Research On Improving Mechanical Properties Of Silk Fiber Based On The Structure And Composition Of The Silkworm Spinning Duct

There are about 100,000 species of spinning animals known on the earth,of which the most studied are silkworms and spiders.The predecessors have paid close attention to these two kinds of silking animals for a long time.There is a poem that reads:“Silkworms make cocoons to wrap themselves,and spiders make nets to catch food.”This is the understanding of the two spinning behaviors from ancient people.Both silkworms and spiders can produce excellent natural protein fiber,but silkworm silk has advantages of high yield,better quality,easier artificial breeding of silkworms and greater adaptability to industrial production.Therefor it has become one of the most important economic insects.Silk fiber is called the"fiber queen"for its excellent comprehensive mechanical properties（such as good toughness,elongation,etc.）,which is widely used in textiles,biomedicine,military industry,environmental development,polymer materials,makeup industry.The silk is formed of the silk protein synthesized and secreted by silk glands in silkworms.For a long time,researchers are interested in the mechanism of silk formation.At present,there is a general consensus on the mechanism of silk formation.First,the liquid silk protein was transported to the spinning pipe.The silk protein was gradually transformed from an irregular coiling structure to aβ-sheet structure under the combined action of stress or shear force,metal ion environment,p H value and other factors.Then,under further compression of the spinneret,the silk protein was further dehydrated,and the structure was highly ordered,thus forming a solid silk fiber.There is a saying that goes:“A handy tool makes a handyman.”The spinning duct is a unique tool for the silkworm to complete the process of fibroin fibrosis.Although people have a certain understanding of the mechanism of natural silk,various biomimetic silk in vitro is still not comparable to natural silk.Synthetic silk cannot completely imitate the remarkable material properties of natural silk.This may be due to the lack of attention to the role of the spinning duct structure in the process of fibroin fibrosis.At present,there is no systematic understanding of the composition and structure of the spinning duct yet,and many details are needed to be explored in depth.It is of great significance to further explore the composition and function of the silkworm spinning duct,which will help us to understand the process of natural fibroin fibrosis,improve the silk formation mechanism,and provide theoretical basis for artificial spinning.Additionally,more and more synthetic materials（such as nylon）are emerged,which have had a huge impact on traditional textile materials including silk.Silk fiber needs to burst with new vitality.The improvement of mechanical properties of silk fiber has aroused the interest of many researchers.However,the existing methods for improving the performance of silk fibers,whether through forced reeling or various physical and chemical treatments,are time-consuming,labor-intensive and costly,which are not suitable for large-scale industrial production and promotion.The mechanical properties of silk fibers are mainly determined by the structure.Studies have shown that metal ions can induce the conformational transformation of silk fibroin,and the study of the metal ion environment that affects the formation of silk fibers has become a potential approach to improve the mechanical properties.Based on the existing research,we use biology,physics,chemistry and other multidisciplinary methods to systematically analyze the structure and composition of the entire silkworm spinning duct.We further improve its spatial structure and deeply explore its role of the process of silk protein transport and fibrosis.At the same time,factors affecting the conformational transformation of silk protein at spinning duct were analyzed.Appropriate targets were selected and a transgenic silkworm strain with improved mechanical properties of silk was obtained through genetic engineering,which was expected to be popularized and applied in large-scale industry.The main research results are summarized as follows.1.Analysis of the composition,structure and function of the anterior silk gland intimaIn order to explore the structure and function of the endometrial layer more accurately,we used the fine microscopic anatomy system to peel off the endometrial layer from the anterior silk gland for the first time and obtained a relatively pure endometrial tissue.The composition of chitin on inner membrane was accurately identified by infrared spectroscopy.β-type chitin is the main component.LC-MS/MS analysis showed that the epidermal protein content on inner membrane was the highest,and elastin was identified for the first time.The autofluorescence experiment proved that elastin uniquely exists on inner membrane.Mechanical tensile test showed that the strength of the inner membrane was 6 Mpa and the elongation was 7%.Then we observed and statistically analyzed the growth and changes of the membrane during the entire larval stage of the silkworm and found that the inner membrane thicken with the development of the larvae and its silk gland.The trend of intimal thickening was consistent with the change of diameter of anterior silk gland cavity,maintaining the same growth rate.Furthermore,we synthesized a specific chitin fluorescent probe,and in situ analyzed the changes of chitin in the inner layer of anterior silk gland during the 4th molt.Chitin undergoes a process of degradation and reconstruction at the early stage of molt,and its basic reconstruction has been completed at the middle stage of molt.It indicated that other matrix proteins further reconstructed the inner membrane on the basis of chitin network before molt renewal.The changes of chitin-related genes in the anterior silk gland were analyzed for the first time from the perspectives of synthesis and degradation.A total of 29 time points were selected from the 4th molt to the end of wandering for analysis.The