| The world’s oldest east-west trade channel was named"Silk Road"because of the great influence of silk products,and China is also called"Silk country"because of its rich silk fabric,which is basically due to the performance of economic insect silkworm cocoon.The silk fiber is composed of two silk fibroins wrapped with sericin.The characteristics of silk are the basis of application and processing,and the characteristics of silk are determined by the structure of silk fibroin and sericin.Among them,the characteristics of sericin will affect the processing of silk and the performance of silk fibers.The advantages of sericin are gradually being discovered.In terms of biological materials,sericin has the advantages of anti-apoptosis,promoting cell proliferation and differentiation.It has high application value and is widely used in tissue engineering,regenerative medicine,cosmetics industry and new composite materials.At present,the basic research on the composition and mechanism of sericin is very weak,and its protein composition and structural characteristics are urgently needed to be systematically investigated.The unique protein composition of silk and its fiber-forming process are the fundamental reasons for the excellent performance of silk.Therefore,a clear study of the role of different sericin in silk fibers has a certain guiding role for the diversified development of the entire silk industry.Three sericins have been found,including sericin1（Ser1）,sericin2（Ser2）and sericin3（Ser3）.The sericins of cocoon silk are mainly consist of Ser1 and Ser3,and the amount of Ser1 is about four times than that of Ser3.The Ser2 exists in silkworm larvae,and it is the primary sericin in young larval silk spun before and after molting.Cocoon sericin mainly adheres silk fibroin tightly to form a cocoon.Since the sericin is difficult to separate and purify,existing studies are based on the protein of entire sericin layer.It was believed that sericin could lubricate and protect silk fibroin.The larval stage before the fifth instar,we call it the young larval instar.Young larval silk mainly plays the role of fixing the molting silkworm,assisting in molting,helping ingestion and preventing falling.Ser2 was considered providing better adhesion performance for young larval silk,but these views were lack of more datas.The three sericin proteins Ser1,Ser2 and Ser3are coded and synthesized by the corresponding sericin genes.The coding genes are located on chromosome 11 and the length of the genes is relatively large.Since Japanese scholars finished the identification of the sericin3 gene 12 years ago,no sericin gene has been discovered.With the help of three generations of genomics and proteomics technology,we have found some sericin-like proteins in the silk glands of larvae before the fifth instar.The characteristics of their coding genes are consistent with the three reported sericin genes,and the previous gene annotations existed errors and missing.After further screening,we listed BGIBMGA011896 and BGIBMGA001819 as candidate sericin genes and named them sericin4 and sericin5.In this study,the two newly discovered sericin genes,sericin4 and sericin5,were selected as the research objects,and a variety of technical methods such as molecular biology,proteomics,transcriptomics,gene editing,materials science,and genetic modification were used to intensely studied.The functions of silk and silk glands and the relationship between silk proteins have been deeply studied.The main results are as follows:1.Identification of novle sericin proteins and analysis of sericin composition.By proteomic analysis of segmented silk glands at different stages of the fourth instar,we found several highly abundant unknown functional proteins.Further screening by expression profile,molecular weight,chromosome location,gene length and gene structure,we listed BGIBMGA011896 and BGIBMGA001819 as candidate sericin genes,named sericin4 and sericin5,and their encoded proteins were abbreviated as Ser4 and Ser5.Sericin4 and sericin5 gene are located in chromosome 11 in the same position with three sericin genes reported.The new sericin genes contain long length and large fragment repeat motifs,and its theoretical molecular weights are more than 100 k Da.Integrating the three generations of genome sequencing,transcriptome sequencing and RACE results,we successfully mapped the complete gene structure of sericin4 and sericin5.The gene length of sericin4 and sericin5 is 40 kb and 22 kb,respectively,and contain 34 and 16exons separately.The period expression characteristics analysis found that the period expression patterns of sericin4 and sericin5 genes were similar with sericin2,with high expression at young larval instars,gradually decreased expression with instar,and no expression at the fifth instar.The results of q PCR and Western blot found that in the fourth instar silk glands,sericins from the outer layer to the inner layer are successively Ser2,Ser5,Ser4 and a small amount of Ser1 proteins;in the fifth-instar silk glands,sericins from the outer layer to the inner layer are successively Ser2,Ser3 and Ser1 proteins.For the first time,we found that Ser2 protein is expressed differently in the fourth and fifth instar silk glands.Through immunofluorescence localization analysis,it was found that Ser4 and Ser5 were located in the same position with Ser1 and co-localized in the sericin layer.By SDS-PAGE and werstern blot of silk,we determined the size and distribution of Ser4 and Ser5 protein bands.Finally,we identified the bands corresponding to Ser4and Ser5 proteins by mass spectrometry and confirmed the presence of Ser4 and Ser5proteins in young larval silks.2.Functional study of new sericins.With the help of CRISPR/Cas9 gene editing technology,we completed the knockout of the sericin4 and sericin5 genes in the silk gland of silkworms.The knockout strains were sequenced and screened by PCR,and the edited forms of deletion of single base and two bases were selected to be homozygous,and finally sericin4 and sericin5 