Study On The Func Tion Of Small Molecule Silk Protein SPI51 And Seroin1 In Silkworm

Silkworm is an important economic insect because it can spin silk and make cocoons.Silk is a kind of natural animal protein fiber.The ancestors of the Chinese nation discovered the value of silk as a textile apparel product as early as more than2000 BC.Since ancient time,silk products have been loved by the public for their gorgeous appearance,elegant luster and comfortable body feeling.Since ancient time,silk products have been loved by the public for their gorgeous appearance,elegant luster and comfortable body feeling.In recent decades,researchers have opened up more extensive space for silk in the fields of biomedical materials,drug delivery carriers,biosensors and cosmetics,because of silk has the advantages of controllable biodegradability,good biocompatibility and strong plasticity.However,the fundamental factor that determines the fine characteristics of silk lies in the unique composition of silk proteins and the process of forming fibers.Therefore,the in-depth study of the important components of silk proteins in the process of forming fibers will play an important role in guiding the development of the silk industry.Current studies have shown that the process of silk fibrosis is essentially the conformation transformation of silk proteins in the lumen of the silk gland and the silk proteins are changing from liquid to solid in this process.There are many factors that affect this process,including p H environment,metal ions,protein concentration and shear force.However,the same important factor in this process is the composition of silk proteins.The Naked pupa?Nd?mutation in silkworm can only make sericin cocoons due to abnormal silk fibroin secretion.When the silk fibroin heavy chain gene was knocked out,the cocoon layer bacame thinner and similar to the sericin cocoons.The lack of silk fibroin protein can cause abnormalities in silk performance.In addition to silk fibroin and sericin,there are many high abundance and small molecular weight proteins in silk,such as protease inhibitors and Seroins proteins.Bm SPI51 is the highest content protease inhibitor in silk,while Seroin1 protein is the highest content Seroin protein in silk.Although studies have found that the protease inhibitor SPI51 can inhibit the activity of trypsin,and that the Seroin1 protein has the activity of inhibiting the growth of bacteria,there have been insufficient experiments to explain the biological functions of these two important small molecular proteins in silk.Therefore,these two small molecule weight silk proteins,SPI51 and Seroin1,were selected as the research objects.The functions of these two proteins in silk fibrosis and cocoon silk were conducted in-depth study by using the various technical means such as molecular biology,biochemistry,material mechanics,and gene editing.The main findings are as follows:1.Expression pattern analysis of high abundance silk proteinsBy analyzing the expression patterns of main silk proteins at the transcription level and protein level,the whole process of silk protein synthesis,transport and secretion can be mapped.It was found that the fibroin gene was only expressed in the posterior silk gland,and the expression level increased gradually in the fifth instar,reaching the maximum before wandering.Moreover,the ratio of transcriptional expression among the three fibroin genes was Fib H:Fib L:P25?6:3:1,which was not identical to the protein level.All the three sericin genes were expressed in the middle silk gland,but the expression patterns were significantly different:Sericin1 gene was expressed in the middle and the posterior of the middle silk gland,and began to be expressed after the third day of the fifth instar,with the expression level is increasing gradually.Sericin2gene was expressed in the anterior of the middle silk gland.The expression level decreased gradually in the fifth instar and stopped after the third day.Sericin3 gene was expressed in the anterior part of the middle silk gland and began to be expressed after the fifth day of the fifth instar,and the expression level increased gradually.It was found that protease inhibitors were mostly expressed in the anterior part of the middle silk gland,which was consistent with the function of protease inhibitors to inhibit pathogenic bacteria in the outer part of silkworm cocoon.It was found that three unknown functional proteins were specifically expressed in the middle silk gland,which was speculated to be related to the assembly of sericin.In general,the three fibroin proteins are specifically expressed in the posterior silk glands and formed silk fibers.There are many proteins specifically expressed in the middle silk gland,including sericin,protease inhibitors and unknown functional proteins,which are involved in the assembly and secretion of sericin and protecting the silk protein from degradation.Seroin proteins which are expressed in both the middle and posterior silk glands may have a more complex functions.It is worth noting that the expression levels of Bm SPI51and Seroin1 genes in silk glands are very high,and both are expressed in the middle and posterior silk glands,suggesting the complexity of their functions.Therefore,we conducted in-depth functional studies on these two small molecule weight proteins.2.Study on the antibacterial function of Bm SPI51 proteinThe expression pattern of Bm SPI51 was analyzed by quantitative PCR and Western blot and it was found that Bm SPI51 was specifically expressed in silk gland,and the expression level was the highest in the anterior of the middle silk gland.The S.cerevisiae,C.albicans and B.bassiana were injected into silkworm on the third day of the fifth instar,it was found that Bm SPI51 gene was significantly up-regulated in the fat body from the18th h to the 24th h after fungal induction.By cloning and prokaryotic expression of the Bm SPI51 with the signal peptide encoding sequence removed,a large number of Bm SPI51 proteins were expressed in the supernatant.The inhibitory activity of Bm SPI51 recombinant protein was detected,and the inhibitory rate of Bm SPI51protein on trypsin activity was found to be up to 100%,but it has no inhibitory activity on chymotrypsin,protease K and other proteases.After incubation with three fungi,Bm SPI51 protein showed good inhibitory activity against three fungi,S.cerevisiae,C.albicans and B.bassiana.Further studies on the inhibitory mechanism of Bm SPI51protein showed that it could inhibit the growth of fungal spores by combining with?