Research On The Mechanism Of Silk Processing In Silkworm And Development Of Novel Silk Gland Expression System

The silkworm（Bombyx mori）is a completely metamorphic lepidopteran insect.Its life cycle includes four metamorphic development stages of egg,larva,pupa,and adult（moth）.The silk fiber of silkworm has excellent mechanical properties,biocompatibility and plasticity.It is widely used in industrial products,national defense and medical supplies.Silk fiber is composed of proteins synthesized by silk glands in vivo,mainly including sericin and fibroin.In recent years,researchers have focused on the mechanism of the silkworm converting silk protein synthesized by silk glands into silk fibers under normal temperature and pressure environments.Previous studies have shown that many factors will affect the fibrosis process of silk protein gradually transforming from the disordered state in the silk gland lumen to the ordered state in the cocoon silk.Including the changes in the physiological and biochemical environment（including p H value,metal ion content）in the silk gland lumen of the silkworm,the types of silk proteins,the interaction between different silk proteins,and the changes in shear force during spinning.At present,the analysis of the mechanism of silk protein from the liquid state in the silk gland lumen to the solid state of theβ-sheet in the silk fibers mainly includes four models:semi-crystallite model,cylindrical fibrils model,string of beads model and micelle model.Among them,the micelle model has gradually become one of the most recognized and most important theoretical models of silk formation.In this model,the hydrophilic conserved N-terminal domains（NTD）and C-terminal domains（CTD）of fibroin heavy chain protein（Fib-H）were located in the outside of micelle,and the intermediate repeating hydrophobic domain was located in the inside of micelle.However,the micelle model was mainly proposed by the results of in vitro simulation experiments assuming that the mixture of sericin and fibroin participates in the formation of silk fibers.And the experiment did not consider that fibroin was composed of the fibroin elementary unit formed by Fib-H,Fib-L（fibroin light chain protein）and P25（glycoprotein）with a molecular ratio of 6:6:1.Therefore,the relationships and roles of sericin and fibroin in the silk processing,and functions of each component of fibroin such as Fib-H,Fib-L and P25 in the silk processing are still needed to be further clarified.Furthermore,the mechanism of silk processing in silkworm was analyzed and improved.In addition,based on the ability of silkworm silk glands to synthesize proteins quickly and efficiently,development of new-type silkworm bioreactors,especially the silk gland bioreactors,has been a research hotspot since the first transgenic silkworm was obtained in 2000.The use of silk glands to express exogenous proteins has been developed so far,and there were still many problems such as low expression levels of exogenous proteins,low or inactive exogenous proteins in transgenic fibroin,and difficulty in purification of exogenous proteins.These problems have directly led to the concept of the silk gland bioreactor being proposed for nearly 20 years,and the real industrialization has not yet been realized.Therefore,based on the analysis of the mechanism of silk processing in silkworm,combined with silkworm transgene and gene editing technology,explore and develop a new-type fibroin expression system that can easy purification and high content of exogenous protein.Solve the above-mentioned problems existing in the silk gland bioreactor,in order to finally promote the industrial development of the silk gland bioreactor of the silkworm.Based on the above considerations,this paper has carried out research in three aspects:（1）Sericin and fibroin expression systems were used to express sericin fusion red fluorescent protein（Ds Red/Ser1）and fibroin Fib-H fusion green fluorescent protein（EGFP/Fib-H）,respectively.By observing the distribution and movement of exogenous fusion fluorescent protein in silk glands and cocoon silk,the relationship between sericin and fibroin,and the role of sericin in the silk processing were analyzed.（2）Use the fibroin expression system to express four exogenous EGFP/Fib-H fusion fluorescent proteins（NGC,containing NTD and CTD of Fib-H;NG,containing NTD of Fib-H;GC,containing CTD of Fib-H;G,without NTD and CTD of Fib-H）.By observing the distribution and movement of these exogenous fusion fluorescent proteins in silk glands and cocoon silks,the role of the NTD and CTD of Fib-H and Fib-L in the silk processing were analyzed and verified.Analyzed the mechanism of fibroin self-assembly and the mechanism of silk processing in silkworm.