homozygous knockout strains were obtained.We found that Ser4 homozygous knockout strains(Sericin4-/-)silkworms,compared with the control g RNA strains（g RNA-Ser4）,have reduced spining silk and smaller silk glands in young larval instar.Compared with the control g RNA strain（g RNA-Ser5）,the Ser5 homozygous knockout strain(Sericin5-/-)also showed smaller silk glands in young larval instar,but there was no significant change in the amount of silk spinning.The young larval silks were observed by scanning electron microscope,it was found that knocking out Ser4 and Ser5 led to a significant reduction in the diameter of silk.Further testing of the mechanical properties of Sericin4-/-and Sericin5-/-strains of silk,it was found that knocking out Ser4 and Ser5 resulted in a significant decrease in the performance of young larval silks,and knocking out Ser4 was even more pronounced.We analyzed the changes of the main silk proteins after knocking out Ser4 and Ser5 through quantitative PCR,Western blot and proteomics analysis.We found that knocking out Ser4 resulted in a significant decrease of three fibroin proteins Fib-H,Fib-1,P25 and Seroin1,but the Ser2 protein was significantly up-regulated.Knockout Ser5 also resulted in a significant down-regulation of Fib-H and Serion1.3.Transgenic overexpression of Ser4 protein to create new silk material.By seamless cloning,we successfully constructed a full-length Ser4 transgenic overexpression vector,and after micro-injecting silkworm eggs,we finally screened positive individuals.Transcription level and protein level test showed that Ser4 protein was successfully overexpressed into cocoon silk.The results of mechanical properties tests showed that overexpression of Ser4 increased the strength and Young’s modulus of cocoon silk.The results of FTIR are also consistent with the mechanical properties,and the silk of the Over-sericin4 strain has moreβ-sheet structures.Observation and statistics on the cocoon silk of Over-sericin4 strain showed that the cocoons were looser and the silk became thinner.Further testing revealed that the silk fibroin Fib-H protein was down-regulated in the silk glands of the transgenic lines,but the silk fibroin P25 protein was significantly up-regulated.In order to determine the reason for the improved performance of Over-sericin4 strain silk,we prepared sericin hydrogels of different strains of silk.The mechanical properties of the hydrogel were analyzed,and it was found that the strength and strain of the Over-sericin4 strain sericin hydrogel were higher than that of the WT sericin hydrogel.In addition,through hormone induction of trimolter silk,we proved that the thinner silk has the stronger modulus and strength.In other words,the improvement of silk properties of the Over-sericin4 strain is the result of Ser4 protein and the reduction of diameter.The detection of the degumming rate of silkworm cocoons showed that the degumming rate of silkworm cocoons of the Over-sericin4 strain was significantly lower than that of the control cocoons,but the total sericin content was not significantly different.Through the analysis of single fiber contact angle experiment,it was found that the silk of the Over-sericin4 strain was more hydrophobic than the control silk,which indicated that the decrease in the cocoon degumming rate of the Over-sericin4 strain might be caused by the more hydrophobic silk.4.Study of sericin repeat motifs reveal the reason for sericin properties difference.The analysis of the deduced amino acid sequence of young larval instar sericins showed that the amino acid sequences of Ser2,Ser4,and Ser5 contained a high proportion of charged amino acids,while the proportion of charged amino acids in the Ser1 and Ser3of silkworm cocoon was very low.We synthesized Ser1-repeat2 peptide（Ser1-rp2）,Ser4-repeat1 peptide（Ser4-rp1）and Ser5-repeat peptide（Ser5-rp）and co-cultured them with NIH/3T3 cells.After 24 hours,the cell proliferation was detected by CCK-8 reagent.The results showed that Ser4-rp1 and Ser5-rp could significantly promote cell proliferation compared with the blank control,while Ser1-rp2 had no significant difference compared with the blank control.Our results preliminarily proved that Ser4-repeat1 and Ser5-repeat endowed Ser4 and Ser5 proteins with better adhesion and proliferation properties.The prediction of the three-dimensional structure of sericin showed that Ser4-repeat2 was composed of a stack of regularly arrangedβ-sheet structures,and a single repeating unit in the repeat region will form an anti-parallelβ-sheet structure.Furthermore,we successfully expressed Ser1-repeat2 and Ser4-repeat2 proteins into cocoon silk by transgenic methods.The results of silk FTIR showed that theβ-sheet structure of the silk of the Over-Ser4-repeat2 strain was significantly higher than that of the wild-type D9L silk,while theβ-sheet structure of the silk of the Over-Ser1-repeat2 strain was not significantly difference compared with that of the control.This showed that the three-dimensional structure prediction of Ser4-repeat2 was accurate.Ser4-repeat2 is more similar to silk fibroin and can form moreβ-sheet structures,so that Ser4 has good mechanical properties.The results of mechanical properties showed that the strength of Over-Ser4-repeat2 silk was increased by 37%and the Young’s modulus was increased by48%compared with WT silk,while the performance between Over-Ser1-repeat2 and WT silk had no significant difference.We preformed a glycoprotein staining with Ser1-repeat2 and Ser4-repeat2.As a result,Ser1-repeat2 was detected glycosylation modification,while Ser4-repeat2 did not detect glycosylation modification.These results further show that Ser4-repeat2 is more similar to silk fibroin.Our research explained the reasons why the Ser4 protein affects the performance of silk and preliminarily studied the function of different repeat regions of sericins,laying a foundation for the subsequent exploration of the performance of different sericins. |
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