Study On Wilforine Insecticidal Action On The Sarcoplasmic Reticulum Calcium Atpase Of The Mythimma Separata Walker

Alkaloids are primary insecticidal compounds isolated from Tripterygium wilfordii Hook.f.,among which wilforine showed the hightest insecticidal activity against Mythimna separata,Plutella xylostella and Pieris rapae,etc.With the breakthrough in the research of biosynthesis of T.wilfordii alkaloids,industrialization production of the insecticidal alkaloids is possible.However,the insecticidal mechanism of wilforine has not been elucidated,which restricted its further development and application.Preliminary observations indicated that M.separata larvae were paralyzed by total alkaloids and wilforine,and muscle tissue was damaged.The target of wilforine maybe located in the muscle tissue of insects.In order to explore the insecticidal mechanism of wilforine,RNAi were used to reveal the function of target gene SERCA,respectively.Finally,the possible binding sites of wilforine were predicted by homologous modeling and molecular docking.Following results were obtained:1.Wilforine had a significant effect on the Ca²⁺-ATPase activity of both larvae and adults.In vivo,wilforine(LC₇₅)could activate the Ca²⁺-ATPase activity of both larvae and adults,and the specific enzyme activity at 36 h was 43.34%higher than that of the control.In vitro,wilforine could also activate Ca²⁺-ATPase in larva and adult at low concentration,but had no significant effect at high concentration in adult Ca²⁺-ATPase.2.Full-length sequence of M.separata SERCA gene（MsSERCA）was cloned by RT-PCR and RACE,and relative expression levels of MsSERCA in different developmental stages and tissues was tested by qRT-PCR.The full-length sequence of MsSERCA was 4139bp,and bioinformatics analysis showed that the ORF of SERCA was 2964 bp,which encoded987 amino acids.The MsSERCA was expressed at a high level in sixth-instar larvae and adults among different developmental stages,and at a high level in head and somatic muscle among different tissues.3.The function of SERCA gene in the insecticidal acivity of wilforine was verified by RNA interference.Four dsRNA（dsMsSERCA1、dsMsSERCA2、dsMsSERCA3and dsMsSERCA4）were synthesized,and the mRNA levels of MsSERCA in the treated larvae was significantly reduced to 54.5%、31.2%、61.2%and 41.8%（48 h）compared to that in corresponding controls.Though the weight of M.separata larvae after injection of dsMsSERCA reduced 52.70%,60.01%and 58.02%（48 h）,the mortality of M.separata larvae did not rise significantly.Wilforine at the dosage of LC₅₀0 showed weak insecticidal activity against M.separata pretreated with dsMsSERCA,with a decrease motality of 32.5%、30.00%、25.00%and 43.77%（48 h）.Thus,SERCA may be the action target of wilforine.4.The possible binding sites of wilforine on SERCA were predicted by homologous modeling and molecular docking.Wilforine could strongly combine with MsSERCA active Pocket composed of amino acid residue Leu-48、Val-49、Leu-240、Phe-243、Leu-247、Pro-295、Leu-298、Pro-299 and Ile-302 and forming a strong hydrophobic interaction.In conclusion,the insecticidal mechanism of wilforine is suggested as follows:wilforine enters the body of insects by feeding,then combines with SERCA in sarcoplasmic reticulum of muscle cells,inducing calcium ions in calcium store unnormally releasing,futher leading to calcium dyshomeostasis;calcium dyshomeostasis causes the damage of cellular structure,and even death or apoptosis,resulting on muscle tissues pathological change andfunction loss;poisoned insects reduce movement and are paralyzed.