20-hydroxyecdysone Upregulates Segmentation Protein RUNT-like Expression To Promote Pupal Cuticle And Wing Development In Helicoverpa Armigera

Research background and scientific questions:Holometabolous insect undergoes morphological remodeling from larvae to adult,including the transformation of larval epidermis into pupae and adult epidermis,and the growth of wing buds turn outward into wings.20-hydroxyecdysone(20E)regulates metamorphosis,but the molecular mechanism by which 20E regulates epidermal remodeling and wing development during metamorphosis is not fully understood.Taking the major agricultural pest Helicoverpa armigera as a model,this thesis screened the highly expressed cuticle proteins and transcription factors during metamorphosis by sequencing the transcriptome of epidermis and wing,studied the functions of cuticle proteins and transcription factors in epidermal morphological remodeling and wing development in insect metamorphosis and development,and revealed the molecular mechanism of ecdysone promoting cuticle protein expression during metamorphosis and then regulating wing development and epidermal remodeling in insect metamorphosis and development.Clarify the molecular mechanism of insect metamorphosis and development in theory,and find new key genes of epidermal remodeling and wing development,so as to provide new target genes for pest control in application.Research results:Immunoblotting indicates that the epidermis in metamorphic development did not undergo autophagy and apoptosis like the midgut.Therefore,through the transcriptome analysis and qRT-PCR detection of the epidermis in feeding and metamorphosis and wing in metamorphosis,and screened out three cuticle proteins that are highly expressed in the metamorphic epidermis and wings,they were named DNA-directed RNA polymerase II subunit RPB1-like(Rpb1-like),Extensin and larval cuticle protein LCP-30(Lcp-30-like)in the H.armigera genome,respectively.The DNA-directed RNA polymerase Ⅱ subunit RPB1-like(Rpbl-like)with the highest expression and multiple up-regulation during metamorphosis was selected for follow-up research.After knocking down Rpb1-like,the larvae had abnormal epidermis,could not pupate normally,died,and could not grow normal wings.RPB1-like is involved in epidermal remodeling and wing formation during metamorphosis.Two transcription factors,segmentation protein RUNT-like(Runt-like)and protein OVOlike(Ovo-like),which are highly expressed in epidermis and wings during metamorphosis,were further screened from the transcriptome.After knocking down Runt-like,the larvae had abnormal epidermis,could not pupate normally,died,and could not grow normal wings,which is similar to the phenotype of cuticle protein Rpb1-like knockdown.After knockdown of Runtlike,it was found that the expression levels of cuticle protein Rpb1-like and Extensin decreased.It indicated that RUNT-like up-regulated the expression of Rpb1-like and Extensin.Luciferase assay was used to detect the transcriptional activity of the Rpb1-like 5’ upstream sequence.The results showed that under the induction of 20E,the overexpressed RUNT-like could express the protein Luciferase-GFP in the reporter plasmid,which proved that 20E upregulated the expression of Rpb1-like through RUNT-like.Injecting 20E into larvae can up-regulate the expression of Runt-like.The expression level of Runt-like decreased after RNAi knockdown of ecdysone receptor(Ecr)and transcription factor forkhead box protein O(Foxo).It shows that 20E promotes Runt-like through EcR and FOXO expression.Conclusion and significance:1.In metamorphic development,the epidermis did not undergo autophagy and apoptosis like the midgut.2.During metamorphic development,a series of cuticle proteins were markedly upregulated.3.Cuticle protein RPBl-like was highly expressed during metamorphosis and involved in epidermal remodeling and wing formation.4.20E induced the transcriptional activity of cuticle protein Rpb1-like 5’ upstream sequence through transcription factor RUNT-like.5.20E promoted the high expression of Runt-like in metamorphic epidermis and wings through EcR and FOXO.Using the lepidopteran H.armigera as a model,this thesis reveals that Runt-like is a key gene regulating epidermal remodeling and wing formation during metamorphosis and Rpb1-like is an important gene involved in epidermal remodeling and wing development during insect metamorphosis.This study provides new theoretical knowledge for 20E to promote epidermal remodeling and wing development during metamorphosis,and provides a new target gene for agricultural pest control.