| Organisms have acquired a variety of survival skills over billions of years of evolution,and animal silk secretion is one of them.For silk-secreting animals,silk fibers play an important role in their survival and reproduction.Silkworm?Lepidoptera?is a kind of silk-secreting animal.Silkworm silk has been widely used in textile,medical,health food and advanced materials because of its excelent performance and environmental protection and non-toxicity.Silk is a kind of natural animal silk fibers.Animal silk is usual y considered as a semi-crystalline fibre material consisting of micro-crystalline zones formed by?-sheet which consist of reverse parallel arrangement dispersed in continuous amorphous zones formed by random coils/helixes.The aggregated structure with certain orientation formed by reassembly of crystal ine and amorphous regions endows silk with excelent mechanical properties.The structure gives silk excellent mechanical properties.The semi-crystalline structure of silk has a clear division of labor when provides mechanical properties.The crystalline region determines the strength and elastic modulus of silk.The total amount of crystals,particle size,orientation and integrity in the crystalline region will affect the strength and modulus properties of silk.The non-crystal ine region mainly determines the ductility of silk.Both of them determine the toughness of silk,which is a comprehensive index for evaluating the properties of silk.Based on the current understanding of silk structure,properties and the relationship between them,many scientists are committed to improving the mechanical properties of silkworm silk to expand its application.At present,the methods of modifying silk include physical modification,chemical modification,modification by adding special substances,development of regenerated silk fibroin fibers and modification by transgenic technology.However,successfully research on improving the mechanical properties of silk is very rare,mainly because our understanding of the relationship between the structure and properties of silk is not deep enough,and the knowledge we have now is not enough to accurately guide us in directional modification of silk.Although scientists have analysed the multilayer structure of silk from a microscopic point of view through nuclear magnetic resonance?NMR?,synchrotron radiation Fourier transform infrared spectroscopy?FTIR?,Raman spectroscopy,wide-angle X-ray diffraction and smal-angle X-ray scattering.However,how to assemble the secondary structure of silk into a higher aggregate structure,how to synergize different levels of structure to give silk excellent comprehensive mechanical properties,and the reasons for the difference in mechanical properties between different types of silk are still unclear.There is still much unknown information waiting for researchers to explore.Based on the above analysis,we explored the effects of bending treatment on the structure and properties of newborn silk,analyzed the differences in structure and properties of different instar silks,and obtained some new rules of structure and properties.Then we used these new rules to guide us to use genetically modified technology to modify the mechanical properties of silk.The main results are as follows:1.The influence of bending treatment on the structure and properties of newborn silkWe first designed a very precise adjustable speed stepping motor system used in forced reeling silk.The observation results of surface and cross-sectional morphology of bending treatment silk with different angles showed that the silk is composed of two brins,bending treatment does not change the number of brins of silk;with the increase of bending angle,the diameter of silk long axis becomes smaller and smaller,that is,the silk becomes thinner and thinner.The results of mechanical properties test showed that with the increase of bending angle,the elongation of silk decreases gradually,and the elastic modulus,strength and toughness of silk increase first and then decrease.The elastic modulus,strength and toughness of silk are the best in the bending range of 67.5°to 90°.The secondary structure of silk with bending treatment is analyzed by synchrotron radiation FTIR.The results showed that with the increase of bending Angle,the content of silk's?-sheet structure increases gradually.This indicates that in the process of forced reeling and bending treatment,the increased angle will increase the interaction force between the silk and the rotor,which will further promote the formation of more?-sheet structure.We used synchrotron radiation wide angle X-ray diffraction technology has carried on the crystal structure analysis to silk with bending treatment,first of al,we calculated the crystal inity of silk with bending treatment,the results showed that with the increase of bending angle,silk crystal inity increase gradually,the results are consistent with FTIR,because crystal structure is mainly composed of?