Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (12): 3720-3730.DOI: 10.1016/j.jia.2023.05.019

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丝氨酸蛋白酶抑制剂LmSPN2和LmSPN3通过Toll信号通路共同调控飞蝗胚胎滞育

  

  • 收稿日期:2023-01-19 接受日期:2023-04-12 出版日期:2023-12-20 发布日期:2023-12-08

Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis (Meyen) via the Toll pathway

FENG Shi-qian*, ZHANG Neng*, CHEN Jun*, ZHANG Dao-gang, ZHU Kai-hui, CAI Ni, TU Xiong-bing#, ZHANG Ze-hua   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

  • Received:2023-01-19 Accepted:2023-04-12 Online:2023-12-20 Published:2023-12-08
  • About author:#Correspondence TU Xiong-bing, Tel: +86-10-82109569, E-mail: tuxiongbing@caas.cn * These authors contributed equally to this study.
  • Supported by:

    This work was supported by the National Key R&D Program of China (2022YFD1400500), the China Agriculture Research System of MOF and MARA (CARS-34-07), the Public-interest Scientific Institution Basal Research Fund, China (Y2022GH12) and the Central Public-interest Scientific Institution Basal Research Fund, China (S2021XM22 and S2022XM21).

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摘要：

飞蝗雌成虫可以感知季节性光周期的变化，从而诱导胚胎滞育，并以此作为越冬的关键策略。丝氨酸蛋白酶抑制剂基因（SPNs）在滞育诱导中发挥重要作用，但只有少数SPNs基因得到了功能验证。LmSPN2在滞育和非滞育卵之间差异表达，然而其生物学功能仍需进一步探究。因此，我们首先对LmSPN2进行了RNAi敲降，结果表明滞育率显著降低了29.7％。使用酵母双杂交、免疫共沉淀和pull-down等技术，我们验证了LmSPN2与LmSPN3之间存在相互作用，且作用位点为LmSPN2的第331位谷氨酸。LmSPN3的RNAi敲降会使滞育率增加14.6％，表明LmSPN2和LmSPN3在滞育调控上具有相反的功能。两个SPN基因的双重敲降导致滞育率降低了26.4％，表明LmSPN2是主导的调节信号。此外，我们发现敲降LmSPN2后，四个Toll通路基因（easter，spätzle，pelle和dorsal）显著上调，而敲降LmSPN3后则下调，因此我们推测这两个SPN基因通过Toll通路调节滞育过程。综上，LmSPN2对蝗虫卵滞育具有正调控作用，而LmSPN3作用相反，二者通过LmSPN2的第331位谷氨酸进行互作，并通过Toll通路影响飞蝗滞育。这种滞育调控机制扩展了我们对昆虫发育调控的理解，并为研发新的蝗虫防控策略的提供了新方向。

Abstract:

Female adults of the migratory locust, Locusta migratoria manilensis (Meyen), can sense seasonal photoperiod changes, which induces embryonic diapause as a key strategy to overwinter.  Serine protease inhibitor genes (SPNs) were thought to play key roles during diapause, while few SPNs were functionally characterized.  LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs; however, its biological function remained to be explored.  So, we conducted RNAi knockdown of LmSPN2, resulting in a significant decrease of the egg diapause rate by 29.7%.  Using yeast two-hybrid assays, co-immunoprecipitation, and pull-down methods, we found an interaction between LmSPN2 and LmSPN3, which was proved to be mediated by a glutamate (E331) binding site of LmSPN2.  RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%, indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation.  Double knockdown of two SPN genes resulted in a 26.4% reduction in diapause rate, indicating that LmSPN2 was the dominant regulatory signal.  Moreover, we found four Toll pathway genes (easter, spätzle, pelle, and dorsal) upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3.  Therefore, we speculate that two SPNs regulate diapause through the Toll pathway.  Our results indicated that LmSPN2 positively regulates locust egg entry into diapause, while LmSPN3 is a negative regulator of embryonic commitment to diapause.  Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway.  This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

Key words: Locusta migratoria ,  insect diapause regulation ,  Toll pathway ,  protein interaction ,  serine protease inhibitor