不同发育阶段牦牛睾丸组织miRNA的分析及鉴定

Analysis and identification of miRNA in yak testis at different developmental stages

选取胎儿期(4~5月龄)、犊牛期(1岁)、性成熟期(3岁)的健康雄性牦牛各2头,分离其睾丸组织,利用Illumina平台对各样本miRNA进行测序,其中同一年龄段的2个样本被视为生物学重复;分析各阶段样本的miRNA数量、碱基偏好性和表达水平,对新miRNA进行预测,对显著差异表达的miRNA的靶基因进行gene ontology(GO)富集分析。结果显示,胎儿期miRNA占smallRNA(sRNA)的比例显著多于犊牛期和性成熟期(P<0.05)。共发现新miRNA成熟体77个,新miRNA前体共80个,已知miRNA的首位具有尿嘧啶(U)碱基偏好性,新miRNA的首位具有胞嘧啶(C)碱基偏好性。差异表达分析发现胎儿期与犊牛期间显著差异表达miRNA共224个,犊牛期与性成熟期间显著差异表达miRNA共36个,胎儿期与性成熟期间显著差异表达miRNA共239个。GO分析发现显著差异表达的miRNA其靶基因的数目在生物学过程排首位的是代谢过程,并从显著差异表达的miRNA的靶基因中筛选出了与精子发生及与精子功能相关的靶基因20个。结果表明,牦牛睾丸发育过程中,胎儿期的miRNA的数量及部分miRNA的表达水平与胚后阶段相比有明显的差异;新miRNA与已知miRNA相比可能存在未知加工机制;不同发育阶段牦牛睾丸之间部分显著差异表达的miRNA可能通过调控其靶基因的表达进而参与精子发生。

A total of six healthy male yaks(two in each group) in the fetal(4-5 months old),calves(one year old),and sexual maturity(3 years old) stages were chosen and testes were separated.miRNAs from each sample were sequenced using Illumina platform and two samples from yaks with the same age were considered as biological duplications.The number, base preference and expression level of miRNA in each sample were analyzed.Novel miRNAs were predicted and target genes of significantly differential expressed miRNAs were analyzed by GO analysis(gene ontology).The results showed that the proportion of miRNA to small RNA(sRNA) in fetal stage was significantly higher than that in calves and sexual maturity stages(P<0.05).A total of 77 novel miRNA mature bodies and 80 novel miRNA precursors were identified.The first position of miRNA has uracil(U)base preference,the 5′end of novel miRNA had cytosine(C)base preference.Expression analysis showed that 224 miRNAs were significantly differential expressed in the fetal and calves stages,36 miRNAs were significantly differential expressed in the calves and sexual maturity stages,and 239 miRNAs were significantly differential expressed in the fetal and sexual maturity stages.GO analysis of differentially expressed miRNAs target genes showed that metabolic process was the top enriched biological process and 20 target genes were found to be associated with spermatogenesis and sperm function.The results suggest that during the development of yak testes,the number of miRNAs and the expression levels of some miRNAs in the fetal stage are significantly different from those in the post-embryonic stage.Compared with known miRNAs,novel miRNAs may have unknown processing mechanisms.Some miRNAs that are significantly differential expressed in yak testes at different developmental stages may participate in spermatogenesis by regulating the expression of their target genes.