Gao Lu, Wang Yanli, Abbas Mureed, Zhang Tingting, Ma Enbo, Xing Shuping, Zhu Kun Yan, Zhang Jianzhen
Institute of Applied Biology, Shanxi University, Taiyuan 030006, China.
College of Life Science, Shanxi University, Taiyuan 030006, China.
Insects. 2021 Sep 10;12(9):812. doi: 10.3390/insects12090812.
Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in . However, the involvement of R2D2 in the siRNA pathway of is still unknown. In the present study, we identified an gene from the transcriptome of . It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of indicated that its transcript level was stable in third-instar nymphs of , whereas the tissue-dependent expression profile exhibited high levels of expression of in the testis and ovary. When was silenced by RNAi, the RNAi efficiency against as a marker gene was significantly diminished, as indicated by the 37.7% increased transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of .
小干扰RNA（siRNA）是长度为21至23个核苷酸（nt）的非编码RNA，几乎存在于所有真核生物中。siRNA的形成是一种由关键蛋白质介导的高度保守的转录后基因沉默机制，这些关键蛋白质包括Dicer2、Argonaute2（Ago2）和R2D2。R2D2已被鉴定为双链RNA（dsRNA）结合蛋白，并被报道为[具体物种1]中siRNA途径的一个组成部分。然而，R2D2在[具体物种2]的siRNA途径中的作用仍不清楚。在本研究中，我们从[具体物种2]的转录组中鉴定出一个[具体物种2]的R2D2基因。它由一个954 bp的开放阅读框组成，编码一个318个氨基酸残基的蛋白质。进一步的序列分析表明，LmR2D2在N端具有两个串联的双链RNA结合结构域（dsRBD）。对[具体物种2]的发育表达谱分析表明，其转录水平在[具体物种2]的三龄若虫中稳定，而组织依赖性表达谱显示[具体物种2]在睾丸和卵巢中表达水平较高。当通过RNA干扰使[具体物种2]沉默时，作为标记基因的[具体物种2]的RNA干扰效率显著降低，[具体物种2]转录水平增加了37.7%表明了这一点。此外，利用原核表达系统获得了LmR2D2上清蛋白。通过将LmR2D2蛋白与生物素-dsRNA孵育，我们发现LmR2D2在体外可以与dsRNA结合，这支持了我们的结论，即LmR2D2在[具体物种[具体物种2]的siRNA途径中起重要作用。
