The Study Of Three Spidroin Gene Structures And Biomimetic Spider Silk Properties

Spider silks have been received much attention from researchers in recent years due to their excellent mechanical properties and good biocompatibility.Orb weaving spiders have seven types of silk glands which can produce 6 types of silk fibers or wet glue,and each type of silk exhibits unique biological functions and material properties.Among these silk types,tubuliform silk has good toughness and breaking strength and is mainly used to form the outer shell of egg case to protect eggs from the interference of the external environment.Aciniform silk has strongest toughness among all types of spider silks,which is used for prey wrapping and inner structure of the egg case.Because of these excellent material properties of spider silk,it has great application value in tissue engineering,aerospace and military.However,due to the cannibalism of spiders and their low yield of silks production,it is difficult to feed spiders on a large scale to obtain silk fibers.Therefore,the preparation of biomimetic spider silks using biotechnology has become the main way to obtain spider silk fibers.With the continuous in-depth research of spider silk proteins by researchers at home and abroad,the studies on spider silk protein genes have been reported successively.However,due to the large spider silk genes,long and complex repetitive sequences and high GC content,the reported spider silk genes are mostly fragmented sequences,and only a few full-length spidroin genes have been sequenced and identified.In addition,these full-length spidroin gene sequences were obtained by constructing genomic or c DNA library.Although this method can identify the complete target gene from genomic DNA,it lags behind the research progress of the full-length spidroin gene sequences due to its shortcomings such as blindness,non-specificity and low efficiency.The scarcity of the full-length spidroin gene sequences not only cannot meet the needs of preparation of high-performance biomimetic spider silk fibers,but also limits the further understanding of the molecular evolutionary mechanism of spider silk genes.In addition,due to the limited understanding of the silk formation mechanism of natural spider silks,the mechanical properties of the biomimetic spider silk fibers prepared by the current spinning methods are much weaker than natural spider silks.Therefore,the improvement of spinning process and selection of suitable spinning reagents are of great significance for improving the performance of biomimetic spider silk.In this dissertation,the Araneus ventricosus tubuliform spidroin?Tu Sp?and aciniform spidroin?Ac Sp?are used as the research objects,using a series of PCR technologies such as degenerate PCR,anchor PCR,long-range PCR?LR-PCR?and RACE to clone and identify the three full-length spidroin coding genes,which provided new gene blueprints for the preparation of high-performance biomimetic spider silk,further uncovered the diversity of spidroins,and provided more details for the molecular evolutionary mechanism of spider silk genes.Meanwhile,this dissertation also constructed and expressed two types of recombinant spidroins.We systematically studied the effect of molecular weight and spinning reagents on the performance of silk fibers and produced high-performance biomimetic spider silk fibers,which laid a solid theoretical foundation for future application in the field of biomedical materials.Part 1.Analysis of the full-length tubuliform spidroin gene structure.As the silk fiber used for the tough outer shell of the egg case,the tubuliform silk produced by tubuliform gland has outstanding mechanical properties,and also can protect eggs from external threats,such as temperature fluctuations and harmful microbes.Therefore,the study of tubuliform silk is great significance for the preparation of new high-performance biomaterials.In this chapter,using the degeneracy PCR,anchored PCR,and LR-PCR technologies,the full-length A.ventricosus Tu Sp1coding gene was successfully cloned and identified from the A.ventricosus genomic DNA.The sequence analysis of the full-length A.ventricosus Tu Sp1 gene showed that the gene is 5763 bp long and encodes 1921 amino acids.No introns were detected.In addition,there are 9 repeat units with high sequence similarity in the repetitive region.Serine?Ser?and alanine?Ala?are the two most abundant amino acids in A.ventricosus Tu Sp1,accounting for 27.9%and 23.8%,respectively.The secondary structure prediction showed that both the NT and CT have five?-helixes.The predicted hydrophobicity of A.ventricosus Tu Sp1 showed that the entire protein displays strong hydrophobic.The identification of A.ventricosus Tu Sp1 gene not only provides more gene resources for the preparation of biomimetic spider silks,but is also crucial for understanding the molecular evolution dynamics of long and repetitive silk genes.Part 2.Exploring the expression of Tu Sp1 recombinant spidroin and the properties of spinning fibers.After the Tu Sp1 gene sequence was obtained,the research is focus on the expression of recombinant proteins in foreign host and preparation of biomimetic spider silks.In this chapter,the NT and CT domains of Tu Sp1 were chimeric with 1?4 Tu Sp1 repeat units?Rp?to construct 4 recombinant spidroins(NTR_1-4CT_{Tu Sp1})with different number of repeat units.The expression of four recombinant proteins in E.coli showed that the yield of proteins decreased with the increase of repeat.In addition,the NTR₄CT_{Tu Sp1} was not expressed,and the NTR_1-3CT_{Tu Sp1}were expressed in inclusion bodies.Due to the low yield and purification difficulty of