The Genome And Spinning Mechanism Of Dragline Silk In The Trichonephila Clavata Spider

Spider dragline silk is a highly sought-after proteinaceous fiber with exceptional mechanical properties and biocompatibility,making it an attractive material for a variety of applications such as biomedicine,defense,and high-strength composites.However,its limited availability due to the difficulty of collecting and processing natural spider silk has led to a significant focus on developing artificial spinning technologies using modified cells or animals.Unfortunately,these technologies often result in fiber properties that fall short of the natural silk’s superior characteristics due to the lack of understanding of the complex biological processes involved in dragline silk synthesis in the Ma gland.Genomic and multiomics approaches have been employed to identify and understand the molecular mechanisms of spidroin synthesis for optimal performance.Using the golden orb-weaving spider,Trichonephila clavata,as a research model,a high-quality chromosome-level genome of the female spider was assembled through third-generation,second-generation,and Hi-C technologies.Multiomics methods were then used to identify key substances and genes involved in the dragline silk synthesis process in the Ma gland and reveal their formation mechanisms.The main findings of this study are as follows:1.Genome assembly and evolution characteristics in T.clavataWe present here the comprehensive assembly and annotation of the female T.clavata spider genome using a combination of ONT,Illumina and Hi-C technologies.The genome size was estimated to be 2.63Gb,with a contig N50 of 1.78Mb and a scaffold N50 of 202.09Mb.The completeness of the assembly was evaluated using BUSCO,with a value of 96%indicating a highly complete genome assembly.Karyotype analysis revealed that female spiders have a diploid chromosome number of2n=26,while male spiders have 2n=24.Notably,female spiders possessed two additional sex chromosomes when compared to their male counterparts,and through Pool-seq analysis,we identified Chr12 and Chr13 as the sex chromosomes,which were also the smallest two.To further enhance gene annotation,we performed RNA-seq analysis across 18 tissues of the spider body,as well as utilized Pac Bio full-length transcriptome evidence from mixed silk glands.Our annotation pipeline,which integrated de novo assembly and homology-based evidence,identified a total of 37,607coding genes in the genome.Phylogenetic analysis revealed that the divergence of T.clavata from T.antipodiana occurred approximately 19.63 million years ago（Mya）.The effective population size of T.clavata reached its maximum（6×1e7）before the Naynayxungla glaciation（⁰.5-0.78 Mya）and subsequently declined gradually.Repeat sequence analysis demonstrated that the T.clavata genome had a repeat content of 53.94%,with DNA transposons（22.9%）being the most abundant,followed by LTR retrotransposons（11.41%）.Extension of repeat sequence analysis to seven other chelicerate animals showed that larger spider genomes contained significantly higher levels of DNA transposons and LTR retrotransposons than smaller spider genomes.LTR insertion time analysis demonstrated that LTRs were continuously inserted into larger spider genomes over the past 10 million years.Mapping DNA transposons and LTR retrotransposons to Hox gene clusters produced similar results,indicating that the frequent insertion of these two transposons contributed to the elongation of Hox genomic fragments in spiders.These findings suggest that the expansion of DNA and LTR transposons played a role in the enlargement of the spider genome.2.The spidroins and their evolution characteristics in T.clavataSpidroins,the primary carriers of spider silk,have long sequences and complex repeat motifs that have posed challenges for researchers.In this study,we identified 28high-confidence spidroins through homology alignment and manual annotation,including 9 Ma Sp,5 Mi Sp,4 Ag Sp,2 Fl Sp,1 Tu Sp,1 Ac Sp,1 Py Sp,and 5 other spidroin types.These spidroins range in length from 194aa（Mi Sp-e）to 7819aa（Fl Sp2）and comprise N-terminal,C-terminal,and repeat motif regions.Amino acid content analysis revealed that glycine（29.0%）,alanine（18.4%）,serine（10.3%）,proline（6.8%）,and glutamine（4.5%）were the most abundant residues.Repeat motif analysis demonstrated thatβ-sheet（（GA）n and poly-A）and 3₁₀ helix motifs（GGX）occurred in Ma Sp and Mi Sp,suggesting that these spidroins possess high strength and extensibility.