A new strategy for identification of highly conserved microRNAs in non-model insect, Spodoptera litura.
A new strategy for identification of highly conserved microRNAs in non-model insect, Spodoptera litura.
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鉴定非模式昆虫斜纹夜蛾中高度保守的 microRNA 的新策略
DOI：
10.3390/ijms13010612
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发表时间：
2012
影响因子：
5.6
通讯作者：
Zhong G
中科院分区：
文献类型：
--
作者：
Gao L;Zuo H;Liu K;Li H;Zhong G
小分子非编码RNA（microRNA，miRNAs）是基因表达的重要调控因子，其中许多在进化上是保守的。茎环RT-PCR作为一种敏感的方法能否用于克隆非模式昆虫中保守的miRNAs尚缺乏信息。本研究采用茎环RT-PCR技术从斜纹夜蛾中克隆了sli-miR-14、sli-miR-2a和sli-bantam三种miRNAs。设计了两组引物，其中一组表现特别好，并证明稳定。两个高度保守的miRNAs sli-miR-14和sli-miR-2a的序列与果蝇中的相同。为了验证该策略的可靠性，在S. litura为代表，与D. sli-miR-14和sli-miR-2a的成熟序列与茎环RT-PCR结果的同源性均为100%。此外，通过实时定量PCR研究这些miRNA的表达模式。Sli-miR-14和sli-miR-2a在模式昆虫中的表达模式与模式昆虫相似，表明茎环RT-PCR技术可用于非模式昆虫中高度保守的miRNAs的鉴定。这些结果为研究高度保守的miRNAs，特别是非模式昆虫中的关键miRNAs的结构和功能提供了一种简单有效的方法。
The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam, were cloned from Spodoptera litura by stem-loop RT-PCR. Two groups of primers were designed, and one of them performed especially well and proved stable. The sequences of two highly conserved miRNAs, sli-miR-14 and sli-miR-2a were identical to those in Drosophila melanogaster. To validate the reliability of this strategy, pre-miR-14 and pre-miR-2a in S. litura as representatives were given as well; this shared high homology with those in D. melanogaster and Bombyx mori, and both mature sequences of sli-miR-14 and sli-miR-2a in their precursors shared 100% identity to the results shown by stem-loop RT-PCR. Moreover, expression patterns of these miRNAs were investigated by real-time quantitative PCR. Sli-miR-14 and sli-miR-2a could be detected successfully and their expression patterns showed similar characteristics with those in model insects, further suggesting stem-loop RT-PCR technology can be used for identification of highly conserved miRNAs in non-model insects. These results provide a simplified and efficient strategy for studying the structure and function of highly conserved miRNAs, especially some critical miRNAs in non-model insects.
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56.9
作者：
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通讯作者：
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影响因子：
3.8
作者：
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