The Queensland Brain Institute, The University of Queensland, St. Lucia, Queensland, Australia.
PLoS One. 2011 Apr 28;6(4):e19350. doi: 10.1371/journal.pone.0019350.
microRNAs (miRNAs) have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum) have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs.
METHODOLOGY/PRINCIPAL FINDINGS: Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates.
CONCLUSIONS/SIGNIFICANCE: Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.
microRNAs（miRNAs）已被报道为真核细胞中转录后水平的关键调节因子。在昆虫中，大多数研究都集中在完全变态昆虫上，而最近只有两种半变态昆虫（Locusta migratoria 和 Acyrthosiphon pisum）鉴定了它们的 miRNAs。因此，研究进化上基本的半变态昆虫德国小蠊 Blattella germanica 的 miRNAs 可能为 miRNAs 的结构和功能进化提供有价值的见解。
方法/主要发现：从小蠊 B. germanica 的整个身体和成年卵巢中构建了小 RNA 文库。高通量 Solexa 测序分别产生了约 1100 万和 800 万个读数。生物信息学分析鉴定了 38 个已知的 miRNAs 以及 11 个已知的 miRNA*。我们还发现了 70 个在其他昆虫中保守的 miRNA 候选物和 170 个专门针对 B. germanica 的候选物。Solexa 数据与实时定量 PCR 之间的正相关表明，读数数量可以用作定量方法。通过 PCR 和测序鉴定了 5 个新的 miRNA 前体，并进行了验证。通过降低 dicer-1 RNAi 敲低个体中其表达水平，已知的 miRNAs 和新的候选物也得到了验证。两个文库的比较表明，全身若虫比卵巢含有更多的已知 miRNAs，而成年卵巢则富含新的 miRNA 候选物。
结论/意义：本研究在基本半变态昆虫 B. germanica 中鉴定了许多已知的 miRNAs 和新的 miRNA 候选物，大多数特定序列都在卵巢中发现。深度测序数据反映了 miRNA 的丰度，并且 dicer-1 RNAi 测定被证明是验证新 miRNAs 的可靠方法。