β-helix motifs and O-glycosylation sites were found in the remaining spidroins,implying that they have high extensibility and viscosity.Notably,Ag Sp and Py Sp contained a higher proportion of O-glycosylation sites,indicating that they exhibit more viscous characteristics.Phylogenetic analysis,co-expression clustering,and silk gland morphology comparison collectively revealed that the silk glands of Ma,Mi,and Fl spiders exhibited higher similarity compared to the remaining four glands,which displayed varying degrees of similarity or functional relevance.Chromosome localization of spidroin genes further showed that Ma Sp genes aggregated into two distinct regions,namely Ma Sp-Group 1 and Ma Sp-Group 2.Specifically,Ma Sp-Group 1 comprised Ma Sp1a–c and Ma Sp2e genes located on Chr4,while Ma Sp-Group 2 comprised Ma SP2a–d genes located on Chr7.Similarly,the Mi Sp-Group included Mi SP-a–e genes located on Chr6.Notably,homology alignment of Ma Sp and Mi Sp genes in closely related T.antipodiana genome showed similar aggregation phenomena.Additionally,genomic collinearity analysis showed that Ma Sp-Group shared higher collinearity inter-species compared to intra-species.Concatenation of the amino acid sequences of Ma Sp-Group of each species into“Supergenes”for phylogenetic analysis showed that T.clavata and T.antipodiana Ma Sp-Group 1 clustered into one branch,while Ma Sp-Group 2 clustered into another branch.Furthermore,synonymous substitution rate（Ks）analysis revealed that the Ks value of the interspecific Ma Sp-Groups was much smaller than the Ks value within species,suggesting that Ma Sp-Groups between species are more closely related than those within species.Collectively,these results indicate that Ma Sp-Group 1 and 2 originated at least before Trichonephila spiders diverged and are more closely related between species than within species.3.Molecular biological processes of dragline silk secreted by Ma glandProteomic analysis of dragline silk identified 28 proteins,including 10 spidroins（9 Ma Sp and 1 Mi Sp）and 18 non-spidroins（1 glucose dehydrogenase,1 mucin-19,1venom protein（Ven）and 15 spider silk elements（Spi CE-DS））.The abundance of Ma Sp exceeded 70%of the total silk protein content.Metabolomic analysis of dragline silk revealed 180 metabolites,with organic acids,organic heterocyclic compounds,and lipids being the three most abundant categories.Transcriptome analysis of the Ma gland identified organic acids and 13 silk proteins as originating from the Tail,where Ma Sp-Group 1 proteins were dominant components.The Sac was the primary site of secretion for the 28 dragline silk proteins and lipids,while the Duct was responsible for the secretion of chitin,ions(Ca²⁺and H⁺),and 13 dragline silk proteins.Single-cell（SC）transcriptome analysis identified ten cell types within the 9,349 cell of Ma gland,with four specific cell types in the Sac and Duct each,while three-sectional tissues shared one cell type,and Tail and Sac shared one cell type.Pseudo-time analysis revealed that the Ma gland originated from Ma gland origin cells,which first differentiated into Tail and then differentiated into specific cell types of Duct and Sac.Spatial transcriptome（ST）analysis captured 597 spots in the Ma tissue,clustered into seven distinct groups,and pseudo-time analysis revealed trajectories of differentiation from Tail to Duct and Sac.We focused on the expression of 28 dragline silk genes in the Ma gland,specifically investigating single-cell expression and spatial location expression.Among the genes,Ma Sp-Group 1 demonstrated high and widespread expression levels in most cell types,particularly in Tail and Sac cells.Conversely,Ma Sp-Group 2 was predominantly expressed in SC clusters 9 and 10 of Sac cells.ST analysis revealed that Ma Sp-Group 1 was expressed in most cell types,with the highest expression levels observed in ST clusters“a-b”.Meanwhile,Ma Sp-Group 2 was mainly highly expressed in ST cluster“d”.The proteomics results indicated that more than 70%of the components in dragline silk were Ma Sp protein.SC expression analysis further supported this finding,suggesting that the abundant presence of Ma Sp in dragline silk