Cloning And Characterization Of Receptor Tyrosine Kinase Gene Torso In Leptinotarsa Decemlineata(Say)

Receptor tyrosine kinases(RTK)constitute one of the largest classes of signaling molecules.It is a receptor and enzyme.RTKs regulate several physiological and biochemical processes,ranging from cell survival and proliferation to differentiation and patterning.All RTKs contain three domains.An extracellular ligand binding domain is usually glycosylated.The ligand binding domain is connected to the cytoplasmic domain by a single transmembrane helix.The cytoplasmic domain contains a conserved protein tyrosine kinase(PTK)core and additional regulatory sequences that are subjected to autophosphorylation and phosphorylation by heterologous protein kinases.Based on the transcriptomic and genome data of L.decemlineata.Using RT-PCR and RACE,16 full-length cDNAs of RTKs were obtained.One of these RTKs,Torso,was further studied by RNA interference.Our results revealed that LdTorso regulated larval-pupal metamorphosis through triggering ecdysone biosynthesis.The main results were listed as follows.1.Molecular cloning,phylogenetic analysis of RTKs in L.decemlineataBased on the transcriptomic and genome data of L.decemlineata and sequences in the GenBank,16 receptor tyrosine kinase(RTK)genes were identified.After verification of the correctness,we obtained 16 full-length cDNAs using RACE method.All these RTKs had typical RTK structural characters.These genes mainly belongs to several families such as epidermal growth factor receptor(EGFR),fibroblast growth factor receptor(FGFR),insulin-like growth factor receptor(IGFR),vascularendothelial growth factor receptor(VEGFR)and nerve growth factor receptor(NGFR).According to the phylogenetic analysis,the 16 RTKs were named as LdHTL,LdPVR,LdOTK,LdCad96Ca,LdRET,LdDDR,LdALK,LdROR,LdROR2,LdDRL,LdTorso,LdEGFR,LdInR1,LdInR2,LdSEV and LdEPHE.Each of the 16 RTKs had a unique homologue in another Coleopteran species Tribolium castaneum.It appears that we identified all RTK members in L.decemlineata.2.Temporal expression profiles of LdTorso,MAPK(LdRas,LdRaf,LdERK)in L.decemlineataThe full length cDNAs of four Torso-MAPK signaling related genes LdTorso,LdRas,LdRaf and LdERK were cloned in L.decemlineata.Sequence alignment and phylogenic analysis revealed that the four genes evolved slowly.They possessed protein-specific conserved domains.The temporal expression profiles of the four Torso-MAPK genes were determined at the embryonic,larval and pupal stages.All ’the four genes were expressed throughout all tested developmental stages.During embryogenesis,their mRNA levels were higher at the early embryonic stage,and were lower at the late embryonic stage.Within the first-,second-and third-instar stages,their expression levels were higher just before and right after the molt,and were lower in the mid instars.During the fourth-instar stage,the expression levels of LdTorso,LdRas and LdERK were higher at the day 1 and day 4 larvae.In contrast,LdRaf mRNA level was higher at the day 3 larvae.The mRNA levels of the 4 Torso-MAPK genes were also tested in the brain,prothoracic glands,ventral ganglion,foregut,midgut,hindgut,hemolymph,epidermis,fat body and Malpighian tubules of the day 1 fourth-instar larvae,as well as female ovary and male testis.The four genes were highly or moderately expressed in the larval prothoracic glands and brain,and in the adult ovary and testis.LdTorso was lowly expressed in other tested tissues.In contrast,LdRas,LdRaf and LdERK were also highly expressed in larval guts.3.Effect of silencing gene LdTorso to L.decemlineataRNA interference-mediated knockdown of LdTorso delayed larval development,increased pupal weight,and impaired pupation and adult emergence.Moreover,knockdown of LdTorso decreased the mRNA levels of LdRas,LdRaf and LdERK,repressed the transcription of two ecdysteroidogenesis genes(LdPHM and LdDIB),lowered 20E titer,and downregulated the expression of several 20E-response genes(LdEcR,LdUSP,LdHR3 and LdFTZ-Fl).Furthermore,silencing of LdTorso induced the expression of a JH biosynthesis gene LdJHAMT,increased JH titer,and activated the transcription of a JH early-inducible gene LdKr-h1,Thus,our results suggest that Torso transduces PTTH-triggered MAPK signal to regulate ecdysteroidogenesis in the PGs in a non-drosophiline insect.