| Natural silk protein(fiber)is a kind of high molecular protein resource produced and secreted by silk-producing animals.Among them,silk and spider silk are the two silk proteins most admired and studied deeply.Although the performance of spider silk is excellent,but limited to the spider group can not be cultured and it is difficult to obtain large-scale.Although the mechanical properties of silk protein secreted by Silkworm(Bombyx mori)are inferior to that of spider silk,it has been completely domesticated and utilized by human beings for a long time.It is the only silk secreting animal that can produce natural silk protein at a large scale and low cost.Domestic silk used to be mainly used in silk spinning.In recent years,studies have found that silk has good biocompatibility,adjustable structure,degradable and other characteristics,in medicine,food,daily fine chemicals,microelectronics and other fields of application potential is very broad,relevant research and development is booming.Meanwhile,genetic manipulation techniques such as transgenic and gene editing for Bombyx mori have become mature.How to use such techniques to precisely transform or endue silk with new properties has become a hot research topic in recent years.According to the literature,Caddisflfly(Stenopsyche marmorata),an insect living in natural water,can spin silk in water and form cocoons by sticking dead branches and cobblestones.Its silk has excellent viscoelasticity and antibacterial properties.The main components of silk gland are Smsp-1,Smsp-2,Smsp-3 and Smsp-4 proteins.Interestingly,the structure and binding mode of Smsp-1,Smsp-3 and Smsp-4 are similar to those of fibroin heavy chain protein(Fib H),light chain protein(Fib L)and glycoprotein(P25).So,can these caddisflfly silk proteins be introduced into the silk gland to transform or give silk new properties? In view of this,this paper carried out the transgenic expression attempt of Smsps protein,and the results are as follows:1.Transfer the production of silkworm material of Smsps Firstly,Smsp-1,Smsp-3 and Smsp-4 were used as target genes,RFP and EGFP were used as controls,and transgenic expression vectors with Fib H and Fib L as promoters were constructed.Then,the corresponding positive individuals were obtained by embryo microinjection and fluorescence screening.Five transgenic materials were obtained by hybridization with the practical silkworm mother "Autumn White".The materials of single Smsps were named H-Sm1,H-Sm4 and L-Sm3,respectively,and the positive control group was named H-RFP and L-EGFP.The results of genomic PCR showed that the transgenic silkworm was successfully prepared.Further,according to the similarity between Smsps and three silk fibroin proteins of silkworm,the single transgenic lines were crossed in pairs and the corresponding positive individuals were screened from their hybrid progenies,which were named H-Sm1/H-Sm4,L-Sm3/H-Sm4 and H-RFP/L-EGFP(positive control).The results of genomic PCR showed that the transgenic silkworm material was successfully prepared and could be used for further research.2.Gene expression analysis of transgenic Silkworm Smsps In order to confirm whether exogenous Smsps were expressed in the silk glands of transgenic silkworms,fluorescent quantitative PCR and Western blot analysis were performed on the posterior silk glands and corresponding cocoon shells of transgenic silkworms on the 3rd and 6th day of the fifth age(L5D3)and(L5D6)respectively.The results showed that:(1)Sm1,Sm3 and Sm4 were all expressed in the posterior silk glands of silkworm.The expression levels of Sm1 and Sm3 increased with the growth of instars,but Sm4 decreased with the growth of instars.(2)The expression levels of endogenous fibroin genes Fib H and Fib L decreased significantly,but P25 did not change significantly,indicating that the introduction of exogenous Smsps into domestic silk glands can affect the expression of fibroin gene.(3)Target proteins Sm1 and Sm3 were detected in the cocoon shell of transgenic silkworm,indicating that Smsps protein was synthesized by posterior silk gland cells and secreted into the cocoon shell,which laid a foundation for further analysis of the effect of transferring Smsps protein on silk properties.3.Analysis of cocoon and silk properties of transgenic Silkworm Smsps In order to analyze the effects of Smsps on silkworm cocoon and silk properties,the pupal cocoons of the transgenic strains were observed on the 5th day of pupal stage(P5D).The results showed that:(1)compared with the control group,the cocoons of the transgenic strains were thinner and smaller;The changes of H-Sm4,H-Sm1/H-Sm4 and L-Sm3/H-Sm4 were obvious and showed thin calluses.(2)Compared with the control group,the total cocoon weight,cocoon layer quantity and cocoon layer ratio of silkworm treated with Smsps decreased;H-Sm4 and H-Sm1/H-Sm4 decreased significantly,which was consistent with the phenotypic observation.These results indicated that the expression of exogenous Smsps inhibited the synthesis and secretion of silk fibroin protein,leading to the decrease of cocoon layer quantity and even the formation of thin skin cocoon.4.Performance analysis of trans Smsps silk In order to investigate whether the transfer of exogenous Smsps would change the properties of silkworm’s silk,we carried out characterization,properties and structure tests on cocoon filaments of transgenic strains at the 5th day(P5D).The results showed that:(1)the diameter of silk transformed with Smsps was smaller and the breaking energy was improved,indicating that the transfer of Smsps significantly improved the comprehensive mechanical properties of silk.Based on the changes in the ductility and stiffness of various strains,it was concluded that Sm1 mainly improved the ductility of silk,Sm3 mainly improved the stiffness of silk material,and Sm4 not only promoted the stiffness of silk,but also contributed to the improvement of ductility.(2)The contents of β-folding and β-turning of spun Smsps decreased,while the contents of random coil increased,suggesting that the comprehensive mechanical properties of spun Smsps could be improved by increasing the contents of random coil.(3)The expression of Smsps inhibited the growth of E.coli and reduced its colony number,indicating that Smsps had good antibacterial effect.Among them,L-Sm3 and L-Sm3/H-Sm4 has the lower number of colonies,which suggest that Sm3 may play a more important role in bacteriostasis.In conclusion,this study confirmed the feasibility of genetic modification technology at the individual level by making and analyzing the silkworm material transformed into Smsps,and then obtained the new silk material with excellent antibacterial and comprehensive mechanical properties through characterization and performance testing.These results provide important clues and theoretical reference for further improving the properties of silk and endow silk with new properties. |
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