| Spiders are a wide variety of chelicerata with different morphological characteristics,which mainly use their chelicerae to prey and defend against natural enemies,and a pair of venom gland(Vg)in their chelicera that secrete venom.The chemical composition of venom is complex and abundant including small molecular substances,peptides,proteins and so on.Currently,transcriptomics and proteomics have been largely used in spider research.The methods allow more and more spider toxin were found,which is useful for discovering interesting biological phenomena and excavating important toxins.Trichonephila clavata is a kind of orb-weaver spiders,they rely mostly on cobwebs for prey,but their chelicerae still play an important role in defending natural enemies,which means that some active toxic substances may still remain in the venom of orb-weaver spiders.In this study,we take T.clavata as the research object,there are many reliable venom components were identified from Vg transcriptome and venom proteome based on venomics techniques,and also unearthed some venom protein related genes by combined with the transcriptome data of other tissues,these results are provide an important data foundation for the development and utilization of toxic substances.Then we analyzed the phylogenetic relationship of the main venom protein family,and several important toxin genes were forecasted by combination with the gene expression patterns of each tissue of T.clavata,we hope to provide support for future research on function mechanism.The results are as follows:1.Reliable components identification of Vg transcriptome and venom proteome in T.clavataAccording to label-free quantification of venom and transcriptome analysis of Vg in T.clavata,746 venom proteins and 3366 Vg expression genes were identified,of which there are 271 overlapping proteins.GO and KEGG enrichment analysis of reliable proteins were mainly related to enzyme activity,glycolysis,salmonella infection and so on.According to the protein domain analysis,reliable proteins were mainly divided into three categories: metabolically functional proteins(67%),venom functional proteins(11%)and unknown functional proteins(22%),Among them,venom functional proteins were divided into nine sub-classes(toxin,CAP,serine proteases,metalloproteases,cysteine proteses,aspartic protease,serine protease inhibitor,cysteine protease inhibitor,and phospholipase),including 17 protein families.This revealed the molecular diversity of the venom components in T.clavata.2.Co-expression network analysis of Vg expression genes in T.clavataSimilar expression patterns may have functionally related between genes.Here,we performed a weighted correlation network analysis(WGCNA)for the transcriptomes of seven tissues in T.clavata(Vg,major ampullate(Ma),minor ampullate,tubuliform,flagelliform,aggregate and aciniform gland).Total 3366 expression genes of Vg were used to calculate co-expression network and the results showed that them were divided into 13 modules.The Meturquoise is significantly related to Vg with 787 genes,of which three typical toxic proteins have high-expression levels in venom proteome:Tc05g085070.1(CAP superfamily protein),Tc05G192610.1(Cystatin superfamily protein)and Tc10G272690.1(Astacin family protein).According to the co-expression network of Meturquoise genes,we found three typical toxic proteins interact with Intraflagellar transport 20,CP2 transcription factor,Sodium: neurotransmitter symporter,Hsp 20 and so on.GO enrichment analysis showed that the genes of the network have the functions of supramolecular polymer,myofibril assembly and peptidase activity and so on.The above results indicated that these toxic proteins may have important functions.3.Expression evolution analysis of the main venom protein families in T.clavataTo explore the evolutionary relationship of venom protein families,we perform phylogenetic analysis and tissue expression analysis for three venom protein families(CAP,Cystatin and Astacin).The Astacin proteins of T.clavata were divided into four main branches(Clade Ⅰ,Clade Ⅱ,Clade Ⅲ and Clade Ⅳ),and genes in the same clade had similar expression patterns,for example,Tc10G272690.1 gene was expressed in Vg and fat body.The Cystatin proteins of T.clavata were divided into two main branches(Stefin and Cystatins),with few gene family members and expressed in multiple tissues.The CAP proteins of T.clavata were divided into four main branches(GAPR-like,CAP-like,CRISP and Ag-like),and there were different expression patterns in the same clade,molecular evolution and sequence characteristic analysis showed that they were tandem genes with purifying selection.Interestingly,we found the three CAP genes of Ag-like clade were detected in the venom of T.clavata.Notably,Tc05G085070.1 has high expression level in Vg,Tc05G087910.1 and Tc13G069900.1 have high expression levels in Vg and Ma gland(the tissue produce dragline silk),among them,Tc05G087910.1 is high expression in anterior and middle of Ma gland;Tc13G069900.1is high expression in middle and posterior of Ma gland.Subsequently,gene structure analysis showed that the Tc05G085070.1 gene has complete CAP domain,Tc05G087910.1 gene lacks CAP2 and some motifs,Tc13G069900.1 gene lacks CAP3 and some motifs.Protein 3D structure prediction revealed that CAP4 and CAP2 are located on the β-sheet,and CAP3 and CAP1 are located on the α-helix.The above results showed that the different structure and motif may lead to different expression and function divergence for CAP genes.4.Preliminary functional study of CAP genes of the T.clavata spiderDue to CAP proteins Tc05G087910.1 and Tc13G069900.1 were detected in the venom of the spider,and also have high expression levels in the Vg and Ma gland,but they also showed gene structure differences with typical CAP protein.Therefore,we doubt these two CAP proteins may have bacteriostatic function.RT-q PCR expression analysis indicated that Tc13G069900.1 gene has high expression level in the venom gland,but low expression in the middle and posterior of the Ma gland,Tc05G087910.1gene has high expression in the anterior and middle of Ma gland.The results are consistent with the RNA-seq results,these phenomena suggest that our results are reliable.Based on the above analysis and previous study,we successfully expressed Tc05G087910.1 and Tc13G069900.1 proteins with a purity of more than 85% by using Bac-to-Bac expression system.Then we carry out the inhibition experiment via Disk Diffusion Test,the Gram-positive bacteria(Bacillus subtilis)and Gram-negative bacteria(Escherichia coli)were selected for the test,the results showed that Tc05G087910.1 and Tc13G069900.1 have no inhibitory effect for these bacteria.Based on the above results,we speculate that the CAP proteins may have more important or complex functions for spiders,but more depth exploration and research are needed. |
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