Study On The Fuction Of The Inositolriphosphate Receptor And Plasma Membrane Calcium Pump Of Mythimna Separata In The Insecticical Effect Of Wilforine

Tripterygium wilfordii Hook.f.is a woody vine of Tripterygium wilfordii.It has the advantages of abundant resources,good insecticidal activity,broad spectrum,and safety to mammals.Wilforine is its main insecticidal active ingredient,and it has great potential for application and development in the development of efficient and safe new biological pesticides.But so far,the target and mechanism of wilforine have not been determined.It is speculated that wilforine acts on the muscle system of Mythimna separata according to the previous research results.Studies on calcium dynamics of Mythimna separata myocytes showed that wilforine could act on the calcium channel of the sarcoplasmic reticulum.In addition,according to the results of transcriptome sequencing,wilforine has a significant impact on the expression of calcium signaling pathway-related genes of armyworm.Based on the above results,this study took Mythimna separata as the research object,inositol1,4,5-trisphosphate receptors（IP₃R）and plasma membrane calcium pump（PMCA）its gene sequence were cloned respectively.The spatiotempora expression of this two genes was tested at the same time.Finally,in order to verify the role of that two calcium channel proteins in wilforine insecticidal function,RNAi was used to analyze the fuction of IP₃R and PMCA.This study has laid the foundation for further elucidation of the insecticidal mechanism of wilforine.Below are main results:1. RT-PCR was used to clone the Mythimna separata IP₃R and PMCA gene sequences,respectively named Ms IP₃R and Ms PMCA.The open reading frame of Ms IP₃R is 8118bp which encodes 2076 amino acids.Its molecular weight and isoelectric point are predicted to be 304784.46 and 6.04,respectively,and the encoded protein has 6 hydrophobic transmembrane regions.The Ms PMCA open reading frame is 3438 bp long which encodes1146 amino acids.The protein molecular weight and isoelectric point are predicted to be125957.46 and 6.59,respectively,and the encoded protein has 10 hydrophobic transmembrane regions.2. The expression of IP₃R and PMCA genes in different development stages and tissues of Mythimna separata was detected by fluorescent quantitative PCR.Ms IP₃R and Ms PMCA are expressed in all developmental stages of Mythimna separata and in the tissues of 5th instar larvae.Ms IP₃R had the highest relative expression in the adult stage of Mythimna separata,which was 21.28 times that of the pupa stage.In different tissues,the relative expression in the somatic muscle was 6.19 and 4.23 times that of the head and gut,respectively.Similar to Ms IP₃R,the relative expression level of Ms PMCA in the adult stage is 17.62 times that of the egg stage,while the somatic muscle are 1.71 and 1.54 times that of the head and gut,respectively.3. The IP₃R of Mythimna separata may not be an insecticidal target of wilforine.RNAi was used to verify the role of Mythimna separata IP₃R in wilforine muscular toxicity.After injection of the ds IP₃R（ds IP₃R1 and ds IP₃R2）,the m RNA level of Ms IP₃R in Mythimna separata was decreased to 39%and 51%（72h）,respectively.There was no significant effect on the growth of test insects.Corn leaf dish with LC₅₀ wilforine was fed after treated with ds IP₃R,the mortality was 52.41%and 57.58%（48h）respectively,but there was no significant change compared with the group fed on LC₅₀ wilforine on Ly,which indicated that the experssion decrease of Ms IP₃R had no significant effect on the sensitivity of M.separata to wilforine.Thus,IP₃R may not be a direct target of wilforine.4. PMCA may be one of the targets of wilforine.After injection,the m RNA level of Ms PMCA in M.separata was reduced to was 43%and 49%（48h）respectively,but there was no significant effect on the weight gain of the treated group.Feeding of poisonous leaves after 48h injection of ds PMCA,the mortality at 48h was 24.68%and 17.82%respectively,which was significantly lower than that group treated with LC₅₀ wilforine on Ly,indicating that repression of Ms PMCA greatly increased the tolerance of M.separata to wilforine.Thus,it is speculated that PMCA may be one of the main target of the wilforine.In summary,the possibility of IP₃R as a direct target of wilforine is ruled out,while PMCA is an important role in insecticidal fuction of wilforine.It is speculated that wilforine affects it and its effect are as follows.After fed on wilforine,the wilforine enters the insect body and combines with the PMCA of the muscle cells,which leads to the disorder of the Ca²⁺concentration intracellular and extracellular.Then,it causes the damage of the muscle cells and even the destruction of muscle tissue structure and abnormal function,which cause the death of insects.