Study On The Aphicidal Mechanism Of Matrine On Pea Aphid

Matrine widely exists in Sophora alopecuroides,Sophora flavescens,Sophora tonkinensis and other leguminous plants.It has a good toxic effect on a variety of aphids and other pests,and is the most registered botanical pesticide product in China.However,its insecticidal mechanism is not clear,which limits the further development and scientific application of this excellent insecticide.Previous studies have shown that matrine may act on the neuro-muscular system of insects and is an inhibitory nerve agent.Based on this,the comparative toxicology method was adopted in this study.Sophocarpine,an analog of matrine,and avermectin,were used as the control agent,and the pea aphid（Acyrthosiphon pisum）was used as the test insect.The action site of matrine was preliminary determined by the symptomatology,physiological and biochemical effects.Further transcriptome sequencing（RNA-seq）technology,RNAi technology,in vitro expression of insect baculovirus expression system,MQAE probe method,homology modeling and molecular docking technology were carried out step by step to identify to preliminarily clarify the insecticidal mechanism of matrine.The main findings are as follows:1.The toxicity of matrine to pea aphid was determined by topical application,and the poisoning symptoms were observed.The results showed that the contact LD₅₀of matrine,sophocarpine and avermectin to pea aphid for 48 hours were 68.88 ng/insect,83.44 ng/insect and 0.72 ng/insect,respectively.The poisoning symptoms of pea aphids after matrine treatment were spasm,paralysis,and death.The toxic symptoms caused by sophocarpine were similar to those of matrine.While after avermectin treatment,the test aphids were in a state of paralysis for a long time,could not feed or move,and finally died.2.The effects of matrine on the physiological and biochemical indexes of pea aphid were detected by various methods.The results showed that matrine and sophocarpine could activate glutamate decarboxylase.Matrine and sophocarpine inhibited alanine aminotransferase in a dose-dependent and time-dependent manner in vivo and in vitro;The content of glutamate in pea aphid treated with two alkaloids increased significantly with time,which was 1.5-2.0 times compared to the control group,and the content of GABA increased by 1.0-1.3 times.Except avermectin,matrine and sophocarpine could inhibit Na⁺,K⁺-ATPase and Ca²⁺,Mg²⁺-ATPase.The three treatments had no significant effect on the contents of acetylcholinesterase or acetylcholine.3.The effect of matrine on the transcription level of pea aphid was analyzed by transcriptome sequencing technology.The results showed that:（1）1025 differential genes were detected,including 295 up-regulated genes and 730 down-regulated genes.Among them,the genes related to glutamate molecular signaling pathway,including glutamate gated chloride channel,kainic acid glutamate receptor,glutamate type 1 receptor and delta type 1 glutamate receptor,have changed significantly.（2）The results of real-time fluorescent quantitative PCR were highly consistent with those of transcriptome sequencing,R²=0.976.（3）Matrine and avermectin have a significant effect on the expression of genes related to glutamate signaling pathway.The expression of glutamate gated chloride channel gene（apglucl）increased significantly with the increase of matrine and avermectin dose and the extension of treatment time.The expression of high concentration was 1.94 and 2.22 times that of the control,and the expression of 72 h treatment was 2.10 and 2.29 times that of the control,respectively.4.Based on RNAi technology,the functions of four glutamate receptor genes（cationic receptor:kainic acid receptor Apgrik,type 1 glutamate receptor Apgr1 and delta glutamate receptor Apgrid;anionic receptor:glutamate gated chloride channel Ap Glu Cl）in the insecticidal effect of matrine were analyzed.The results showed that:（1）after 36 hours of injection,the interference efficiency of apglucl gene and apgrik reached the highest,64.50%and 37.10%,respectively.After 48 hours of injection treatment,the interference efficiency of Apgrid and Apgr1 reached the highest of 41.68%and 35.24%,respectively.（2）Treated with LD₅₀doses of matrine and avermectin for 36 hours after ds Ap Glu Cl injection,the sensitivity decreased significantly by 35.08%and 40.64%,respectively,indicating that matrine can act on the glutamate gated chloride channel（Ap Glu Cl）of pea aphid.However,the sensitivity of test insects after injection of ds Apgr1,ds Apgrid and ds Apgrik did not change significantly.5.Ap Glu Cl was expressed by insect baculovirus expression system,and the effect of matrine on the function of Ap Glu Cl was analyzed by MQAE probe method.The results showed that:（1）the recombinant p Fast Bac vector was constructed by two-step ligation,and transformed by electric shock.After obtaining the recombinant Bacmid,it was transfected into sf9 cells and successfully expressed.（2）The intracellular（sf9 cells expressing Ap Glu Cl protein）chloride ion concentration decreased after matrine treatment,which was consistent with avermectin,indicating that matrine can act on the Ap Glu Cl protein,thereby affecting the intracellular concentration of chloride ions.6.Homology modeling and molecular docking techniques were used to predict the possible binding modes and binding sites of matrine in Ap Glu Cl.The results showed that matrine,sophocarpine and ivermectin could bind to Ap Glu Cl protein of pea aphid,and the binding sites were located in the deep cleavage interface of C-terminal and N-terminal domains.However,matrine and sophocarpine bound to Leu270,Ile581,Phe254,Ser257and Asn274 through hydrophobic and polar effects,while ivermectin bound to Arg261（arginine）and Ser 257（serine）through hydrogen bonds.The results showed that matrine and ivermectin had the same target,but their effects on the target were different.In summary,Ap Glu Cl may be one of the targets of matrine,and the mechanism of action of matrine is speculated as follows:after matrine enters the insect body,it binds to Ap Glu Cl through hydrophobic interaction,electrostatic interaction,etc.,resulting in its conformational change.Disruption of the balance of intracellular and extracellular chloride ions further causes inhibitory signal transmission,resulting in paralysis of the test aphids and gradual death;in addition,direct or indirect effects on cationic glutamate receptors and Na⁺,K⁺-ATPase and Ca²⁺,Mg²⁺-ATPase may lead to symptoms such as spasms..