results showed that both chitin synthase enzymes were expressed in the anterior silk glands.Combined with the results of infrared identification,the anterior silk glands were dominated by chitin synthase B.In addition,the only chitin deacetylase（Bm CDA1）in the silk glands has specific transcriptional changes during the 4th dormant,but no transcription at the 5th instar.It indicates that Bm CDA1participates in the intimal remodeling during the 4th molt.Endochitinase and exochitinase are two different kinds of chitin degrading enzymes.The results show that 7 endochitinase genes are transcribed in the anterior silk glands,most of which are endochitinase genes mainly expressed during molt.Eight exochitinase genes are all transcribed in anterior silk gland,and they are mainly expressed during molt.The overall expression trends of the two chitinase genes are similar.To degrade chitin,they are mainly expressed during molt and wandering.Based on the changes of genetic level and the content of chitin,the chitin layer in the anterior silk gland intima mainly has three stages:renewal during molt,stable and thicken at each growth stage in larvae,and degradation during latter wandering.Among them,CHT-7 and CHS-2-3 are in accordance with the objective law of the existence of the chitin layer,and their expression in the 3rd molt is completely consistent with the expression in the 4th molt and the 5th instar.It shows that CHT-7 and CHS-2-3 can be used as potential targets to analyze the influence of intimal chitin layer on silk fiber formation.Most importantly,based on the anterior silk gland-specific high-expressing green fluorescent protein transgenic silkworm species constructed by our team,we directly observed the spatial position and movement of the anterior silk gland of the silkworm during wandering through a fluorescence microscope.It updated our previous understanding of the state of anterior silk gland in the body.The results showed that the front silk glands were twisted and folded and moved with the swing of the silkworm,and could be stretched or twisted at any angle.The front silk glands are rigid and flexible,combining soft and hard,and providing a stable spatial structure for the transportation of silk protein and the formation of silk fibers.2.Research on the regional spatial structure of the silk pressing areaOn the basis of the predecessors,we further analyzed the spatial structure of the silk pressing area,and found that there was a unique transition structure between the silk pressing area and the common tube for the first time,which was verified by section and electron microscope.The specific function of the unique transition structure remains to be further studied.The actual stretching frequency of the silk pressing area was calculated to be about 2Hz by using the transgenic strain of silkworm with a specific high expression of green fluorescent protein in the anterior silk gland.At the same time,the whole head of the silkworm was sectioned and observed,and it was found that the muscular system in the silk pressing area was developed and delicate,and there were 7 muscle bundles controlling the silk pressing area.3.Analysis of morphological structure and local mechanical characteristics of spigotThe last tissue in the silkworm spinning duct is the spigot.There is currently a lack of understanding of this silk-forming organ.First,we determined the basic morphology of the silkworm spigot through optical microscope and scanning electron microscope.The spigot has a conical shape as a whole,a complete curved structure at back,and three bearing skeletons at ventral.We summarize it as a three-axis belt surface structure.At the same time,we compared the spigot morphology of various silkworm strains and found that the morphology was conservative.The morphological changes of the spigot during the whole growth were observed by scanning electron microscope,and unique growth and development pattern was found.The shape of the spigot varies at different instar.At 1st-3rd instar,the structure of it is still a three-axis belt surface structure,but it is relatively slender as a whole.At the 1st instar,the shape of the spigot is very different from the profile of it at 5th instar.The morphology of spigot at the5th instar is finally stable and mature.During the growth of the silkworm,the spigot will continuously grow and adjust to serve the spinning behavior at each instar.Through a three-dimensional microscope with ultra-depth of field,we found that the spigot has a phenomenon of coloring and hardening.At early stage of wandering,the color of most areas of the spigot changed from white to brownish yellow,and the texture hardened.Among them,the changes of the three bearing skeletons at ventral were the most obvious.Through partial destruction experiment of the spigot,we proved that the it is a necessary functional organ for silkworm to spin.In order to further understand the composition of the spigot,the elemental composition of it was analyzed by scanning electron microscopy and energy spectroscopy（EDS）.There are C,N,O,Al,K,Ca,Fe,Cl,Ni and other elements in it.We believe that Al,K,Ca,Fe,Cl and other elements play an important role in the structure,composition and function of it.Furthermore,we analyzed the microstructure of the spigot by environmental scanning microscope.There are multi-level microstructures in the entire area of the spigot,and the hierarchical structure of the bearing skeleton area is more refined and changeable.The local stiffness of the spigot can be tested by an atomic force microscope,and it is found that the elastic modulus of different test areas is quite different,indicating that it has hierarchical mechanical properties.It is proved that different hierarchical structure of the spigot gives it different mechanical properties.On the whole,the spigot adopts components such as chitin protein complex,metal ions and water to build a rich multi-level microstructure from bottom to top,and to construct a rich multi-level microstructure from nano to macro,which endows it with high-strength classification mechanical properties and serves for long-term silking