| Bombyx mori is an important Lepidopteran model insect and economic insect,which has played an important role in earning foreign exchange for export and poverty alleviation of our country for a long time in the past.However,with the continuous development of social economy,the application field of silk is gradually expanding,and the market also puts forward higher requirements for silk,especially in its performance.In this context,some researchers have proposed to use gene editing and transgenic technology to improve the properties of silk,which has attracted people’s attention.In this thesis,the transgenic spider silk protein gene silkworm,JBmSp6-T and HBmSp6-T homozygous strain containing the MaSp6(major ampullate spidroin-6)gene of Nephila clavipes was used as the research object,to explore the cocoon silk characters and properties of JBmSp6-T and HBmSp6-T strain from four aspects,namely phenotypic detection,MaSp6 insertion site,tissue stage expression pattern and mechanical properties of silk protein,respectively,in order to find a new breakthrough point for breeding new silkworm strains with stable genetic and high mechanical properties of transgenic silk.The main research contents and results are as follows:First,by fluorescence microscopy observation of JBmSp6-T and HBmSp6-T,we found that the red fluorescent protein(Ds Red)was expressed in the compound eyes of the late embryonic and adult stages.enhanced green fluorescent protein(eGFP)can not only be expressed in silk gland,but also be secreted out of vitro together with silk protein to form complex silk protein fibers,which can be presented in the form of cocoon.qRT-PCR showed that eGFP gene was specifically expressed in the posterior silk gland(PSG)and showed an increasing trend in the PSG of the fifth instar larvae,reaching the highest level at L5D8.As MaSp6 gene is expressed in a fusion mode with eGFP gene,these results indicate that MaSp6 gene has been successfully translocated into JBmSp6-T and HBmSp6-T genome and expressed normally.Second,JBmSp6-T and HBmSp6-T silk lines were tested and analyzed for cocoon weight,cocoon quality and mechanical properties.The results showed that the total cocoon weight and cocoon layer weight of JBmSp6-T strain decreased by 17.86% and 24.32%,respectively,compared with that of Jing Song strain,indicating that the cocoon weight of JBmSp6-T strain did not reach the high level of parent Jing Song strain,and the cocoon weight was relatively low.Compared with Haoyue,the cocoon layer weight of HBmSp6-T in spring and autumn silkworm stages was significantly increased by 16.67% and 44.4%,respectively,indicating that the cocoon weight of the transformed HBmSp6-T had reached the level of the parent Haoyue,which was slightly higher than that of the parent Haoyue.The analysis of the data of relaxation filament length,the total weight of raw silk and the amount of cocoon silk per grain showed that compared with Jingsong,JBmSp6-T had lower results in cocoon silk,and the weight was reduced by 55.82%,The results showed that the filament length of JBmSp6-T was poor and the filament yield was low.Compared with Haoyue,the three indexes of cocoon silk production of HBmSp6-T increased,among which the relaxation filament length increased by 27.03%,indicating that the relaxation filament length of HBmSp6-T was better and the rate of filament production was higher.At the same time,the average diameter of single silk of HBmSp6-T strain(5.12μm)was 21.71% less than that of its parent(6.54μm),which might be due to the expression and secretion of MaSp6 protein together with cocoon silk protein,which changed the original conformation of cocoon silk protein,leading to the decrease of the diameter of single silk.The analysis of the mechanical properties of single filament showed that the average breaking strength of HBmSp6-T was 5.18% lower than that of Haoyue,indicating that the average breaking strength of cocoon silk of HBmSp6-T was weaker than that of Haoyue.The average elongation at break and the average energy at break of HBmSp6-T were 24.52% and20.08% higher than those of Haoyue,respectively,indicating that the silk fiber of HBmSp6-T had good elasticity and toughness.In the results of the data analysis of the mechanical properties of the monofilament fiber the average breaking strength of cocoon filaments of HBmSp6-T strain was 5.18% lower than that of Haoyue strain,indicating that the average breaking strength of cocoon filaments of HBmSp6-T strain was weaker than that of Haoyue strain.The average elongation at break and the average energy at break of HBmSp6-T strain were 24.52% and 20.08% higher than those of Haoyue strain,respectively,indicating that the silk fiber of HBmSp6-T strain had good elasticity and toughness.In conclusion,the study of this project shows that the homozygous strain HBmSp6-T has good cocoon silk properties and mechanical properties.The results of this study will provide a reference for the research and development of biomaterials required for the development of high-end fields,and will be of great value and significance to promote the production and development of emerging industries. |
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