| Silkworm silk is made up of silk fibroin(about 75%)and sericin(about 25%).As the source of structural foundation and mechanical properties of silk,silk fibroin plays an important role.The secretion mechanism,yield and application of silk have always been the focus of attention.Since it was proposed that silk fibroin was composed of fibroin heavy chain(Fib H),fibroin light chain(Fib L)and P25 protein with a molecular ratio of 6:6:1,thesse three proteins in silk fibroin were considered to be important or even necessary for silk secretion.Currently,more studies have been conducted on Fib H and Fib L proteins in silk fibroin protein,and they have been demonstrated to have important effects on the composition and secretion of silk fibroin protein.However,research on P25 protein,another component of silk fibroin protein,is relatively scarce,and its role in silk secretion and mechanical properties is worth exploring.Moreover,according to the needs of basic research and disease treatment,it is of great significance to use transgenic technology to introduce exogenous functional genes to modify silks.Although the production of silk has proved the amazing capacity of protein synthesis and secretion of silk glands,it is a great challenge to reduce or slow down the competition for endogenous silk protein resources in order to become an ideal bioreactor for the production of exogenous proteins.It is worth exploring whether the loss of P25 protein can bring notable improvement to the expression of foreign protein.Therefore,in terms of the above problems,the relevant researches were carried out and the results are as follows in this thesis:1.The mutation efficiency of the first exon of P25 gene was 86.84% by the CRISPR/Cas9 gene editing system.The results of q PCR and Western blot displayed that the homozygous mutant of P25 gene was obtained.The truncated mutant protein with a molecular weight of about 10.4 k Da produced by the early termination of translation caused by the base deletion did not appear in the cocoon layer proteins,which further confirmed the successful deletion of the P25 protein.The transcriptional termination of P25 gene occurred in advance and the P25 protein was successfully deleted.Phenotypic observation showed that the silk glands and cocoons of the mutant were normal in appearance.However,further study showed that the weight of posterior silk glands,cocoon layers and pupae decreased significantly.The results of mechanical properties test indicated that the mechanical properties of mutant cocoon silks were significantly weaker than that of wild type.Among them,the strength of female and male cocoon silk decreased by 14.82% and 11.99%,the elongation decreased by 30.06%and 28.16%,and the toughness decreased by 38.20% and 41.51%.In addition,the content of β-folded conformation in the secondary structure of the mutant cocoon filament decreased significantly,which was consistent with the results of mechanical properties test.These results indicate that the loss of P25 protein leads to a decrease in silk production and a decrease in the overall mechanical properties of silks.On the contrast,compared with Fib H and Fib L gene mutants,the silk secretion function of silkworms with P25 protein deletion still retained a relatively normal state.The result showed that P25 protein was dispensable for silk secretion.2.The transcriptome analysis was carried out using the self-built database.The results showed that after the mutation of P25 gene,the transcription level of P25 and Fib L gene was significantly down-regulated,which was the direct reason for the decrease of the silk yield of the mutant.The transcription level of some ribosomal protein genes was down-regulated,which affected the synthesis of centromere protein.The P25 gene mutation causes DNA damage,and the cell activates the DNA damage repair mechanism and apoptosis mechanism,thereby inhibiting the synthesis of silk protein.On the other hand,the enrichment of differentially expressed genes in related signal pathways such as folding and degradation suggested that the truncated mutant proteins and misfolded Fib H proteins which might be produced due to P25 gene mutation might trigger the endoplasmic reticulum stress response and then started the self protection mechanism of cell,leading to the inhibition of protein synthesis and the degradation of abnormal proteins.At the same time,the AMPK signaling pathway was activated to balance the energy consumption caused by the cellular defense process,and these metabolic responses ultimately reduce silk protein synthesis and secretion.3.Human epidermal growth factor(hEGF)was set as the target protein.Two piggy Bac transgenic vectors driven by Fib L and Fib H gene promoters were constructed to express Fib L-hEGF and Fib H-hEGF fusion proteins in the posterior silk glands of Lan10,and then were microinjected into Lan10 silkworm eggs.Fluorescence screening showed that 8 Fib L-hEGF and 4 Fib H-hEGF transgenic positive lineages were obtained,with a positive rate of 10.3% and 36.36%,respectively.Routine PCR,reverse PCR and Western blot results confirmed that two kinds of transgenic silkworms were successfully obtained.The fusion proteins were expressed and secreted into the cocoons,and the proteins expression level varied with the insertion sites.The phenotypic observation of cocoons and cocoon silk filaments showed that transgenic silkworms had normal silk-spinning and cocoon-forming ability,and the phenotypic changes of cocoons and cocoon silk filaments were not significant.The mechanical test results showed that the strength and Young’s modulus of the two transgenic recombination cocoons were significantly higher than those of the wild type.The results of cell proliferation test showed that the two groups of transgenic recombinant cocoon silk after degumming had good activity to promote cell proliferation and had little toxicity to cells.The above results indicated that the recombinant hEGF silks with good mechanical properties and biological activity could be obtained by piggy Bac transposon-mediated transgenic technology effectively.Therefore,it may have the potential to manufacture wound dressings for medical use,and also provide reference for different modification and multi-field application of silks.4.Based on the previous studies on P25 gene mutation and hEGF transgene,the effect of P25 protein deficiency on the expression level of hEGF protein in transgenic silks was further explored.Firstly,the hEGF transgenic vector driven by the P25 gene promoter was constructed on the basis of the transgenic vector driven by the Fib H gene promoter.A total of 5 transgenic lineages were obtained by fluorescence screening,with a transgenic positive rate of 14.29%.Routine PCR,insertion site detection and Western blot results confirmed the successful preparation of hEGF transgenic silkworms.Transgenic silkworms with wild type,heterozygous and homozygous mutation of P25 gene were obtained by mating transgenic silkworm moths with P25 gene mutants.Proteins in cocoon layer confirmed that hEGF was successfully expressed and secreted into cocoon layers.The quantitative results of protein electrophoresis band gray analysis showed that the expression level of hEGF in the transgenic silkworm with homozygous mutation of P25 gene was 2.2 times higher than that of the common transgenic silkworm.These results demonstrate that the loss of endogenous P25 protein can improve the expression level of foreign proteins,and indicate that the homozygous mutant of P25 gene has the potential to act as an improved bioreactor for high expression of foreign protein.In conclusion,the study firstly mutated P25 gene using Cas9 technology and explained that the P25 protein was not a necessary factor for silk secretion,except for reducing silk production to a lesser degree.Combined with transcriptome analysis,the mechanism involved in P25 protein decreasing silk yield was explored,which laid a foundation for optimizing the yield of foreign protein to better improve silks.Secondly,piggy Bac mediated transgenic technology was used to modify the silks,and the recombinant silks with improved mechanical properties and biological activity was obtained,which has a good reference significance for the development of new materials.Finally,hEGF transgenic silks were prepared by using P25 gene mutant as bioreactor.These results demonstrated that the deletion of endogenous P25 protein can improve the expression level of hEGF in silk,indicating that the homozygous mutant of P25 gene has the potential to improve the silkworm bioreactor. |
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