| Tetranychus cinnabarinus,a serious agricultural mite pest that harms crops.Its strong reproductive ability and serious resistance to pesticides make it extremely difficult to control.Plant secondary metabolites have shown great potential in the prevention and control of agricultural mites.The natural coumarin compound scoparone can be extracted from medicinal plants（Artemisia capillaris）and chemically synthesized.It has high acaricidal activity,is safe for non-target organisms and the environment,and has broad application prospects in the control of agricultural mites.micro RNA（miRNA）is an important factor that regulates gene expression in organisms.It can regulate reproduction,development,growth,metabolism,and even induce death of agricultural mites by regulating the transcription of key genes in the body of agricultural mites.It is a potential target for efficient control of harmful mites.It is a potential target for efficient control of agricultural mites.The purpose of this study is to analyze the molecular targets and mechanism of miRNA-mediated scoparone’s acaricidal action against T.cinnabarinus,providing theoretical guidance for exploring efficient lead acaricides and creating green,safe,and friendly natural product acaricides.The main research results were as follows:1 Screening of candidate miRNAs related to the acaricidal mechanism of scoparone against T.cinnabarinusRNA-Seq results showed that after 48 hours of scoparone stress treatment,179 miRNAs were significantly differentially expressed,of which 128 were up-regulated and 51 were down-regulated.At the same time,GO and KEGG analysis found that the functions related to"calcium signaling pathway"were significantly enriched entries,indicating that miRNAs that mediate the differential expression of calcium signaling pathway may be the key target for the acaricidal effect of scoparone,and miR-263b-5p＿1 was selected as a candidate miRNA for further research.2 Validated the different expression pattern of miR263b-5p＿1 under scoparone stressqPCR was used to detect the expression of miR-263b-5p＿1 under scoparone stress.The results showed that scoparone significantly activated the expression of miR-263b-5p＿1,indicating that the miRNA mediated the acaricidal mechanism of scoparone against T.cinnabarinus.3 Proved that miR-263b-5p＿1 targeted regulation of TcVGCC gene expressionA total of 18 candidate m RNAs,such as tetur08g04060（named TcVGCC）,were obtained by predicting the target gene of miR-263b-5p＿1.Further,the miRNA mimic feeding method and q RCR method were used to verify the targeting relationship between miR-263b-5p＿1 and the predicted m RNA.The results showed that after feeding miR-263b-5p＿1 mimic,the expression of miR-263b-5p＿1 in T.cinnabarinus increased by 47.12%compared to before treatment,and only TcVGCC among the candidate target genes decreased by 39.27%,indicating that TcVGCC may be the target gene of miR-263b-5p＿1,and presents a negative regulatory relationship in targeting.4 Identified the targeted regulatory site of miR-263b-5p＿1 on TcVGCCAfter feeding T.cinnabarinus with miR-263b-5p＿1 mimic,the expression of TcVGCC decreased by 41.14%,in addition,after feeding miR-263b-5p＿1 inhibitor,the expression of TcVGCC increased by 59.46%,indicating that miR-263b-5p＿1 targeted negative regulation of TcVGCC;And the results of dual luciferase validation showed that the 2 to 20 bases of miR-263b-5p＿1 and the 112 to 134 bases of the 3’non coding region（3’UTR）of TcVGCC complement each other to play a regulatory role.5 It is confirmed that the target gene TcVGCC of miR-263b-5p＿1 mediates the acaricidal mechanism of scoparone against T.cinnabarinusEvolutionary tree analysis showed that TcVGCC had the closest genetic relationship with Tetranychus urticae,but had a distant genetic relationship with mammals;qPCR and RT-PCR analysis showed that the TcVGCC gene was expressed at all developmental stages of T.cinnabarinus.The RNAi results showed that after feeding TcVGCC-ds RNA,the expression of TcVGCC decreased by 40.32%,after TcVGCC was silenced,the sensitivity of T.cinnabarinus to scoparone increased,and the LC50 decreased by 23.38%,indicating that TcVGCC mediated the acaricidal mechanism of scoparone against T.cinnabarinus.Sensitivity testing showed that after feeding miR-263b-5p＿1 mimic,its LC50 decreased by60.43%,and after feeding miR-263b-5p＿1 inhibitor,its LC50 increased by 87.05%,further proving that miR-263b-5p＿1 mediated the acaricidal mechanism of scoparone against T.cinnabarinus.These results confirm that miR-263b-5p＿1 negatively regulates the expression of TcVGCC gene by targeting,thereby mediating the acaricidal activity of scoparone against T.cinnabarinus.In summary,this study clarified that the action of scoparone involves the targeting of miR-263b-5p＿1 to negatively regulate the expression of TcVGCC gene;The target site of miR-263b-5p＿1mediated acaricidal action of scoparone against T.cinnabarinus was clarified.This study will provide a theoretical basis for developing target specific green acaricidal compounds based on miR-263b-5p＿1. |
