB1 gene can promote the differentiation of granulosa cells and stimulate the synthesis and secretion of the steroid hormones estradiol(E2)and progesterone(P4)Features.(2)NDUFAB1 gene promotes follicular development and inhibits follicular atresiaAfter using shRNA interference to knock down the NDUFAB1 gene,RT-qPCR and Western Blot were used to detect the expression levels of the CCND1,Caspase-3,and Bcl-2 gene in the granulosa cells.The results showed that CCND1 and Bcl-2 gene mRNA and protein expression levels were significantly down-regulated(P<0.05),and Caspase-3 gene mRNA and protein expression levels were significantly up-regulated(P<0.01).These results indicate that NDUFAB1 gene can promote the proliferation of granulosa cells,inhibit the apoptosis of granulosa cells,and has a potential molecular role in promoting follicular development and inhibiting follicular atresia.EDU kits and flow cytometry were used to detect the proliferation and apoptosis of granulosa cells at the cellular level in samples with silenced NDUFAB1 gene.The results showed that the NDUFAB1 gene can significantly promote granular cell apoptosis and inhibit granular cell proliferation(P<0.01).These results indicate that NDUFAB1 gene can promote follicular development and inhibit follicular atresia.Meanwhile,NDUFAB1 gene has a potential biological function of promoting egg laying traits,and can be used as a candidate molecular marker gene for egg-laying traits.3.Molecular role of GABRA1 gene in inhibiting the development of chicken follicles(1)GABRA1 gene inhibits the synthesis and secretion of E2 and P4 hormonesAfter using shRNA interference to knockdown GABRA1 gene,RT-qPCR and Western Blot technology were employed to detect St AR and CYP11A1 gene expression levels in granulosa cells.The results showed that St AR and CYP11A1 mRNA and protein expression levels were significantly up-regulated(P<0.01),which indicated that GABRA1 gene has the function of inhibiting granular cell differentiation and steroid hormone E2 and P4 hormone synthesis and secretion.(2)GABRA1 gene inhibits the growth and development of folliclesAfter Silencing GABRA1 gene using shRNA,the expression levels of CCND1,Caspase-3and Bcl-2 in granulosa cells was checked using RT-qPCR and Western Blot technology to detect.The results showed that CCND1 and Bcl-2 gene mRNA and protein expression levels were significantly up-regulated(P<0.01),while Caspase-3 gene mRNA and protein expression levels were significantly down-regulated(P<0.01),These results indicate that GABRA1 gene has a potential molecular role in inhibiting granulosa cell proliferation and promoting granulosa cell apoptosis.EDU kits and flow cytometry were used to detect the proliferation and apoptosis of granulosa cells at the cellular level.The resulted showed that it can significant promotion of the proliferation of granular cells and inhibit the apoptosis of granular cells(P<0.01).These results indicate that GABRA1 gene has a potential molecular role in inhibiting granulosa cell proliferation and promoting granulosa cell apoptosis.In summary,for the first time in this study,RNA-seq transcriptome analysis was performed on the follicular tissues of three different developmental stages(LWF,SYF,and LYF)in highand low-yield hens.A total of 1166 DEGs related to egg-laying traits were screened,among which 143 candidate genes were closely related.two key candidate genes co-expressed from LWF,SYF,and LYF follicles,NDUFAB1 and GABRA1 were further verified by functional analysis.The study aimed to verify their molecular regulatory effects on the development of chicken follicles.The results not only to lay an omics foundation for analyzing the molecular mechanism of laying traits but also to provide a reference for molecular breeding or genetic improvement of local chickens targeting egg-laying traits. |
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