-D-glucan and mannan on the cell wall of the fungus.A commercial soy trypsin inhibitor?Gm STI?was used as a parallel control,and Bm SPI51 was found to have significantly stronger inhibitory activity against S.cerevisceris and C.albicans than Gm STI.Through amino acid sequence analysis,we found some differences between the Bm SPI51 protein of silkworm and the homologous protein of Wild SPI51 at the active site and C-terminal of the sequence,which was speculated to be the mutation caused by domestication.The active site P₁-P₅ of SPI51 was mutated from"RGGFR"of Bombyx mandarina to"KGSFP"of Bombyx mori,and the"Y"amino acid coding sequence of nine amino acids at the C-terminal of SPI51,"YNTCECSCP",became the termination codon after the mutation,leading to the early termination of the translation of silkworm Bm SPI51,with nine amino acids missing.Three vectors were constructed,including Bm SPI51-PPsumo,Bm SPI51+tail?tail was nine amino acids at the C end of Wild SPI51?-PPsumo,and Bm SPI51M-PPsumo?M represents the mutation of active site of SPI51from silkworm to the active site of silkworm?.Studies on the enzyme inhibition and fungal activity of these three proteins showed no significant difference in their stability and only had inhibitory activity on trypsin,but Bm SPI51+tail protein and Bm SPI51M protein were significantly stronger than Bm SPI51 protein in inhibiting the growth of fungi.The above results indicate that the 9 amino acids at the active site of Wild SPI51and the C-terminal are the main reasons for the higher antifungal activity of Wild SPI51protein.In other words,the safer environment was created by the domestication process and caused the degradation of SPI51,and further resulting in that the antifungal activity of the silkworm Bm SPI51 protein was significantly lower than that of the wild silkworm Wild SPI51 protein.3.Seroin1 proteinwas involved in the intracellular replication process of silk glandsBy CRISPR/Cas9 gene editing technology,Seroin1 gene was knocked out.The results showed that when the Seroin1 gene was knocked out,the silk gland became smaller,the dry and wet weights of the silk gland were significantly reduced,and the development of the silk gland was delayed,especially from the third day of the fifth instar.Two methods were used to analyze the factors affecting the silk gland.1.The number of silk gland cells was analyzed,and no significant changes were found compared with the control group.2.Through analysing of the volume of silk gland cells,it was found that the silk gland cells volume of Seroin1^-/-strain were significantly smaller than that of control gourp.The above results indicated that the key ofsilk gland size change was the decrease of silk gland cell volume.DNA contents of Seroin1^-/-silk gland cells was significantly reduced,which was only about 1/4 of that of the control group at the wandering stage.Further using the 5-ethynn-2'-deoxyuridine?Ed U?to label the silk gland cell in the S phase of the cell cycle?DNA replication?,and it showed that a significant decrease in DNA replication in Seroin1^-/-strain.We further examined the expression changes of key genes in regulating the cell cycle and found that E2F1,cyclin E and CDK2 genes were significantly up-regulated after Seroin1 was knocked out.Next,Seroin1 was overexpressed in Bm E cells,we found that E2F1,cyclin E and CDK2geneswere significantly down-regulated,which meaned that Seroin1 could indeed affect the expressions of E2F1,cyclin E and CDK2 genes.In conclusion,the expression of the E2F1,cyclin E and CDK2 genes were significantly up-regulated after Seroin1 knocked out,which slowed down the DNA replication process of silk gland cells and caused the smaller cells.4.Seroin1 protein affected the spinning behavior of silkwormBy investigating the silk secretion behavior after Seroin1 gene knocking out,it was found that the silk fiber secretion of the Seroin1^-/-strain from the baby silkworm was less than that of the normal silkworm,and the silk fiber was no longer secreted from the third to the fifth instar,resulting in naked pupae.The morphology of the silk glands was observed at the wandering stage.it was found that the silk gland contents of the Seroin1^-/-strains became lighter in color and softer when compared with the normal silkworm.The contents of the silk glands were unable to form silk fibers when it was subjected to external forces.It indicated that the key factor of no spinning silk of Seroin1^-/-strains might due to the difference in the contents of the middle silk gland.The silk glands were divided into five sections and then sliced.By observing their cross-sections,we found the silk gland contents of Seroin1^-/-strains had no sericin layer when compared with normal silkworms.After q RT-PCR detection and proteomics analysis,we found that the expression of sericin was significantly reduced,resulting in only fibroin but no sericin in the silk gland lumen of the Serolin1^-/-strain.In the absence of sericin,the silk fibroin of the Serolin1^-/-strain still migrated to the anterior silk gland,but could not be secreted out of the spinneret orifice.The secondary structure of silk glands contents were analyzed by infrared spectroscopy,it showed that the secondary structure of the silk gland contents of Seroin1^-/-strains changed a lot.The random coil and helical structures increased significantly,while the?-sheet and?-turn content obviously decreased.Through the rheological analysis,we found that the protein of silk gland lumen displayed a lower viscosity than the control after knocking out the Seroin1gene.The initial storage modulus was also lower than that of the control,and the yield point appeared earlier than the control group during the dynamic strain scanning process.These results indicated that the silk protein structure of Seroin1^-/-strain is more susceptible to break.The above results showed that the sericin layer had an important protective effect on silk fibroin,and silk fibroin could not be folded and secreted correctly in the absence of sericin.