Huifang Song1,2, Yunhe Fan1,2, Jianqin Zhang1,3, Anastasia Mw Cooper4, Kristopher Silver4, Daqi Li1,5, Tao Li1, Enbo Ma1, Kun Yan Zhu4, Jianzhen Zhang1. 1. Research Institute of Applied Biology, Shanxi University, Taiyuan, China. 2. College of Life Science, Shanxi University, Taiyuan, China. 3. Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan, China. 4. Department of Entomology, Kansas State University, Manhattan, KS, USA. 5. Institute of Plant Protection, Shanxi Academy of Agricultural Science, Taiyuan, China.
Abstract
BACKGROUND: The efficiency of RNA interference (RNAi) varies considerably among different insect species, and there is growing evidence to suggest that degradation of double-stranded (dsRNA) prior to uptake is an important factor that limits the efficiency of RNAi in insects. In Locusta migratoria, RNAi is highly efficient when dsRNA is delivered by injection, but not by feeding. However, detailed mechanisms causing such differential RNAi efficiency are still elusive. RESULTS: We identified and characterized the full-length complementary DNAs (cDNAs) of two new dsRNA nuclease (dsRNase) genes from L. migratoria, which were named LmdsRNase1 and LmdsRNase4. Transcript analyses revealed that LmdsRNase1 and LmdsRNase4 were highly expressed in hemolymph with relatively lower expression in other tested tissues. Our study using heterologously expressed LmdsRNase1 and LmdsRNase4 fusion proteins showed that LmdsRNase1 can degrade dsRNA rapidly at an optimal pH of 5, whereas LmdsRNase4 had no activity at any of the pH values examined. In comparing the substrate specificity of the four LmdsRNases, we found that only LmdsRNase1 and LmdsRNase2 digested dsRNA; however, our experiments suggested that the physiological pH of hemolymph (7.0) suppresses LmdsRNase1 activity permitting significant dsRNA stability in this tissue. Conversely, the physiological pH of midgut juice (6.8) is ideal for LmdsRNase2 activity, resulting in degradation of dsRNA in midgut. CONCLUSION: The physiological pH of different insect tissues or compartments can significantly alter the stability of dsRNA by influencing LmdsRNase activity in L. migratoria. Thus, new strategies to overcome such obstacles are expected to help implement RNAi-based technologies for insect pest management.
BACKGROUND: The efficiency of RNA interference (RNAi) varies considerably among different insect species, and there is growing evidence to suggest that degradation of double-stranded (dsRNA) prior to uptake is an important factor that limits the efficiency of RNAi in insects. In Locusta migratoria, RNAi is highly efficient when dsRNA is delivered by injection, but not by feeding. However, detailed mechanisms causing such differential RNAi efficiency are still elusive. RESULTS: We identified and characterized the full-length complementary DNAs (cDNAs) of two new dsRNA nuclease (dsRNase) genes from L. migratoria, which were named LmdsRNase1 and LmdsRNase4. Transcript analyses revealed that LmdsRNase1 and LmdsRNase4 were highly expressed in hemolymph with relatively lower expression in other tested tissues. Our study using heterologously expressed LmdsRNase1 and LmdsRNase4 fusion proteins showed that LmdsRNase1 can degrade dsRNA rapidly at an optimal pH of 5, whereas LmdsRNase4 had no activity at any of the pH values examined. In comparing the substrate specificity of the four LmdsRNases, we found that only LmdsRNase1 and LmdsRNase2 digested dsRNA; however, our experiments suggested that the physiological pH of hemolymph (7.0) suppresses LmdsRNase1 activity permitting significant dsRNA stability in this tissue. Conversely, the physiological pH of midgut juice (6.8) is ideal for LmdsRNase2 activity, resulting in degradation of dsRNA in midgut. CONCLUSION: The physiological pH of different insect tissues or compartments can significantly alter the stability of dsRNA by influencing LmdsRNase activity in L. migratoria. Thus, new strategies to overcome such obstacles are expected to help implement RNAi-based technologies for insect pest management.
Authors: Nina Hahn; Luca Büschgens; Nicola Schwedhelm-Domeyer; Sarah Bank; Bart R H Geurten; Pia Neugebauer; Bita Massih; Martin C Göpfert; Ralf Heinrich Journal: Front Mol Neurosci Date: 2019-10-11 Impact factor: 5.639
Authors: Fabricio Barbosa Monteiro Arraes; Diogo Martins-de-Sa; Daniel D Noriega Vasquez; Bruno Paes Melo; Muhammad Faheem; Leonardo Lima Pepino de Macedo; Carolina Vianna Morgante; Joao Alexandre R G Barbosa; Roberto Coiti Togawa; Valdeir Junio Vaz Moreira; Etienne G J Danchin; Maria Fatima Grossi-de-Sa Journal: RNA Biol Date: 2020-12-31 Impact factor: 4.652
Authors: Jiaxin Lei; Yongan Tan; Fabian List; Robert Puckett; Aaron M Tarone; Edward L Vargo; Keyan Zhu-Salzman Journal: Front Physiol Date: 2022-01-28 Impact factor: 4.566