is due to the large number of Ma Sp-secreting cells in the Ma gland.Additionally,GO functional analysis of the Ma gland tri-section indicated that organic acid-related genes were highly expressed in the Ma Sp-Group 1 synthesis cell and cluster“a”of Tail,suggesting that organic acid synthesis and Ma Sp-Group 1 secretion occur in the same cell type.In contrast,lipid-related genes were highly expressed in lipid synthesis cells and ST cluster“e”of Sac,indicating that Ma Sp-Group 2 and lipids are secreted by different cells.Ion-and chitin-related genes were highly expressed in the Chitin synthesis cell,p H adjustment cell,and ST cluster“f-g”,suggesting that ion-related genes primarily act in the Duct.Together,these results indicate that the secretion and synthesis of liquid silk protein solution is a well-organized and hierarchical process within the silk gland lumen.4.Convergent characteristics of silk and silk glands between silkworm and spiderThe spider occupies a distinct phylogenetic position from the silkworm（Bombyx mori）within the phylum Arthropoda.However,the T.clavata Ma gland（Tail,Sac,and Duct）shares a similar morphology and structure with the B.mori silk gland（PSG,MSG,and ASG）.To characterize the molecular functional similarity between the silk glands of B.mori and T.clavata,we conducted a segmented GO function analysis,which revealed that the two glands share the fatty acid metabolism process in the middle（MSG vs.Sac）,calcium ion binding,chitin binding and signal transduction in the anterior（ASG vs.Duct）,but no shared GO function in the posterior（PSG vs.Tail）.Both PSG and Tail are the primary positions of silk protein production.We identified50 pairs of orthologous genes with convergent expression,including two pairs of orthologous genes corresponding to B.mori PSG and T.clavata Tail,five pairs of orthologous genes corresponding to MSG and Sac,and 42 pairs of orthologous genes corresponding to ASG and Duct.Notably,seven V-type H⁺transport ATPase genes were found in the anterior of silk glands,which have been reported as key factors controlling silk formation in the anterior.Proteomic analysis revealed that mucin-19and GDH enzyme were common protein components between B.mori cocoon and T.clavata silk.Metabolomics identified six shared metabolites among the top 90%metabolites between B.mori cocoon and T.clavata silk,with choline and DL-Malic acid being the most abundant.Choline is a substance in the phospholipid bilayer in the cell membrane,which is related to silk protein secretion.DL-Malic acid may act as a p H controller on silk,giving it anti-corrosion and antibacterial properties.There are many homologous substances and convergent expression genes between silkworms and spider silk and silk glands.We selected the marker gene Hsp20（Tc04G175120）of the ST cluster"f",which encodes a small heat shock protein that acts as a protein chaperone to protect other proteins against misfolding and aggregation.Homology alignment revealed a high degree of conservation（identity=87.1%）between the Hsp20 protein sequences of T.clavata and B.mori,particularly within the Hsp20 structural domain and signal peptide region.We used CRISPR/Cas9-based knockout（KO）of the Hsp20 gene in B.mori and successfully identified indels（96.46%）at the target site of the Hsp20 sg RNA.The results indicated that the silk production（cocoon weight and cocoon layer rate）of the Hsp20-KO strain was significantly lower than that of the wild type,suggesting that Hsp20 is related to silk yield.In this study,we present a high-quality genome sequence of the T.clavata spider and its spidroins.Our findings suggest that the size of the spider genome is primarily influenced by DNA transposons and LTR retrotransposons.Through multiomics analysis,we demonstrate that the formation of insoluble solid silk fibers in the silk gland lumen involves a series of complex biochemical processes that include exposure to organic acids,lipids,p H,and ionic shear forces,followed by tensile forces.Interestingly,we observe convergent silk characteristics in the silk glands of silkworms and spiders,suggesting the existence of shared key processes and substances during silk formation.Taken together,our study provides an important reference for spider genome research and contributes to our understanding of the silk spinning process and its evolution.