Fisheries College, Jimei University, Xiamen, China.
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, China.
Mol Reprod Dev. 2018 Jun;85(6):519-531. doi: 10.1002/mrd.22989. Epub 2018 May 7.
The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identification of 108 miRNAs (out of 1,417; 7.6%) that showed significant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem-loop real time quantitative PCR (RT-qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3'-untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.
miRNA 在生殖调控中的作用正受到越来越多的关注。本研究分别从中国明对虾卵巢和精巢文库中获得了 9643114 条和 15498999 条原始reads。经过数据挖掘，剩余用于分析的可比对小 RNA 序列分别为 4096464 条和 11737973 条。通过比对溞属 Daphnia pulex 和其他蟹类的参考基因组和表达序列标签（EST），共鉴定出 1417 个 miRNA。在这 1417 个 miRNA 的基础上，在雌雄文库中共同表达的有 514 个（36.3%），在雌文库中特异表达的有 336 个（23.7%），在雄文库中特异表达的有 567 个（40%）。文库测序数据的分析导致鉴定出 108 个 miRNA（1417 个中的 7.6%）在两个样本之间存在显著的差异表达。其中，13 个 miRNA 仅在精巢中表达，两个 miRNA 仅在卵巢中表达，93 个 miRNA 共同表达：57 个（61.3%）上调（卵巢/精巢），36 个（38.7%）下调（卵巢/精巢）。为了验证预测 miRNA 的表达模式，我们从 108 个差异表达 miRNA 中随机选择了 14 个候选 miRNA，在五个卵巢发育阶段进行茎环实时定量 PCR（RT-qPCR）检测。五个 miRNA 的表达模式与差异基因鉴定（IDEG6）分析中显示的几乎每个阶段都相似。上述五个 miRNA 被预测与已发表的中国明对虾基因的 3'-非翻译区匹配。其中 4 个 miRNA 对许多基因有调节作用，特别是与性腺发育相关的基因。
