| In order to explore the regulatory mechanism of chicken ovarian development, in this study, Jining Biri chicken ovarian tissue was sampled to identify the difference of expressed miRNAs and genes at different developmental stages by small RNA high-throughput sequencing and transcriptome high-throughput sequencing techniques, construct regulatory networks between the target gene and differential expressed miRNA, and verify quantitatively the accuracy of different expressed miRNAs and genes. The results were as follows:(1) Small RNA sequencingFour small RNA libraries were constructed at four developmental stages, including 60 days(S_A), 100 days(S_B), 140 days not starting production(S_C) and 140 days already starting production(S_D). In the present study, 8156581, 6926412, 8906171 and 10510049 clean readings were obtained from S_A to S_D by Hiseq high-throughput sequencing, accounting for 98.56%、 97.67%、98.72%、98.00% of the raw reads, respectively. The number of mapped known miRNA precursors were 3181902、2751006、3107415、1459649, accounting for 73.74%、72.56%、67.38%、and 30.12%, respectively. In addition, 35 novel miRNA were predicted. The analysis of different expressed miRNAs found that there were 14、22 and 74 miRNAs significantly differentially expressed in the S_B、S_C and S_D library, compared with S_A library, respectively(qvalue<0.01 and |log2(foldchange)|>1). Compared with S_B library, there were 8 and 78 different expressed miRNAs in S_C and S_D library. There were 51 significantly different expressed miRNAs between before and after starting laying eggs. Nine significantly different expressed miRNAs selected from Hiseq high-throughput sequencing were validated by qRT-PCR. The results indicated seven miRNAs of them were consistent with high-throughput sequencing, and the correlation coefficient of linear fit was 0.8878. The functional enrichment analysis of different expressed miRNA’s target genes showed that the target gene mainly enriched to the biological processes and cellular components GOterms; however, only SNARE interactions in vesicular transport KEGG pathway were significantly enriched.(2) Analysis of transcriptome sequencingIn our study, four transcriptome libraries were constructed at four different developmental stages, including 60 days(T_A), 100 days(T_B), 140 days not starting production(T_C) and 140 days already starting production(T_D). 62285300, 75609458, 87655702, 83921800 high-quality reads were obtained through Hiseq high-throughput sequencing, which accounting for 96.20%, 95.96%, 95.73%, 95.80% of the original reading, respectively. There were 53248104, 64794485, 74926383, 70651433 reads mapped to the reference genome in these four libraries, accounting for 85.49%, 85.7%, 85.48%, 84.19% of high-quality reading, respectively. The analysis of different expressed genes found that there were 51, 118 and 2,284 genes significantly differentially expressed in the T_B、T_C and T_D library, compared with T_A library, respectively(qvalue < 0.005 and |log2(FoldChange)| > 1. Compared with T_B library, 20 and 2,882 different expressed genes were found in T_C and T_D library. There were 2,579 different expressed genes between before and after starting laying eggs. Nine significantly different expressed genes selected from Hiseq high-throughput sequencing were validated by qRT-PCR. The results showed that seven genes were consistent with the high-throughput sequencing, and the correlation coefficient of linear fit was 0.8662. Functional enrichment analysis for all sets of different gene expressions found that there were 567 GOterm and seven KEGG pathways enriched which were significant.(3) Integration analysis between mi RNAs and genesTo further explore the regulation networks between different mi RNA expressions and genes, integration analysis were conducted between miRNA and mRNA from the same comparison combination. The results showed that 99 different miRNA expressions regulate 1,656 different gene expressions in all combinations. |
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