NTR_2-3CT_{Tu Sp1},the NTR₁CT_{Tu Sp1} was used as the representative protein to prepare recombinant spider silk fiber.Previous studies have demonstrated that postspin drawing can improve the quality of the biomimetic silk fibers.Although the molecular weight of NTR₁CT_{Tu Sp1} protein is only 50 k Da,the mechanical properties of the postspin silk fibers spun using HFIP?1,1,1,3,3,3-hexafluro-2-propanol?showed that the fibers have a breaking strength of 400 MPa,which are almost equivalent to that of natural wrapping silk,and their toughness can reach 100MJ/m³.In this chapter,the biomimetic spider silk prepared by NTR₁CT_{Tu Sp1} displays high performance,demonstrating that the A.ventricosus Tu Sp1 is ideal spidroin gene to produce high-performance biomaterials.Meanwhile,in order to create superior silk fibers,the future works will focus on the codon optimization and expression host to improve the yield of the other three recombinant proteins.Part 3.Cloning and analysis of the full-length aciniform spidroin 1?Ac Sp1?gene.Aciniform silk plays an important role in spider reproduction and predation and is used for wrapping prey and forming inner layer silk of egg case.Meanwhile,it is toughness silk fiber of all types of spider silks,because of its high strength and extensibility.In this chapter,the full-length Ac Sp1 gene was successfully cloned and sequenced by using the above PCR technology,showing that the Ac Sp1 gene is 10338 bp long and encodes 3445 amino acids.The Ac Sp1 repetitive region contains 15 long and complicate repeat units with extremely high similarity.Compared with Tu Sp1,the content of Ser?18.0%?and Ala?12.6%?in Ac Sp1 are lower,while the content of glycine?Gly?increases to 14.5%.The secondary structure prediction of Ac Sp1 showed that there are 5?-helixes in NT domain and 4?-helixes in CT domain.The result of phylogenetic tree showed that Ac Sp1 and Tu Sp1 are likely to originate from a common ancestral silk gene.Following hundreds of millions of years of evolution,they formed highly differentiated silk genes.The cloning and analysis of the A.ventricosus Ac Sp1 gene not only help to better understand the evolutionary history of different spider silk genes,but also provides usable genetic resources for the preparation of high toughness biomimetic silk fibers.Part 4.Characterization of the novel complete spidroin gene?Ac Sp2?structure.Although spiders can produce seven types of silk fibers,the silk proteins are extremely diverse as the main component of spider silks.The whole genome sequencing of Nephila clavipes showed that there are at least 8 major ampullate spidroin?Ma Sp?genes and 4 minor ampullate spidroin?Mi Sp?genes.However,only one type of aciniform spidroin genes?Ac Sp1?were identified.Although the Ac Sp1 gene sequences in different species are different,the NT,CT,and repeat still have relatively high homology.In this chapter,RACE technology combined with degenerate primers and LR-PCR were used to successfully identify the second type of full-length aciniform spidroin gene which was named Ac Sp2.The full-length Ac Sp2 gene is 14238 bp and encodes4745 amino acids.The Ac Sp2 has a predicted size of 476 k Da.The repetitive region of Ac Sp2 is composed of 25 repeat units.The first 24 repeats are 183?185 amino acids in length,and the last truncated repeat has only 11 amino acids.Meanwhile,these repetitive sequences display very low similarity with those of Ac Sp1,indicating that these repeats are new repeat module,and the two amino acids?Serine and aspartic acid?were deleted in some repeat units.However,its NT and CT sequences have high homology with Ac Sp1.The result of genetic evolution analysis showed that although Ac Sp2 is distantly relate to other Ac Sp1,the Ac Sp2 is still grouped in Ac Sp clade,suggesting that the Ac Sp2 may be differentiated from the Ac Sp1 in the distant past,and then evolved.In this chapter,the research firstly demonstrates the existence of the second type of aciniform spidroin,indicating the differentiation of spidroin genes was commonly happened in the evolutionary history of spiders and different types of spider silk proteins.Part 5.Effects of spinning solvent and the number of repeat units on the properties of recombinant Ac Sp2 silk fibers.The mechanical properties of natural spider silk fibers are mainly determined by the repetitive sequences in spidroins.The type and size of repetitive sequences can greatly affect the mechanical properties of silk fibers.For biomimetic spider silks,both the spinning solvent and the number of repeats can greatly change the quality of silk fibers.In this chapter,based on the full-length Ac Sp2 gene sequence,the NT,CT,and 1?6 repeats of Ac Sp2were genetically recombined to construct 6 Ac Sp2 recombinant proteins with different numbers of repeats,and all of them were expressed in E.coli.The purified recombinant proteins were used as protein materials to produce biomimetic silk fibers by wet spinning.The mechanical property test results showed that the increase for the number of repeats can improve the extensibility of the silk fibers,but has little effect on the strength of silks.Meanwhile,the results of spinning with different solvent showed that when HFIP is used as the solvent and water is used as the coagulation bath for spinning,the silk fibers are thinner.Despite their weak breaking strength,their extensibility has been greatly improved,up to 161%,and exceeds that of natural wrapping silk.In this chapter,biomimetic spider silk fibers with high extensibility beyond natural spider silks are not only prepared,but also provided a new idea for the preparation of biomimetic spider silk with excellent mechanical properties.