-sheet.Then we used Herman orientation factor method to analyze silk with bending treatment orientation,the results showed that with the increase of bending angle,silk orientation factor gradually reduced.Finally,we calculated the crystal size of silk with bending treatment at various angles by using the Scherrer formula,and the results showed that with the increase of bending angle,the crystal particles of silk gradually increased,which was consistent with results of FTIR and crystallinity analysis.The influence of bending treatment on the mechanical properties of newborn silkworm silk can be summarized as follows:with the increase of bending Angle,the crystallinity and crystal size of silkworm silk increases gradually,while the orientation factor decreases gradually.In the early stage,the increase of mechanical properties due to the increase of crystallinity plays a major role,in the later stage,the decrease of mechanical properties due to the decrease of orientation factor and the increase of crystal size play a major role.Therefore,comprehensive mechanical properties of silk with bending treatment at different angles increased first and then decreased.The range of bending angle from 67.5°to 90°is the boundary.When the bending angle is in this region,silk shows the best mechanical properties.Then we used the three-dimensional space coordinate system and space vector angle calculation formula to establish the analysis method of natural bending angle of silk.The results showed that the range of natural bending angle of silk is 70.23°to 87.98°.This result is just in the best bending angle range of mechanical properties.The natural bending angle of silk coincides with the best bending angle range of mechanical properties during silkworm spinning.We believe that this is the result of evolutionary selection:Because of the characteristics of silk protein,silk fibers need bending treatment in the range of67.5°-90°to achieve the best mechanical properties.In order to obtain the best performance silk to construct the best protective cocoon,The silkworm's silk-spinning behavior has gradually evolved into its present form.This may also be one of the reasons why the head of silkworm spins“8”-shaped.Of course,there is another possibility.Because of its own physiological structure,the silkworm can only spin in the“8”-shaped path,and the newborn silk will inevitably bend in the range of 70.23°to 87.93°.In order to avoid the influence of bending on the mechanical properties of silk and the protective properties of cocoon,through evolution,silkworm selected a silk protein with the best mechanical properties after bending treatment in the range of 70.23°-87.93°.The silk and cocoon made of this silk protein has the best mechanical properties,which are most conducive to the protection of pupae and the survival and reproduction of silkworm.2.Structural and mechanical properties of silk from different instars of Bombyx moriFirstly,we collected 10 kinds of silk from different developmental stages of silkworm,I-B silk,I-E silk,II-B silk,II-E silk,III-B silk,III-E silk,IV-B silk,IV-E silk,scaffold silk and cocoon silk.Then we measured the mechanical properties of 10 kinds of silk accurately.The results showed that 10 kinds of silk can be divided into three categories:instar beginning silk,instar end silk and mature silk?scaffold silk and cocoon silk?.The mechanical properties of larva silk?instar beginning silk and instar end silk?are better than that of mature silk,and the instar end silk has super mechanical properties.In terms of biological functions,it is inevitable that the mechanical properties of larva silk are better than that of mature silk,because in the process of foraging,feeding and moving,in order to resist harsh natural environment?mainly wind blowing and vibration caused by it?,silkworm larva need to spit out larva silk with excelent mechanical properties to ensure that they do not fall from mulberry leaves or trees.Especially at the end of the instar end stage,the silkworm larva also needs more excelent mechanical properties of instar end silk to fix itself and help itself molt.Therefore,larva silk is very important for the growth and development of silkworm larva,so larva silk has excelent mechanical properties.For mature silk,a large number of mature silk gathered together,its comprehensive mechanical properties are enough to protect the pupa,so it does not need single mature silk mechanical properties are also excellent.Then we characterized the secondary structure of 10 kinds of silk by ATR-FTIR.The results showed that the content of?-sheet of instar end silk was higher than that of mature silk and instar beginning silk.This is the reason why instar end silk have high mechanical properties.Then we analyzed the crystal inity of 10 kinds of silk by wide-angle X-ray diffraction?XRD?.The results showed that the crystallinity of silk decreased gradually with the increase of instar,which is the reason why the elongation of larva silk is lower when the instar is smaller.Comprehensive mechanical property of IV-E silk is the best,but its crystallinity is not the highest.Therefore,the crystal inity is not completely positively correlated with the mechanical properties.Only when the balance of the structure of?-sheet crystal,?-helix crystal,?