Screening Of Insect Juvenile Hormones And Nuclear Polyhedrosis Virus Membrane Receptors

1.Screening of insect juvenile hormone membrane receptorsJuvenile hormone is a kind of sesquiterpene hormone,which is synthesized and secreted by the lateral body of the pharynx located at the back of the insect brain.The function of JH depends on the genome transcription pathway mediated by its nuclear receptor Met(Methoprene-tolerant)and the non-genomic signal transduction pathway that is membrane dependent.In the transcription pathway of JH nuclear genome,JH binds to the nuclear receptor Met and forms a transcription complex with Taiman to regulate the transcription of downstream genes,such as Cdc,Mcm,kr-h1,Ago,and so on,and then regulates Locusta migratoria.Kr-h1 is an early response gene of the JH pathway,which can respond to the induction of JH and mediate the synthesis of L.migratoria Vitellogenin(Vg).Recent studies have shown that JH can trigger non-genomic signal transduction pathways through G protein-coupled receptor(GPCRs)and receptor protein tyrosine kinase(RTKs),and coordinate with nuclear genome transcription pathway to regulate the transcription of JH response genes.However,relevant studies are not in-depth.Accordingly,in this paper,JH-dependent migratory locust yolk generation was taken as the research object,and the downstream kr-h1,Vg A,and Vg B gene transcription mediated by the transcription pathway of JH nuclear genome and the intracellular second messenger system triggered by JH were analyzed after RNAi silencing of GPCR and RTK in the worms,so as to screen the membrane receptors mediating the non-genomic signal transduction pathway of JH.By using RNAi and q RT-PCR methods,25 GPCR genes were silenced and 2 RTK genes could significantly inhibit the up-regulated expression of kr-h1,Vg A and Vg B genes by JH.These genes include:GPCR class A members: NYR-1,adrenergic receptor-1,CCKR-1,oxytocin-1,oxytocin-2,NTSR-1,5-HTR-1,5-HTR-2,5-HTR-3 and 5-HTR-4;Class B members: PTHR-1,PTHR-2,PTHR-3,PTHR-4,Methuselah-like-1,Methuselah-like-2,Methuselah-like-3,Methuselah-like-4,Methuselah-like-5 and ADHR-1;Class D member: p H regulator-1;Class F members: Frizzled-1,Frizzled-2,Frizzled-3;RTK family members: FGFR-like-1 and FGFR-like-2.The function of FGFR-like-1 and FGFR-like-3 may be involved in the Akt pathway.Adrenergic receptor-1,CCKR-1,5-HTR-1,5-HTR-2,5-HTR-3,5-HTR-4,PTHR-1,PTHR-2,PTHR-3,PTHR-4 and ADHR-1 may participate in the c AMP-PKA signaling pathways;oxytocin-1,oxytocin-2,NYR-1 and NTSR-1 may be involved in the DAG-PKC pathway.Frizzled-1,Frizzled-2,Frizzled-3 belong to the membrane receptor Fz and are directly involved in the Wnt-?-caterin signaling pathway.The else types and functions of the other genes are unclear.ELISA of JH-induced intracellular c AMP concentration showed that ds RNA injection silencing FGFR-like-1 and FGFR-like-2 significantly inhibited the up-regulated intracellular c AMP concentration of JH,indicating that FGFR-like-1 and FGFR-like-2 could mediate the JH-triggered intracellular c AMP second messenger pathway.c AMP variation was used as the standard and two genes were screened by ELISA.The results of this thesis have initially screened 25 GPCR genes and 2 RTK genes that can mediate JH signaling pathways,which can lay the foundation for further research on JH non-genomic signaling pathways and enrich the research progress of JH signaling pathways regulating insect reproduction.2.Screening of nuclear polyhedrosis virus membrane receptorsNucleopolyhedrovirus(NPV)is a baculovirus,a type of double-stranded circular DNA virus with a capsule envelope.In nature,arthropods are used as specific hosts for infection and transmission.Not only can baculoviruses be widely used as pesticides because of their specificity and harmlessness to humans,they also have important practical applications in eukaryotic expression systems,gene therapy,and genetically engineered vaccines.NPV has a biphasic replication cycle and produces two types of virions with different morphologies and functions,namely Occlusion derived virus(ODV)and Budded virus(BV).ODV is contained in the polyhedron and is responsible for the infection process between host individuals;BV is produced by ODV and is responsible for the infection process between various tissues in the host.The infection process begins when the polyhedron enters the host.The polyhedron releases ODV in the hosts intestinal alkaline environment.ODV enters the host epithelial cells and performs viral transcription and translation in the nucleus.The new BV particles sprout from the basement membrane and cause systemic infection.In the process of secondary infection,the envelope protein of BV particles plays a vital role,and the BV envelope protein binds to the host cell surface membrane receptor to enter the host cell.This envelope protein is GP64 in group I nuclear polyhedrosis virus and F protein in group II.At present,there are still doubts about how BV enters host cells,and the membrane receptors that bind to BV envelope proteins have also been unknown.Type C lectin is a type of calcium-dependent sugar-binding protein that functions as a pattern recognition receptor to recognize non-self components,regulates cell phagocytosis and adhesion response,enhances cell melanogenesis and encapsulation,and inhibits or acceleratse viral multiplication in the body.In the previous work of this experimental group,it was shown that BV can induce the expression of Helicoverpa armigera Lectin(Ha-lectin)in cotton bollworm.BV capsule fusion protein F(NPVF)interacts with secreted C-type lectin.It is inferred that there may be a specific receptor protein on the cell membrane of Helicoverpa armigera,which can interact with C-type lectin.By recruiting C-type lectin,BV is presented to the cell surface receptors,which enhances or inhibits endocytosis of virions.In summary,in this paper,the screening of nuclear polyhedrosis virus membrane receptors was carried out based on the relationship between BV membrane protein,C-type lectin and host cell membrane receptor by H.armigera.The Helicoverpa armigera nucleopolyhedrovirus(Ha NPV)used in this paper belongs to group II.Based on the early research on the relationship between Ha-lectin and NPVF in this laboratory,this paper conducted an initial screening of its mass spectrometry results and screened out a suspected membrane receptor VHDLR.VHDLR was then identified and analyzed using cell biology,molecular biology,and genetics methods such as immunofluorescence,Co-IP,Western blot,RNAi,and more.VHDLR was identified as a host cell surface membrane receptor that binds to the BV envelope protein NPVF.It is speculated that the mechanism of action between the three is that Ha-lectin captures BV virus particles in hemolymph tissue,presents it to the surface of the host cell membrane,binds the BV envelope protein NPVF to the host cell membrane receptor VHDLR,and allows BV disease particles to enter the host cell.And by preparing expression vectors for each component of VHDLR,the relationship between each component of VHDLR and Ha-lectin and NPVF was verified in H-O cell lines.It is determined that NPVF combined with the C domain in VHDLR,but Ha-lectin is not combined with VHDLR.It is a new study to reveal the mechanism by which baculovirus enters host cells,and it also lays the foundation for the next screening of the membrane receptors of GP64 in group ?.