The Analysis Of Differentially Expressed Genes Of The Posterior Silkland And Studies On Improvement Of The Mechanical Properties Of Silkworm Silk Fiber,Bombyx,Mori

The silkworm, Bombyx mori, is a lepidopteran economic insect, and the silk gland is a highly specifically differentiated silk-producing organ determining the silk production and quality. The silk gland has been used as a bioreactor to express recombinant proteins because of its impressive ability to synthesis, secrete and store huge amount silk protein. However, the extremely low production of the various heterologous proteins expressed by the silk gland bioreactors was reported by previously research. To improve the mechanical properties of the silkworm silk fiber through expressing the dragline silk protein of the black window spider (Latrodectus hesperus) into silk cocoons, we explore the gene expression and regulation in the posterior silk gland (PSG) using transcriptomics and proteomics techniques, and preliminarily reveal the molecular mechanism of highly efficient synthesis of silk protein and lay the foundation for further highly efficient expression of the recombinant dragline silk protein. In this study, high-performance silk was obtained by transgenic technology to express the dragline silk protein of the black widow spider(Latrodectus hesperus) into silk cocoons, the main results are as follows:1. The metabolic process associated with the synthesis of silk protein is revealed by transcriptomics technique.To explore systemic gene expression and comprehensive molecular regulatory mechanisms underlying the transcription profiles for silk protein synthesis amomg different genotypic silkworm mutations, the transcription profiles of the three samples of the PSGs from the Nd, Nd-s, and Qiufeng strains on the third day of the fifth instar were sequenced individually. The main composed genes of fibroin showed generally decreasing expression levels at the transcriptional level, which result in low-yield of the silk. Some key genes playing regulatory roles in development were reduced, so the silk gland is hindered during the process of development stage. Tissue destruction, abundant misfolded and unfolded proteins exist in PSG which result in increase of energy-wasting metabolism process including the ubiquitin-proteasome system (UPS), autophagy driven vacuolar (lysosomal) proteolysis and antioxidant metabolism, so the synthesis of fibroin which is also the energy-consuming process are greatly hindered.2. The metabolic process associated with the synthesis of silk protein is revealed by proteomics technique.To further explore the comprehensive molecular mechanism of silk protein synthesizing system amomg different genotypic mutations B.mori at the translational level, we also compared the proteomic profiles of these samples used in transcription profiles analysis using isobaric tags for relative and absolute quantification (iTRAQ) technique in this study. The main composed genes of fibroin show generally decreasing expression levels at the protein level and have the same trend with the transcriptional level, which result in low-yield of the silk. Some key genes playing regulatory roles in development, the synthesis of fibroin and the pathway associated with protein synthesis were reduced. Moreover, abundant energy is used for useless metabolic process. Thus, the synthesis of fibroin is greatly hindered.3. Improvement of the mechanical properties of the silkworm silk fiber using transgenic technique.We designed two series of piggyBac vectors containing key element of MaSpl basic repeat (two sequential Ⅰ/Ⅱ/Ⅲ/Ⅳtypes) or MaSp2 basic repeat (three sequential I/IVtypes) based on the dragline silk gene originating from the black widow spider, Latrodectus hesperus (Theridiidae), respectively, and integrated them into silkworm genome by microinjection technique. The positive-transgenic lineages were identified sequentially by Inverse-PCR, Q-PCR and Western blotting, and 16 transgenic lineages were obtained. The transgenic silkworm silks have significantly improvement compared with the wide-type silk fiber,71.92% for the maximum value of stress, 34.508% for the strain,57.819% for the Young’s modulus and 131.673% for the toughness.4. This study has proved that there is a greatly significant linear correlation between the protein size and the mechanical properties of the black window spider dragline silk.According to the transgenic silkworm lineages containing different re-MaSp1 sizes and two-kind of genotypes including heterozygote or homozygote, we can come to a conclusion that the mechanical properties of silk fiber will be better along with the increasing of the protein sizes. This result indicate that the outstanding mechanical property of the black widow spider dragline silk attribute to the large molecular weights of the proteins. Thus, the protein-based silk fibers in nature with outstanding mechanical properties all adopt this strategy, i.e. composed of highly repetitive and large protein, and this phenomenon is inevitable result of biological evolution.In conclusion, we performed an integrated analysis upon the transcriptome and proteome of the PSG of Qiufeng, Nd and Nd-s strains, and revealed the molecular mechanism of high-efficiency fibroin syntheses which lay the foundation for remould the silk gland for high-efficiency expression of heterologous proteins. On the other hand, we obtained various transgenic lineages expressing the recombinant dragline silk protein, which have significantly improvement on the mechanical properties of silk fiber. Meanwhile, a significant linear correlation between the mechanical properties and protein sizes were proved, which lay key foundation for improving the mechanical property of silkworm silk. This study illustrates that the mechanical property silkworm silk fiber would be further improved through transforming the gene expression pattern of the PSG and introducing larger molecular weight spider silk protein.