| The Asian corn borer,Ostrinia furnacalis(Guenee)(Lepidoptera: Pyralidae),is an important pest of maize,mainly distributed in East Asia,Southeast Asia,Australia and other regions.The occurrence of Ostrinia furnacalis has caused a large reduction in corn production.According to statistics,the damage has been reduced by about 30 %.Because of its perennial emphasis on occurrence,in 2023,Ministry of Agriculture and Rural Affairs of the People’s Republic of China included it in the ’ first-class crop pest list ’ for management.Chemical pesticides are the main means to prevent and control the Ostrinia furnacalis.The perennial use of chemical pesticides has led to a strong resistance of the Ostrinia furnacalis.At the same time,it also poses a threat to the farmland ecology and natural environment,and poses a huge risk to the quality and safety of agricultural products.Therefore,finding new green environmental protection control measures has become an urgent need for the prevention and control of the Ostrinia furnacalis.The development of nucleic acid pesticides based on RNAi and the research of transgenic crops have become the development trend of pest targeted control,and the screening of high-efficiency molecular targets is the primary condition that needs to be considered.20-hydroxyecdysone(20E)is involved in a series of physiological activities such as insect growth,development,molting and diapause,and plays an important role in the growth and development of insects.Therefore,in this study,ecdysone biosynthesis genes(Ofnvd、Ofspo、Ofphm、Ofdib、Ofsad and Ofshd)and receptor genes(Of Ec R and Of USP)were studied by molecular biology techniques,and explored the function of ecdysone biosynthesis and its receptor genes in the growth and development of Ostrinia furnacalis.This is of great significance for elucidating the growth and development of pests,screening target genes for pest control,and providing theoretical basis for the development of new strategies for pest control.The main results are as follows :1.Cloning and bioinformatics analysis of 20 E biosynthesis and its receptor genes.In this study,eight 20 E biosynthesis and its receptor genes were cloned,including the open reading frames of Ofspo,Ofphm and Of USP and the main open reading frames of Ofdib and Ofsad.The lengths were 1605 bp,1617 bp,1233 bp,1551 bp and 1314 bp,encoding534,538,410,516 and 437 amino acids,respectively.Through the analysis of the above genes by relevant bioinformatics software,it can be seen that Ofspo,Ofphm,Ofdib and Ofsad belonging to the Halloween family genes have typical conserved domains of P450 proteins :Helix-C,Helix-I,Helix-K,PERF motif and heme-binding domains.Of USP has a central highly conserved DNA binding domain(DBD)and a C-terminal ligand binding domain(LBD).Through sequence alignment and phylogenetic tree analysis with other insects,it can be seen that the Ostrinia furnacalis is closely related to Lepidoptera insects such as Bombyx mori and Chilo suppressalis,and is far related to Coleoptera,Diptera and Hymenoptera insects.2.Spatial and temporal expression patterns of 20 E biosynthesis and its receptor genesRT-qPCR was used to detect the expression of 20 E biosynthesis and its receptor genes in different instars and different tissues of Ostrinia furnacalis.The results showed that the expression of Halloween family genes and Of USP in different instars of Ostrinia furnacalis was basically the same : the expression level of the first day of the same instar from 2nd to4 th instar was higher than that of the third day,and the expression level of the third day of the 5th instar was higher than that of the first day.The expression level of Of Ec R on the 3rd day of the 3rd and 4th instars was significantly higher than that on the first day of the same age,and the expression level on the third day of the 5th instar was slightly higher.Except Ofphm,Of Ec R and Of USP,other genes were highly expressed in prepupa stage.In the expression distribution of different tissues of 4th instar larva and 5th instar larva,20 E biosynthesis related genes were not expressed in the brain,and Ofnvd,Ofspo,Ofphm,Ofdib and Ofsad were highly expressed in fat body and epidermis.The expression of Ofshd in peripheral tissues such as fat body increased significantly,indicating that 20 E activation was carried out in peripheral sissues such as fat body and midgut.Of Ec R and Of USP were highly expressed in midgut and fat body,indicating that these two tissues play an important role in20 E signal transduction.3.Using RNAi to explore the function of 20 E biosynthesis and its receptor genes.The function of these genes was explored by silencing 20 E biosynthesis and its receptor genes by microinjection.The results showed that the expression of all genes except Ofnvd,Ofphm and Ofshd was inhibited.The expression of Ofsad was significantly down-regulated by 71%,and the expression of Of USP was significantly down-regulated by 48%.After ds RNA injection,the expression of Of Ec R and Of USP in different treatment groups also decreased.After dsnvd,dssad,dsshd and dsusp treatment,the expression of Ec R decreased significantly,which was 50%,47%,60% and 52%,respectively.After dsdib and dsshd treatment,the expression of USP was significantly down-regulated by 34% and 40%.Silencing Ofphm resulted in a decrease in the survival rate of larvae.Silencing Of Ec R and Of USP resulted in a significant prolongation of larval development duration.Silencing Ofnvd,Ofphm,Ofsad,Ofshd and Of Ec R resulted in the inability of the old epidermis of the larvae to completely molt,the normal activity of larvae is blocked,and eventually death.It shows that 20 E biosynthesis and its receptor genes play a very important role in the molting process of Ostrinia furnacalis larvae.In summary,in this thesis,eight 20 E biosynthetic metabolic pathway genes of Ostrinia furnacalis were cloned,and the functions of 20 E biosynthesis and its receptor genes were explored by microinjection.It was found that inhibiting the expression of 20 E biosynthesis and its receptor genes(especially Ofsad and Of USP)would lead to the normal growth and development of Ostrinia furnacalis larvae,significantly prolonged development duration and molting failure.This study provides a theoretical reference for the study of insect growth and development and the exploration of molecular targets for pest control. |
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