| Sex determination and differentiation are key to the normal development and species continuation of sexually reproducing animals.Arthropods are a typical group of sexually reproducing animals among invertebrates,with significant sexual dimorphism in behaviour,morphology and life history.In-depth studies of some species of the insect order Arthropoda have revealed that the Dmrt gene family,which is the underlying sex-determining gene in arthropods and is highly conserved,is essential for the development of sexual dimorphism.Spiders also have a highly complex sexual signal,but little research has been conducted on sex determination and differentiation.In this paper,we performed gene identification and gene family analysis of doublesex(dsx);analyzed the spatio-temporal dynamic expression characteristics of dsx using quantitative PCR;and finally used microinjection technique to inject ds RNA and validate the biological functions of dsx using RNAi.The main findings are as follows:1.Potential sex determination and differentiation genes in spiders were identified and basic bioinformatic analysis of the sequences of the encoded proteins was carried out.A comparative analysis of the Dsx protein sequences from Drosophila melanogaster and Daphnia magna revealed that they all contain a conserved DM domain.Clustering analysis of dsx revealed that the pt-Dsx3,pt-Dsx A2 and pt-Dsx1 genes of D.melanogaster clustered into three subfamilies of the Dmrt gene family,Dmrt93 B,Dmrt99B and Dsx(dmrt1-like).2.The results of real-time quantitative PCR showed that dsx was differentially expressed between males and females.And the gene was mainly differentially expressed in the pedipalps and brain of spider.pt-Dsx3 and pt-Dsx A2 were specifically and highly expressed in male pedipalps tissues and pt-Dsx A2 was specifically and highly expressed in female brain tissues.3.The transcriptome sequencing results demonstrated that many genes in the functional enrichment analysis of the gait and haptic apparatus are involved in processes and pathways related to growth,reproduction and gonadal development.For example,the genes that are down-regulated in the gaiter are mainly TGF-β signaling pathway,MAPK signaling pathway,Purine metabolism,Hedgehog signaling pathway,etc.It is suggested that doublesex may be involved in the development of the musculo-neural system and growth hormone regulation in Parasteatoda tepidariorum,which in turn affects their locomotion,development and even reproductive functions.The main functions of the genes down-regulated in the haptic apparatus are Hippo signaling pathway,Insect hormone biosynthesis,Neuroactive ligand-receptor interaction,etc.It is suggested that this gene may be involved in the regulation of muscle motor neural signalling and sexual behavioural signalling associated with the sexual organs of adult greenhouse spiders.4.RNAi was performed on subadult spiders using a microinjection method and the target gene was down-regulated by 88% after interference.After microinjection,subadult spiders with interference of the pt-Dsx3 gene were reared to adulthood,male adult spiders showed asymmetry on both sides of the pedipalps,shortened acrosomal structures consisting of the guide and insertion,low mating success and abnormal offspring development;and showed significant morphological changes such as bending and folding in the morphology of the gaiters;supporting the final results of transcriptome sequencing analysis.In this study,the spatio-temporal dynamics of dsx gene expression in male and female were clarified,and the important role of doublesex gene in the locomotion,development,mating and reproduction of was revealed.The results of this study fill a gap in molecular research in chelicerates and provide a reference to enrich the role of dsx in spider sex determination. |
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