Peng Yingchuan, Wang Kangxu, Fu Wenxi, Sheng Chengwang, Han Zhaojun
The Agricultural Ministry Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
Front Physiol. 2018 May 28;9:624. doi: 10.3389/fphys.2018.00624. eCollection 2018.
Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (, and ) from different orders were characterized using a modified assay method. The results revealed that all insect dsRNA degrading nucleases tested showed high activity in alkaline environments at optimal Mg concentrations and elevated temperatures. We also found that enzymes from different insects varied in terms of their optimal reaction conditions and kinetic parameters. Whole body enzyme activity differed dramatically between insect species, although enzymes with higher substrate affinities (lower ) were usually balanced by a smaller to maintain a proper level of degradative capacity. Furthermore, enzyme activities varied significantly between the four tested tissues (whole body, gut, hemolymph, and carcass) of the insect species. All the insects tested showed several hundred-fold higher dsRNA degrading activity in their gut than in other tissues. Reaction environment analysis demonstrated that physiological conditions in the prepared gut fluid and serum of different insects were not necessarily optimal for dsRNA degrading nuclease activity. Our data describe the biochemical characteristics and tissue distributions of dsRNA degrading activities in various insects, not only explaining why oral delivery of dsRNA often produces lower RNAi effects than injection of dsRNA, but also suggesting that dsRNA-degrading activities are regulated by physiological conditions. These results allow for a better understanding of the properties of dsRNA degrading nucleases, and will aid in the development of successful RNAi strategies in insects.
双链RNA（dsRNA）降解核酸酶负责dsRNA分子的快速降解，因此导致了昆虫物种间RNA干扰（RNAi）效率的差异。在此，我们使用改良的检测方法，对来自不同目的四种昆虫（ 、 和 ）中dsRNA降解核酸酶的生化特性和组织特异性活性进行了表征。结果显示，所有测试的昆虫dsRNA降解核酸酶在碱性环境中、最佳镁离子浓度及升高的温度下均表现出高活性。我们还发现，来自不同昆虫的酶在最佳反应条件和动力学参数方面存在差异。尽管具有较高底物亲和力（较低 ）的酶通常会通过较小的 来平衡，以维持适当的降解能力水平，但昆虫物种间的全身酶活性仍存在显著差异。此外，在所测试的昆虫的四种组织（全身、肠道、血淋巴和胴体）中，酶活性也存在显著差异。所有测试的昆虫在其肠道中的dsRNA降解活性均比其他组织高出数百倍。反应环境分析表明，不同昆虫的肠道液和血清中的生理条件不一定是dsRNA降解核酸酶活性的最佳条件。我们的数据描述了各种昆虫中dsRNA降解活性的生化特征和组织分布，不仅解释了为什么口服dsRNA通常比注射dsRNA产生更低的RNAi效应，还表明dsRNA降解活性受生理条件调控。这些结果有助于更好地理解dsRNA降解核酸酶的特性，并将有助于开发成功的昆虫RNAi策略。