and cocooning of silkworm.4.The influence of the overall metal ion environment on the structure and performance of silk fiberThrough the ICP-AAS test,we found that the silk gland of the silkworm contains a variety of metal elements,the content of which is different in different sections,and it is relatively high at anterior silk gland.In addition,the content of various metal elements in silk glands changed greatly before and after wandering,indicating that the transport and conformational change of silk protein in the anterior silk gland depended on a certain metal ion environment.Through degumming experiments,it is confirmed that most of the metal elements are located in silk fibroin.The qualitative analysis of metal elements on the cross section of silk and cocoon was carried out for the first time by using a scanning electron microscope energy spectrum combined system.Furthermore,the metal ion chelating agent EDTA is used to disturb the overall metal ion environment during the formation of silk.Changes in the overall metal ion environment will disrupt the structure of silk fibers and reduce the mechanical properties of silk.Through iron ion injection,we found that iron ions can increase the content ofβ-sheets in silk fibers and improve the mechanical properties of silk fibers,which can be used as a target for subsequent improvement of the mechanical properties of silk fibers.5.A high-performance silk fiber transgenic silkworm strain was created using Fe³⁺Based on investigations above,we use iron ions as the target to modify the mechanical properties of silk fibers.The iron-related protein gene in anterior silk gland was screened,and ferritin was selected.Based on the stable overexpression transgene system of the anterior silk gland,an overexpression vector was constructed.Microinjection was carried out in the practical breed FR,and transgenic positive individuals were screened in the G₁ generation.The molecular level detection（Western Blot and q RT-PCR）of genetically modified individuals showed that the target protein was successfully overexpressed in anterior silk gland.The morphological observation of transgenic cocoons,cocoon nets and silk fibers showed no significant difference with the control groups.The basic statistics show that the genetically modified silkworm does not affect the basic economic traits such as cocoon layer rate and pupa weight of the species.Synchrotron radiation infrared spectroscopy analysis showed that the secondary structure content of transgenic silk changed significantly,and the content ofβ-sheet increased from 26%to 32%.The results of X-ray diffraction analysis also found that the content of crystals in the transgenic silk increased,which was consistent with the secondary structure of infrared analysis.The mechanical properties are measured and analyzed.The results showed that the strength and ductility of the genetically modified silk was significantly enhanced,and the toughness was greatly improved,making it stronger and tougher than the control group.The results above show that we have successfully improved the mechanical properties of silk fibers by genetically interfering with the ionic environment without changing the gene sequence of silk protein.More importantly,genetically modified individuals are practical varieties,which is suitable for subsequent industrial large-scale breeding and promotion.6.Research on the mechanism of Fe³⁺improving the mechanical properties of silkThrough turbidity analysis and CD analysis,it is found that iron ions can promote the conformation transformation of the regenerated silk protein solution.As the concentration of iron ions increases,the absorbance of the regenerated silk fibroin solution increases.CD analysis found that the secondary structure of the regenerated silk fibroin solution changed.Initial random coil and helical conformation gradually decreased,and theβ-sheet conformation gradually increased.By adding iron ions in the process of regenerating silk fibroin in vitro,it is found that its mechanical properties gradually increase with the increase of iron ion concentration.Both in vivo and in vitro experiments have proved that iron ions can change the secondary structure of silk protein.Through fluorescence spectroscopy analysis,we found that iron ions can specifically interact with tyrosine in regenerated silk protein,and the interaction with tyrosine alone will produce the same effect.In order to further study the interaction between iron ions and tyrosine,we synthesized（GAGAGY）₅ peptide fragments based on the amino acid sequence of silk protein.The fluorescence intensity analysis also showed that the fluorescence intensity of the peptide fragments gradually weakened with the increase of iron ion concentration.When we added Fe³⁺to serine-rich（Ser）polypeptide fragments,there was no obvious phenomenon.While flocky precipitate gradually appeared when Fe³⁺was added to tyrosine（Tyr）-rich polypeptide fragments.It was confirmed that iron ions specifically interact with tyrosine.At the same time,through software modeling,kinetic simulation of the interaction process between iron ions and tyrosine is carried out.In vitro experiments and molecular dynamics simulation results show that one of the mechanisms by which iron ions affect the structure and performance of silk fibers is that iron ions can interact with the tyrosine in the silk protein peptide chain promoting more production ofβ-sheets.In summary,this study systematically explores the structure and function of the chitin layer,the silk press and the spigot.Now we have a deeper understanding of the structure of silkworm’s natural spinning duct and its role played in the process of protein transport and silk fiber formation.At the same time,a genetically modified silkworm strain with improved silk fiber mechanical properties was obtained through genetic engineering methods without changing the silk protein sequence.It provides a new theoretical basis for subsequent silk protein solution flow and silk formation mechanism,silk fiber biomimetic research and the improvement of the mechanical properties of silk fiber through metal ions.