Molecular Mechanism Of Amorphigenin Inhibiting Mitochondrial Complex Ⅰ Of Oriental Armyworm Mythimna Separata (Walker)

The main insecticidal active ingredient Amorphigenin was isolated from the fruit of Amorpha fruticosa.Chemically,it belongs to rotenone compounds.Laboratory bioassay results showed that Amorphigenin had toxic effects on the larvae of Pieris rapae,Plutella xylostella,Aphis gossypii and Culex pipiens pallens.Previous studies showed that Amorphigenin had inhibitory effect on the mitochondrial complex enzyme I of Culex pipiens pallens.Previous studies have shown that eukaryotic mitochondrial complex enzyme I has 44 different subunits,and only 14 subunits in prokaryotic cells can normally exercise the function of mitochondrial complex enzyme I.Therefore,these 14 subunits are considered to be the key subunits of mitochondrial complex enzyme I.Among them,7 subunits are encoded by nuclear genes,and the NDUFV1,NDUFV2,NDUFS1 and NDUFS2,NDUFS3,NDUFS7,and NDUFS8 subunits are mainly responsible for NADH oxidative dehydrogenation and electron transport,and the other 7 are encoded by mitochondrial genes.In order to explore the effect of Amorphigenin on seven nuclear gene-encoded subunits of Mythimna separata（Walker）,seven key subunits of M.separata were cloned,identified and bioinformatics analyzed.The effect of Amorphigenin on seven key subunits of M.separata was explored by real-time fluorescence quantitative PCR and RNAi technology.The research process and results are as follows:1.The indoor toxicity test and weight inhibition test of Amorphigenin were carried out,and the results showed that the LC₅₀ of Amorphigenin for 48 h in the 3rd instar larvae of M.separata was 491.56 mg/L,and the toxicity regression equation was y=（3.93±0.89）x-（5.58±2.38）.Amorphigenin has a significant inhibitory effect on the weight of M.separata.2.The total RNA of M.separata was extracted,c DNA was synthesized by reverse transcription,and primers were designed.The key subunits encoded by seven nuclear genes in mitochondrial complex enzyme I of M.separata were cloned and identified by PCR.According to the genome data of M.separata published in NCBI and the sequencing data of 2+3 transcriptome of M.separata determined by previous research group,the distribution of seven subunits on chromosomes and the length of genes,the number of exons and introns were obtained,and the gene structure was plotted by TBtools software.Phylogenetic analysis and motif analysis of the seven key subunits were carried out.It was found that the branches of the seven subunits were clustered together,indicating that the naming and biological classification of the seven subunits were not problematic,and there were many conserved regions,indicating that the seven key subunits remained basically unchanged in the evolution process.3.Transcriptome sequencing analysis was performed after treatment with sublethal dose of Amorphigenin.The results showed that the expression of NDUFV2,NDUFS3 and NDUFS7 genes was significantly inhibited,and the expression of NDUFS1 gene was significantly activated.Inhibition of NDUFS2 gene expression;activate NDUFV1,NDUFS8 gene expression.Real-time quantitative PCR was used to detect the relative expression of NDUFV1,NDUFV2,NDUFS1,NDUFS2,NDUFS3,NDUFS7 and NDUFS8 genes in the third instar larvae of M.separata after 48 h treatment with low,medium and high concentrations（350 mg/L,550 mg/L and 750 mg/L）of Amorphigenin.The results showed that Amorphigenin significantly inhibited the expression of NDUFV2,NDUFS3 and NDUFS7 genes.Low concentration of Amorphigenin significantly activated the expression of NDUFV1,while high concentration significantly inhibited the expression of NDUFV1.Low concentration of Amorphigenin significantly inhibited the expression of NDUFS2 gene,while high concentration of Amorphigenin activated the expression of NDUFS2 gene.Amorphigenin activates the expression of NDUFS1 and NDUFS8 genes.The primers were designed and ds RNA was synthesized.The ds NDUFV2 was screened according to the pre-test results.The 3rd instar larvae of M.separata were RNAid by injection method and feeding method.The results showed that the feeding amount of M.separata fed with ds NDUFV2 and injected with ds NDUFV2 was significantly lower than that of M.separata fed with blank control.The expression of NDUFV2 gene in M.separata fed with blank control was 2.7 times and 1.6 times of that fed with ds NDUFV2.After injection of ds NDUFV2 for 12 h,24 h and 36 h,the gene expression of M.separata decreased by89.4%,77.0%and 78.0%,respectively.