（3）Based on the exploration of the mechanism of silk processing in silkworm,explore and develop a fibroin Fib-H expression system that was easy to purify exogenous protein targeting silkworm cocoons;Use CRISPR/Cas9 gene editing technology to develop a fibroin Fib-H expression system for high-efficiency expression of exogenous proteins targeting silk glands.The main research results of this paper are as follows:1.The function of sericin in silk processingThroughing the microinjection of silkworm embryos,the constructed vector of silk gland expression system was introduced into the silkworm genome and the following transgenic lines were obtained:1,The transgenic line Ts-R containing the expression cassette"Ser1P---Ds Red---SV40"containing a sericin-specific promoter expresses the exogenous protein R（Ds Red/Ser1 fusion fluorescent hydrophilic protein,the grand average of hydropathicity was-0.578（a negative value indicates that the protein was hydrophilic））.2,The transgenic line Th-NGC containing the expression cassette"Fib-H P---NTD-EGFP-CTD---poly（A）"containing a fibroin-specific promoter expresses the exogenous protein NGC（EGFP/Fib-H fusion fluorescent hydrophilic protein containing NTD and CTD of Fib-H,the grand average of hydropathicity was-0.537）.3,Transgenic lines Ts-R&Th-NGC were obtained after crossing the transgenic lines Ts-R and Th-NGC,which expressed R and NGC at the same time.By observing the distribution of R and NGC in different parts of the silk glands and cocoon silk fibers,it was found that R only existed in the outer sericin layer and NGC only exists in the middle fibroin layer from the silk glands to the cocoon silk.The distribution characteristics of R and NGC indirectly proved the location of sericin and fibroin,indicating that sericin and fibroin have never been mixed during the silk processing in the silkworm.Sericin was wrapped around the fibroin from beginning to end,providing a relatively closed environment for fibroin.The sericin of middle silk gland（MSG）as aquasorb,to ensure the fibroin in MSG lumen has certain water content,to prevent the premature and crystallization of fibroin.The sericin of anterior silk gland（ASG）as a dehydrant,similar to a sponge,and absorbs and stores dissociative water from the fibroin layer in the ASG.Sericin was a kind of gel protein,which acts as a lubricant in the process of forward advancement in the silk gland,which will reduce the shear force in the silk gland cavity,especially the shear force from the ASG of filamentous duct and the spinneret.In this chapter,we also found that the expression of R in the sericin layer and NGC in the fibroin layer significantly reduced the mechanical properties of silk fiber,which indirectly indicated that both sericin and fibroin could significantly reduce the mechanical properties of cocoon silk.The influence of the fibroin layer NGC was far greater than that of the sericin layer R.The results show that fibroin was the main factor and sericin was the secondary factor for the mechanical properties of silk fiber.It was speculated that the fusion of NGC and fibroin will destroy the aggregation,assembly and crystallization of fibroin,thereby affecting theβ-sheet structure during the silk processing,and ultimately reducing the mechanical properties of cocoon silk.The fusion of R and sericin changes the content of sericin,thereby affecting the function of sericin,which changes the spinning conditions of the transgenic silkworm in silk processing,which in turn affects the speed and progress of fibroin crystallization,and ultimately reduces the mechanical properties of cocoon silk.2.Research on the mechanism of silk processing in silkwormUsing the fibroin Fib-H expression system,four transgenic silkworm lines（TS-H-NGC,expressing the exogenous hydrophilic protein NGC fused with NTD and CTD of Fib-H,the grand average of hydropathicity was-0.499;TS-H-NG,expressing the exogenous hydrophilic protein NG fused with NTD of Fib-H,the grand average of hydropathicity was-0.55;TS-H-GC,expressing the exogenous hydrophilic protein GC fused with CTD of Fib-H,the grand average of hydropathicity was-0.416;TS-H-G,expresses the exogenous hydrophilic protein G containing only the signal peptide of Fib-H,the grand average of hydropathicity was-0.486）expressing different fusion green fluorescent proteins（EGFP/Fib-H）were obtained.The distribution of fusion fluorescent protein in silk gland and cocoon silk was different,indicating that NTD and CTD have different functions.The fibroin elementary unit are assembled by Fib-H,Fib-L and P25 with a molecular ratio of 6:6:1.These exogenous proteins fused with NTD or CTD can form different fibroin elementary units of exogenous.According to the primary structure of Fib-H and the hydrophilicity and hydrophobicity of different components of the fibroin elementary unit,fibroin easily forms a micelle structure with internal hydrophobic and external hydrophilic.The micelle model provides a theoretical model for silk processing.However,the micelle model does not consider the existence of the fibroin elementary unit composed of Fib-H,Fib-L and P25,especially the combination of the CTD of Fib-H and Fib-L through disulfide bonds（CTD==L,"=="means disulfide bond）.The results of the distribution of several fusion fluorescent proteins containing NTD or CTD in silk glands and cocoon silk showed a difference from the previous micelle model.No matter whether the fibroin elementary unit was hydrophilic or hydrophobic,as long as CTD==L was contained,it exists in the hydrophobic fibroin layer,especially in the