| The use of snakes in traditional Chinese medicine has a long history dating back to the Spring and Autumn Period.In the Compendium of Materia Medica written by Li Shizhen in the Ming Dynasty,the medicinal value of snakes is also richly recorded.Snakes are mainly used in medicine,food,and tanning,among which there are more than 70 species of medicinal snakes.The Bungarus multicinctus(belonging to Elapidae)has been recorded as the animal resource of Jin Qian Bai Hua She in the 2020 edition of the Chinese Pharmacopoeia.Because its venom is both presynaptic and postsynaptic,the B.multicinctus is considered one of the most lethal snakes in the world.In addition,snakebites remain a major public health threat,resulting in an estimated 81,000 to138,000 deaths and approximately 400,000 permanent disabilities each year.Therefore,it is urgent to develop safe and efficient snake venom inhibitors to remedy this dilemma.Snake venom is an important source of new drug research and development,with at least eight snake venoms already on the market and many more in the pipeline.Therefore,the B.multicinctus can be used as an ideal neurotoxic protein model organism of snake,providing more possibilities for the development of safe and efficient snake venom inhibitors.Optical mapping is considered an indispensable tool for high-precision genome assembly.However,there are far fewer tools available for genome assembly using optical mapping data compared to existing assembly tools with rich sequencing techniques.Since the optical map of each enzyme label contains its own independent internal errors,some researchers have speculated that there is great potential to improve the final assembly result by using multiple optical maps.Here,we report a multi-enzyme optical map scaffolder(MOMS)with uncapped enzyme tolerance in chromosome-level optical map scaffolding.This tool imparts limitless potency to the optical maps-based scaffolding approach.For local positioning of contig labels and prevention of cumulative error of coordinate transformation,together with the aim of representing adjacency between genome regions,a data structure called Directed Node Graph(DNG)was used to organize positional information.To enable path traversal and conflict resolution,we used a heuristic algorithm,namely,weighted minimal spanning tree.For gap filling,we introduced a new gapped map alignment and a multiple-restriction-enzyme site alignment.To leverage single molecules optimally,we used MOMaligner and a SSPACE translator to directly scaffold sequences by molecules.For NA12878 human genome results showed that MOMS significantly improved the contiguity and completeness of the initial assembly,reducing the number of contigs/scaffolds and incorporating more data than those from the standard hybrid scaffolding.Besides,scaffolding contigs constructed from Pacbio data,MOMS were also used to scaffold primary assemblies constructed from Illumina paired end and mate pair data(PE+MP),10 X Genomics,Hi-C or Oxford nanopore data.Experiments showed that in all circumstances,MOMS robustly improved contiguity.The MOMS pipeline improved the genome-assembly completeness,thereby enabling high-fidelity chromosome assembly,large fragment structural variation detection and chromosome-level evolutionary analysis.Using the MOMS,we performed a high-quality chromosome-level assembly of B.multicinctus genome.The genome size is about 1.58 Gb,including 18 chromosomes,and a total of 20,246 protein-coding genes have been annotated,including 270 toxic protein-coding genes from 34 families.Cluster analysis showed that different toxic protein-coding genes were distributed in clusters according to their respective families,and these gene clusters were independent of local tandem repeats.Based on this continuous genome,we analyzed the gene expression,chromatin three-dimensional structure and histone modification of B.multicinctus by transcriptome,proteome,Hi-C and Ch IP-seq.Transcriptome and epigenetic analysis of different tissues showed that toxin-coding genes were specifically expressed in the venom gland,and the production of venom was regulated by complex mechanisms(chromatin three-dimensional structure,histone modification,etc.).By analyzing the amino acid energy and composition of the encoded protein,it was found that in the process of expression and synthesis of venom proteins,B.multicinctus usually prefers amino acids with high energy cost.Three-finger toxins(3FTxs)are the main components of venom in cobra species.In this study,the origin of 3FTxs was studied.Tracing its origin and evolutionary history is one of the most important discoveries in this study.α-bungarotoxin(α-Bg Tx),γ-bungarotoxin(γ-Bg Tx)and κ-bungarotoxin(κ-Bg Tx)belong to the 3FTxs protein family.In the genome of B.multicinctus,we identified 33 3FTxs genes,including seven pseudogenes.We found that all venom 3FTxs originate from a single human LY6 E paralog with a featured “LYEWLN” motif followed the termination codon.As deduced from its sequence,this tail was responsible for membrane tethering.Variations in the remains of the truncated tail could be considered as a scale for 3FTx evolution.Secretability owing to the loss of the membrane tethering tail is the first key step for modern 3FTx formation.α-Bg Tx is a special type of 3FTx with a unique long-tail and was the first discovered member of this family.Sequence analysis showed that its long tail was derived from a subsequent deletion event after truncation but not obtained from another ancestor.This observation in sequences corroborates the speculations of evolutionists who using phylogenetic analysis and is more perceptible and conclusive.We deem the identification of 3FTx ancestor a fundamental discovery for 3FTx evolutionary research.Accordingly,we speculate that the emergence and expansion of3 FTxs could be traced back to 45 million years ago before the divergence of colubrids and viperids.Contrary to popular belief,3FTx emergence was not an Elapidae-specific event.Hence,this discovery moves up the date of 3FTx origination.If we consider the expansion of LY6 E as the onset of 3FTx’ evolution,the origination can be dated back to the divergence of amniotes.β-Bg Tx(β-bungarotoxin,β-Bg Tx)is a presynaptic peptide neurotoxin,which is a dimer formed by covalent binding of two subunits through disulfide bonds.The PLA2 active center exists on the larger molecular weight A subunit,which has certain sequence homology with the smaller molecular weight B subunit protein kinase inhibitor(Kunitz).The A-chain of β-Bg Tx(a peptide from the PLA2 family)is the functional core of this covalently linked heterodimer.In B.multicinctus,nine Asp49-type PLA2 encoding genes were identified including four elapid group PLA2(GIPLA2)clustering on Chr2 and two viperid group PLA2s(GII-PLA2s)on Chr17.The coexistence of GI-and GII-PLA2 s in the krait genome implies that the divergence of elapid and viperid PLA2 toxins may have originated from the different duplications of PLA2 ancestral genes.The unit-B of β-Bg Tx belongs to the kunitz gene family with a characteristic Cys55 in the C-terminus.We found a large tandem arrayed kunitz family gene cluster located on Chr5,including four unit-B type genes.Cys55 is a typical result of a nonsynonymous mutation with a nucleotide transition from C to T and an amino acid(AA)transition from arginine to cysteine.Surface plasmon resonance experiments confirmed the necessity of this cysteine to form a dimer of β-Bg Tx.Our study found that the B chain has been formed after the differentiation of the genus Cobra and the genus Cyclops,which is marked by the B chain-specific Cys55 cysteine.Then a point mutation of the A chain to form a cysteine is the covalent binding of β-Bg Tx becomes possible,so the formation of the A chain may be after the B chain.Venom production is complex and sophisticated at molecular,morphological,behavioral,and functional levels.The expression of toxins is correlated with chromatin topological organization and histone modification.Except for toxin-coding genes,other genes involved in muscle contraction,protein folding,and proteolysis,were found to be expressed during venom replenishment.Analysis of the origin and regulation of snake toxins will facilitate the treatment of snakebite in animals and the development of endogenous antipsychotic,antitumor,and antiviral drugs. |
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