Bai Man, Sun Limin, Jia Chao, Li Jiarong, Han Yue, Liu Hang, Chen Yang, Jiang Huaizhi
College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China.
College of Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China.
G3 (Bethesda). 2019 Feb 7;9(2):523-533. doi: 10.1534/g3.118.200947.
Small Tail Han Sheep is a highly valued local breed in China because of their precocity, perennial estrus, and high fecundity. The average annual lambing rate of ewes is as high as 180-270%, the semen of ram has characteristics of high yield, high density, and good motility. To reveal the key miRNAs and miRNA-targets underlying testis development and spermatogenesis in male Small Tail Han Sheep, integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old testes was performed by RNA-seq technology and bioinformatics methods. The results showed that total of 153 known sheep miRNAs and 2712 novel miRNAs were obtained in 2-,6 - and 12-month-old Small Tail Han Sheep testes; 5, 1, and 4 differentially expressed (DE) known sheep miRNAs, and 132, 105, and 24 DE novel miRNAs were identified in 2- 6-, 6- 12-, and 2- 12-month-old testes, respectively. We combined miRNA results of this study and the mRNA results obtained in our previous study to predict the target mRNAs of DE known sheep miRNAs; 131, 10, and 15 target mRNAs of DE known sheep miRNAs and 76, 1, and 11 DE miRNA-targets were identified in the three groups, respectively. GO and KEGG analyses showed that: in 2- 6-month-olds, the target genes of DE known sheep miRNAs were involved in 100 biological processes and 11 signaling pathways; in 6- 12-month-olds, the target genes of DE known sheep miRNAs were involved in 4 biological processes; and in 2- 12-month-olds, the target genes of DE known sheep miRNAs were involved in 17 biological processes and 4 signaling pathways. Three miR-target regulatory networks were constructed based on these DE miRNA-targets. The key miRNA-Targets involved in testis development and spermatogenesis were screened. 6 known sheep miRNAs and 6 novel miRNAs were selected to validate the accuracy of miRNA sequencing data by qRT-PCR. The binding sites of oar-miR-379-5p with was validated by a dual luciferase reporter gene detection system.
小尾寒羊是我国备受重视的地方品种，因其早熟、常年发情和高繁殖力。母羊的年平均产羔率高达180 - 270%，公羊精液具有产量高、密度高和活力好的特点。为揭示雄性小尾寒羊睾丸发育和精子发生过程中的关键miRNA及其靶标，采用RNA-seq技术和生物信息学方法，对2月龄、6月龄和12月龄睾丸中的miRNA和mRNA表达谱进行了综合分析。结果表明，在2月龄、6月龄和12月龄的小尾寒羊睾丸中，共获得153个已知绵羊miRNA和2712个新miRNA；在2 - 6月龄、6 - 12月龄和2 - 12月龄的睾丸中，分别鉴定出5个、1个和4个差异表达（DE）的已知绵羊miRNA，以及132个、105个和24个DE新miRNA。我们将本研究的miRNA结果与之前研究获得的mRNA结果相结合，预测DE已知绵羊miRNA的靶标mRNA；在三组中分别鉴定出131个、10个和15个DE已知绵羊miRNA的靶标mRNA，以及76个、1个和11个DE miRNA-靶标。GO和KEGG分析表明：在2 - 6月龄中，DE已知绵羊miRNA的靶基因参与100个生物学过程和11条信号通路；在6 - 12月龄中，DE已知绵羊miRNA的靶基因参与4个生物学过程；在2 - 12月龄中，DE已知绵羊miRNA的靶基因参与17个生物学过程和4条信号通路。基于这些DE miRNA-靶标构建了三个miR-靶标调控网络。筛选出参与睾丸发育和精子发生的关键miRNA-靶标。选择6个已知绵羊miRNA和6个新miRNA，通过qRT-PCR验证miRNA测序数据的准确性。通过双荧光素酶报告基因检测系统验证了oar-miR-379-5p与……的结合位点。
