SHh Gene Expression And Function Research In Tracheal Gill And Analysis Of Dpp,Hh,and Wg Signaling Pathways In Simulium Xingyiense

Conserved patterns of appendage development may exist in the animalia,and some molecular mechanisms of appendage development are highly conserved in insects and vertebrates.The appendages of Drosophila are currently the most extensively studied because of their simple structure and easy genetic manipulation,but there exist some limitations.In contrast,the tracheal gills of Simuliidae insects are simpler in structure than the wings and develop in a unique manner.Previously,our laboratory confirmed that tracheal gill develops in an appendage manner,which has the potential to be a new model for appendage development studies.Objective:It is proposed to study the expression of Hh homolog gene(SHh)in larvae tracheal gill and the effect of silencing SHh gene on the morphological development of tracheal gill;to analyze the specific members of Dpp,Hh and Wg signaling pathways in Simulium xingyiense.Methods:(1)Establish a localized BLAST database,search for homologs of Drosophila Hh(tentatively named SHh),design primers using Primer 5,obtain target fragments of SHh gene using RT-PCR and RACE technique to amplify the 3’-and 5’-end sequences of SHh gene,and use DNAMAN to splice the full-length c DNA sequence;use NCBI ORF Finder,DNAMAN,Ex Pa Sy,SMART,Deep TMHMM,MEGA X,etc to analyze and predict its coding protein sequence,amino acid composition,protein molecular mass,isoelectric point,hydrophilicity/hydrophobicity,conserved structural domains,The amino acid sequences of homologous genes were compared for homology;the exons and introns were clarified by BLAST sequence comparison and annotation of gff files in S.xingyiense genomic data,and the gene structure was schematically mapped using GSDS.(2)A frozen sectioning was performed on the 7th instar larvae of S.xingyiense,and SHh gene expression pattern on tracheal gills was detected by RNA FISH(design of RNA probes labeled with FITC using SHh gene)and observed by ortho-fluorescence microscopy.(3)Silencing of the SHh gene by RNAi(design of si RNA using SHh gene)and direct stereoscopic microscopic observation of the pupal stage results;to study its effect on tracheal gill development.(4)The IGV software was used to explore the major genes related to Dpp,Hh,and Wg signaling pathways among the genomic data of S.xingyiense measured in the laboratory,and the protein sequences of their CDs sequences were compared by NCBI BLAST,and multiple sequence comparison and phylogenetic trees were constructed using DNAMAN and MEGA X.Results:(1)The homologous sequence of D.melanogaster Hh gene was searched from the localized BLAST database,and the full-length c DNA sequence of SHh gene was obtained by RT-PCR and RACE amplification of 2310 bp,including an ORF sequence of 1233 bp,encoding 410 amino acids,5′-UTR 713 bp,3′-UTR 363 bp,with 66%similarity to Aedes albopictus;the full length of the gene was 29685 bp,with four exons and three introns,obtained by BLAST comparison of S.xingyiense whole genome sequencing database.The theoretical isoelectric point of SHh protein is about 6.40,and the relative molecular mass is about 45.66 k Da.further analysis of its instability coefficient shows that the total number of Arg+Lys is 42;the total number of Asp+Glu is 46;the instability coefficient in solution is 37.64,and the GRAVY is-0.290.the transmembrane structure of SHh amino acid sequence was analyzed,and the protein has one signal peptide and the rest is all outside the membrane.The conserved structural domains of SHh protein were predicted,and the protein was found to have two complete structural domains of the Hh protein family,Hh-N and Hh-C,respectively.(2)RNA FISH showed that SHh hybridization signals could be seen in the respiratory filaments of tracheal gills of S.xingyiense late 7th instar larvae emanating from the stalk and along the wall of the tube are displayed;The efficiency of interference with this gene after 24 h and 48 h of RNAi treatment respectively was53.65% and 25.18%;after RNAi treatment the larvae were stunted(body length of 7th instar larvae did not differ much from that of 5th instar larvae,4-5 mm)or even died;in pupalized individuals,the respiratory filaments lower-outer filaments(a2)became shorter and the upper-inner filament(d2)growth position was changed(emitted from the same short stalk as the upper filament(d1)).SHh hybridization signals were also observed in the intestinal wall of S.xingyiense larvae in the middle of the 7th instar;RNAi results also showed that the ventral segments of the pupal stage became fewer,developed abnormally,and did not fledge normally into adults.(3)The main related genes in Dpp,Hh and Wg signaling pathways were obtained by IGV in S.xingyiense genome database,4 genes in Dpp signaling pathway,7 genes in Hh signaling pathway and 7 genes in Wg signaling pathway,among which all the main genes in Hh signaling pathway were obtained.The amino acid sequence identity of the obtained genes ranged from 39.98% to 85.74%.By building an evolutionary tree of these genes with homologs of six different insect species(Aedes aegypti、A.albopictus、Hermetia illucens、D.melanogaster、Anopheles stephensi and Culex quinquefasciatus)from three families,it showed that S.xingyiense was evolutionarily more closely related to Culicidae.Conclusions:The SHh gene of S.xingyiense is 29685 bp in length,encoding 2310 bp of m RNA and 410 amino acids;it has four exons and three introns;the SHh protein is a stable hydrophilic secretory protein with two complete structural domains of the Hh protein family,thus confirming that the amplified gene belongs to the Hh family.The SHh gene was expressed in the tracheal gill development of S.xingyiense,and the length and growth position of the respiratory filaments changed after RNAi,suggesting that the SHh gene is involved in tracheal gill development.The d2 enabled the new short stalk,suggesting that the gene affects the branching development of the respiratory filaments.The expression of the SHh gene in the intestinal wall of S.xingyiense,RNAi indicates that the abdomen of S.xingyiense pupae becomes shorter and the larvae are stunted or even die.The morphogenetic-related Dpp,Hh and Wg signaling pathways are conserved in Simuliidae.This experiment elucidated the structure of S.xingyiense SHh gene for the first time and confirmed its involvement in the branching development of tracheal gills.This experiment provides a detailed bioinformatics analysis of the signaling pathways related to appendage development,and lays the foundation for the further study of tracheal gill development mechanism in Simuliidae.