-helix amorphous structure and random coil structure is reached,can the silk obtains the best mechanical properties.Finally,we compared and analyzed the difference in main silk protein content between high performance silk—IV-E silk and cocoon silk by LC-MS/MS technology.The results showed that the content of P25 protein increased about 2.9 times in IV-E silk.Because P25 protein is a small molecule protein with molecular chaperone function,it plays an important role in the assembly and secretion of the fibroin elementary unit.It is speculated that the addition of P25 protein in IV-E silk can promote the formation of more?-sheet structure of Fib-H protein,connect macromolecules and enhance the stability of molecule network,thereby enhancing the mechanical properties of IV-E silk.3.Creation of high performance silk materials by transgenic technologyWe selected Tenebrio antifreeze protein AFP,Drosophila melanogaster heat shock protein HSP?molecular chaperone protein?and spider silk small molecule rich in cysteine protein CRP as target proteins,next constructed three fusion recombinant protein expression vectors,and then obtained transgenic silkworm strains AFP,HSP and CRP by injection and screening of positive individuals.The results of insertion site analysis showed that the target gene AFP insertion site was 341298–3412373 of scaffold 45 in chromosome 7 of the silkworm genome,the HSP insertion site was 6140556–7406261 of scaffold 43 in chromosome 24 of the silkworm genome,and the CRP insertion site was9968206–11378158 of scaffold 16 in chromosome 11 of the silkworm genome.RT-PCR results showed that three target protein genes were successfully expressed in the posterior silk gland cels of Bombyx mori.Immunofluorescence results of the posterior silk gland showed that 3 target proteins were successfully expressed in the posterior silk gland cells of the silkworm and secreted into the lumen of the silk gland.Western blot results of transgenic cocoon silk showed that the 3 target proteins were successfully incorporated into silk.The phenotypic observation of silk gland and transgenic silkworm cocoon showed that the three fusion recombinant proteins had no significant effect on silk gland development,cocoon size,cocoon shape and color.However,AFP and HSP can make the transverse section of the transgenic silkworm cocoon loose and porous,and the last three fusion recombinant proteins have no significant effect on the cocoon shell percentage and the average diameter of the cocoon silk.The results of mechanical properties test showed that the mechanical properties of AFP and HSP transgenic silk were improved comprehensively,but the mechanical properties of CRP silk were not improved obviously.The order of comprehensive mechanical properties of silk was AFP>HSP>CRP>D9L.Comparing the dynamic mechanical properties storage modulus-temperature curves of three transgenic silk strains,the results showed that the molecular tightness of the amorphous region of the transgenic silk was lower than that of the control D9L,and the molecular tightness of the amorphous region of the HSP transgenic silk was the lowest because of its maximum molecular weight.The loss factor-temperature curve shows that two low-temperature phase change loss peaks of the transgenic silk T?1 and T?2 move to the high-temperature region,it indicated that the ability of binding water in the amorphous region of transgenic silk was enhanced.The loss factor-temperature curve shows that there is an additional phase loss peak of T?x in transgenic silk than in D9L silk.We speculate that this is caused by the interaction between foreign fusion protein and silk protein.In addition,the main phase transition loss peak Tg2?glass transition temperature?of the transgenic silk shifted to the high-temperature region,which indicated that the activity of the whole molecular chain in the amorphous region of the transgenic silk was affected by the cross-linking of the recombinant fusion protein,and the interaction between the molecular chains was strengthened.It can be confirmed that the incorporation of the fusion protein into the amorphous region of transgenic silk affects the dynamic mechanical properties of silk.The results of synchrotron radiation Fourier transform infrared spectroscopy?SR-FTIR?showed that the content of?-sheet in silk of three transgenic strains was increased,and the order of content was AFP>HSP>CRP>D9L,which was consistent with the results of mechanical properties.Results of synchrotron radiation wide-angle X-ray diffraction showed that the crystal inity of silk of three transgenic strains was improved,and the order of crystallinity was AFP>HSP>CRP>D9L,which was consistent with the results of FTIR and mechanical properties.The crystal size of silk of three transgenic strains decreased,and the order of crystal size was D9L>CRP>HSP>AFP.The orientation factor of the silk of the transgenic strains decreased,and the order of orientation factor was D9L>CRP>HSP>AFP.The reasons for the significant improvement of mechanical properties of AFP and HSP can be attributed to the increase of crystal inity,grain refinement,slightly decrease of orientation factor,and the cross-linking effect of fusion recombinant protein. |
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