The Study Of Araneus Ventricosus PySp Gene Diversity And MaSp1 Spliceoforms

To date,more than 42,000 spider species are known,nearly all spiders can spin silks and orb-weaving spiders even can produce up to seven types of silks for different purposes.These spider silk fibers are mainly composed of silk proteins which are called spidroins,and their gene structures are highly conserved.The dragline silk is the highest-strength type of spider silk,which is considered to be one of the toughest native biopolymers.The dragline silk is used for the framework of spider web and the lifeline when the spider jumps from a height to avoid predators.The pyriform silk produced in pyriform gland is a special silk type,which is actually a combination of dry silk and wet glue,and is main component of attachment discs.The attachment discs can bond dragline silk to make spider move quickly and safely,and firmly attach the web to surface of various objects.Spider silk is a biocompatible and biodegradable protein-based material.Since the most spiders cannot be fed on a large scale,the genetic engineering become effective approach to produce silk proteins,and using the artificial biotechnology to prepare artificial spider silk fibers.In many industrial fields,the silk fibers with excellent mechanical properties,good biocompatibility and soft texture are more favored.The excellent quality of artificial spider silk fiber will play a role in different fields,such as tissure engineering,military,and aerospace.To date,artificial spider silk fibers have low yield and are expensive,and the material properties are still much weaker than those of natural spider silks.The main reason is that there are still many key issues that need to be resolved in the process of artificial silk preparation.For example,the full-length genes of most spidroins have not yet been obtained,the studies of the structure and function of spidroin genes are few,and the mechanism of silk formation is still unclear.The spinning process of natural spider silk is very complex and precise,in which spider silk proteins are assembled from highly soluble storage states into ordered and insoluble fibers.In fact,In fact,the orderly arrangement of spider silk proteins during spinning is the basis for high performance of spider silk.However,the factors that promote the assembly of spider silk fibers and the mechanism for spidroins respond to these factors are poorly understood,which limit the development for the research of artificial spider silks.Part 1.Cloning and identification of full-length PySp1 gene from A.ventricosus.The first report for full-length spidroin gene sequences are the black widow spider?Latrodectus hesperus?major ampullate spidroins?MaSp1 and MaSp2?.After that,other types of spidroin gene sequences were also described,such as A.ventricosus MiSp and Latrodectus hesperus AcSp1.However,due to the large repetitive region in spidroin gene and the cloning strategies favor amplification of 3'-regions,there are numerous spidroin genes have not been obtained.As a combination of dry fibers and wet glue,the existing knowledge of pyriform spidroins is rare and there is only one pyriform spidroin gene was identified from Argiope argentata.To better understand the diversity of PySp1 molecular structure and its relationship to other spidroin family members.In this chapter,we obtained the full-length PySp1 gene sequences from A.ventricosus by degenerate PCR and long-distance PCR?LD-PCR?.The A.ventricosus PySp1 gene is 11,931 bp in length and encodes 3976 amino acids,and the central repetitive region is composed of 16highly homologous repeat units.Similar to A.argentata PySp1 sequence,A.ventricosus PySp1 also has a long N-linker with a repetitive sequence,which is used to bridges the amino-terminal and repetitive regions.The prediction of the secondary structure and hydrophobicity of A.ventricosus PySp1 showed that the?-helix dominate the terminal and repetitive regions while the N-linker is mainly random coil and displays extreme hydrophilcity.Phylogenetic analysis results showed that A.ventricosus PySp1 and other PySp1 genes are grouped in an independent evolutionary branch which together with other types of spidroin genes form a large spider silk gene family.Part 2.Cloning,identification and spinning of the novel full-length Pyriform spidroin?PySp2?.With the development of genome sequencing,as many as dozens of MaSp genes have been described in the specific species,while only one type of pyriform spidroin?PySp1?was uncovered.To expand our knowledge of spidroin family,the discovery and research of new pyriform spidroin genes are necessary.In this chapter,we identified the second type of pyriform spidroin?PySp2?gene from A.ventricosus by RACE and LD-PCR.Compared with PySp1_A.vrepetitive sequence,the repetitive sequence of PySp2_A.v is more complex and can be divided into four types of repetitive regions,including three repetitive sequences that are completely different from other PySp1 and one repetitive region with high similarity to PySp1_A.v repeat units.However,the