hydrophilic[H==L]_（1）[NGC==L]_（5）---[P25]_（1）,[NGC==L]_（6）---[P25]_（1）and GC==L.The exogenous protein in the sericin layer obtained by immersing TS-H-NG cocoons in PBS was hydrophilic[NG]_（6）---[P25]_（1）.Studies have shown that hydrophilic NTD can be negatively charged in weakly alkaline PSG and neutral MSG to prevent premature crystallization of silk protein in vivo.It can be speculated that the hydrophilic NTD was located at the periphery of micelle.Observation results of fusion proteins containing CTD:The fibroin elementary unit containing CTD==L were all present in the fibroin layer,no matter whether it was hydrophilic or hydrophobic.Therefore,we speculate that CTD==L was located inside micelle.The protein dot hybridization experiment confirmed:NTD was on the outside of micelle,CTD==L and the middle repeat hydrophobic domains of Fib-H were on the inside of micelle.Finally,based on the experimental results in this chapter,a dynamic formation model of silk protein from PSG to cocoon silk fiber was proposed.The fibroin elementary unit was secreted into the silk gland lumen to form micelle.The exterior of micelle was hydrophilic NTD.The weakly alkaline PSG and neutral MSG environment make NTD have negative charge,that is,a large number of negative charge on the surface of micelle,which causes micelle to repel each other,thus effectively preventing them from agglomerating into larger micelle and resulting in premature crystallization.The hydrophilic CTD==L and Fib-H repeated crystallization regions were located inside the micelle,which increases the solubility of the micelle and effectively avoids the premature crystallization of fibroin.The emergence of sericin provides a relatively closed environment for fibroin,which absorbs and stores dissociative water in the fibroin layer and other hydrophilic protein molecules such as G and[NG]_（6）---[P25]_（1）that do not contain CTD.When fibroin reaches the weakly acidic environment of ASG,the acidic amino acids of NTD were protonated,which reduces the negative charge and weakens the electrostatic effect,which further promotes the folding of NTD intoβstructure,making silkⅠ（usually including random coil andα-Helix）was transformed into silkⅡ（a regular array of anti-parallelβ-sheet structures connected by hydrogen bonds）,and finally the silk fibers were stretched out by the squeezing of the spinneret and the"8"swing of the silkworm itself to form a cocoon shell.3.Research and development of an easy-purify,high-content silk gland expression systemThrough the exploration of the mechanism of silk processing in silkworm,it was found that the exogenous protein without NTD and CTD obtained by the fibroin Fib-H expression system was located in the sericin layer of cocoons.In this chapter,using PBS or H2O to directly soak cocoons can dissolve the exogenous protein in the sericin layer,and the acquisition rate was more than 80%,which was very convenient for the purification of exogenous protein and effectively solves the problem of purification of proteins expressed by the fibroin Fib-H expression system.Studies have reported that the use of gene editing technology to knock out Fib-H will increase the expression of exogenous proteins.Combined with the exploration of the mechanism of silk processing in silkworm and CRISPR/Cas9 gene editing technology,this chapter uses the EGFP protein without NTD and CTD of Fib-H as the target protein to explore the development of a fibroin Fib-H expression system with high content of exogenous protein.The exogenous protein does not fuse the NTD and CTD of Fib-H,which guarantees the activity of the exogenous protein to the greatest extent.This chapter successfully obtained a transgenic line TS-3C expressing Cas9protein and a transgenic line TS-g-G expressing EGFP with g RNA targeting Fib-H.Molecular experiments and protein analysis have identified transgenes.For knockout mutants:（1）The sequence alignment of the target site reveals that there were different degrees of base deletion and insertion,and the total knockout rate was about 55%;（2）The weight of silkworm pupa increases（regardless of female or male pupa）;（3）PSG has obvious tumor-like appearance and surface was not smooth;（4）Fib-H gene expression level was significantly reduced,and GFP gene expression was significantly increased;（5）The pupation rate was only 22%.The cocooning rate and emergence rate were 0,and no heritable offspring can be produced,and there was no concern about the safety of genetically modified organisms;（6）The length of PSG was reduced by 18%.The fresh weight of silk glands was reduced by 10%,and the dry weight was reduced by 38%.In this chapter,H₂O was used to directly soak the silk gland grinding powder of knockout mutants.The highest rate of obtaining exogenous EGFP protein was 59.7%.On average,each silkworm can obtain 51.6 mg of exogenous protein.In this chapter,the use of CRISPR/Cas9 technology to knock out Fib-H and use the optimized fibroin Fib-H expression system to express exogenous proteins（non-fusion protein,to ensure the activity of exogenous protein to the greatest extent）effectively solves the problem of low exogenous protein content and promotes the industrialization of silk gland reactors.