The Role Of Guanosine Triphosphate Cyclase Hydrolase In Epidermal Melanin Formation And Immunity

The physiological processes of cuticle tanning and innate immunity are indispensable for insect survival and reproduction.The former is associated with insect body plan integrity,maintaining normal molt development and protecting from exogenous physical injury,while the latter can induce a large number of immune-related proteins to synthesize and activate melanin and participate in a series of reactions including phagocytosis,pathogen coating and wound healing.Therefore,the melanin metabolism pathway is closely linked to these two fundamental physiological processes through provision of Dopa and other supporting metabolites.In this process,tetrahydrobiopterin is a necessary cofactor in the synthesis of dopa by hydroxylation of tyrosine.While,GTPCH is the only rate-limiting enzyme in the metabolic pathway of BH₄ synthesis.Guanosine triphosphate cyclohydrolase?Punch?is highly conserved in vertebrates and invertebrates.Previous studies have shown that Guanosine triphosphate cyclohydrolase plays an important role in melanin formation in the epidermis of dipterans and lepidopterans,but it has not been reported in coleoptera.In order to explore the function of Guanosine triphosphate cyclohydrolase in Coleoptera,we chose Tribolium castaneum as the research object.The ORF sequence of Punch gene was obtained by PCR.The gene has a total length of 771bp and four exons,encoding 256amino acids.Multiple sequence analysis showed that Punch was highly similar to other insect homologous proteins.In order to explore the important role of Punch in melanin formation and its regulation mechanism,the spatio-temporal expression pattern of Punch gene was analyzed by q RT-PCR.The results showed that there was a significant high expression of Punch in the early adults,and the expression of Punch decreased gradually with the completion of pigmentation.Further tissue expression patterns showed that Punch was highly expressed in the Elytra.These results suggested that Punch gene may be related to cuticle tanning.After knocking down Punch gene expression by RNAi,it was found that Pupae developed normally and molted to the adult stage,but tanning of the adult cuticle was delayed by several hours with the cuticle eventually becoming slightly darker than the normal rust-red color.And ELISA results showed that the content of dopamine decreased significantly.The later tetrahydrobiopterin complementary experiment could restore the phenotype of adults.To determine the elementary immune biological functions of Punch,the expression pattern of Punch gene in the larva was analyzed by q RT-PCR.The results showed that it was highly expressed not only in epidermis,but also in immune-related tissues such as fatbody and hemolymph.These results implied that Punch may be involved in the immune process of Tribolium castaneum.The expression of Punch gene in larvae was significantly induced at 3 hours after stimulation with Escherichia coli and Staphylococcus aureus,and the expression of Punch reached the highest at 6 hours,and then the expression multiple decreased rapidly to the same level as that of the control group.It was suggested that Punch played an important role in immune defense.Further detection of hemolymph spontaneous melanization showed that there was no obvious spontaneous melanization of hemolymph in interference group compared with the control group.Finally,the mortality statistics after E.coli and S.aureus stimulated were performed.In the control,after E.coli and S.aureus stimulated 72 hours,the cumulative mortality was 30%.While,the larval mortality reached 60%in the interference group.The above results showed that Tribolium castaneum,which interfered with Punch expression,was more susceptible to infection by Staphylococcus aureus and Escherichia coli,indicating that Punch plays an important role in the immune process of Tribolium castaneum.In order to further study the regulatory mechanism of Punch in Tribolium castaneum,we analyzed its downstream related genes and pathways by RNA sequencing high-throughput sequencing.The results of sequencing showed that there were 797 differentially expressed genes between RNAi interference group and control group,of which 506 genes expression increased and 191 genes decreased.The enrichment and analysis of related genes by GO term and KEGG pathway showed that not only the metabolic pathways of tyrosine,phenylalanine and tryptophan were disordered,but also the process of DNA replication and Insect hormone biosynthesis were hindered.