linker regions of PySp2_A.v is shorter and simpler that those of PySp1_A.v.Phylogenetic analysis results showed that PySp2_A.v and PySp1_A.v are grouped in independent branch,but are divided into two sub-clades,and are relative distance to each other.Furthermore,we construct recombinant protein PySp2-R1B clone that contains patrial repeat and expressed in E.coli,studying the expression of the new type recombinant spidroins and spider fiber formation mechanism.The results showed that the yield of PySp2-R1B is very high when the cells were induced in LB medium at 16?for 16 hours.Then,silk fibers were prepared by wet spinning and hand-drawing.Although the PySp2-R1B only contains partial repeat unit,it can still self-assemble to silk fibers.The full-length PySp2 gene sequence not only provides new template for artificial spider silk fibers,the spinning research laying a steady foundation for large-scale production of high-performance spider silk.but also approve the presence of multiple pyriform spidroin genes.Part 3.The identification,structure and spinning mechanism of two spliceforms of A.ventricosus MaSp1,and reveal unique functions of N-linker region.Alternative splicing widely exists in eukaryote.However,only two spliceoforms of MaSp were discovered in orb-weaving spider,Nephila clavipes.The spidroins are mainly composed of three domains,a large central repetitive region that flanked by non-repetitive and relative conserved N-and C-terminal regions.The terminal regions were considered to be responsible for assembly during silk formation,and the repetitive region determines the mechanical properties of silk fibers.However,as a structure used to bridge terminal and repetitive regions,the functions of linkers are still unclear.To better understand the linker functions and expand our knowledge of alternative splicing events of spidroins,in this chapter,we obtained two MaSp1 spliceoforms from A.ventricosus c DNA by reverse transcription PCR?RT-PCR?.The primary structure for the two MaSp1 spliceoforms showed that both of them are lack of almost entire central repetitive regions and have NT and CT.The main difference between the two spliceoforms is whether contain an N-linker.Therefore,the different properties of two spliceoforms can reflect the function of N-linker.The prediction of hydrophobicity and secondary structure of two spliceoforms showed that N-linker is dominated by random coli and display extreme hydrophilicity.Moreover,the two MaSp1 spliceoforms and N-linker proteins were expressed in E.coli?BL21?and then were spun into fibers by hand-drawing.Western blot results showed that the protein containing N-linker was more likely to form dimers in solution.The CD results at different temperature showed that N-linker can effectively increase the thermal stability of silk proteins.The mechanical properties results showed that the mechanical strength of the major spliceoforms silk is also significantly improved,indicating the N-linker may promote the formation of intermolecular disulfide bond,thereby increasing the breaking strength of the self-assembled silk fiber.Our findings not only approved that the N-linker of MaSp1 has important functions in the process of silk protein aggregation and silk formation,which fills the gaps in the research of N-linker function,but also demonstrate the spidroin without repetitive region still can self-assemble into silk fibers with high breaking strength.This research expands the design idea of constructing the optimal recombinant spider silk protein sequences,and lay a good foundation for high-performance artificial fibers.Part 4.Identification for A.ventricosus PySp1-MiSp fusion transcripts and characterization of A.ventricosus genomic codons.In this study,we firstly discovered the existence of natural spidroin fusion transcripts by transcriptome sequencing,which provides a theoretical basis for the preparation of artificial spider silk fibers with high-performance.In order to understand the characteristics of codon usage of A.ventricosus,we firstly analyze the codon usage and influencing factors,and evaluate the role of natural selection and mutation pressure in shaping the codon usage pattern.We screening 184,982 high-confidence CDS from the A.ventricosus genome data.Following a series of analyses,the results showed that the codon usage bias is weak,which is affected by nucleotide composition,mutation pressure,natural selection,gene length,and gene expression level and mutation pressure may play a major role in codon bias.19 optimal codons were selected,which the third position of codon is A or T.The results of comparison of the five model organisms showed that A.ventricosus has the smallest codon difference from tobacco and Bombyx Mori.In addition,the spidroin genes were clustered according to the codon usage,and the spidroins were clustered in spidroin types.We firstly used bioinformatics to analyze the codon usage bias of A.ventricosus,and calculation the high-frequency codon and the optimal codon,which provides new perspectives on spider evolution and new gene discovery.