Titanium Nanoparticles Influences Silk Protein Synthesis In The Silkworm,(Bombyx Mori)

Silkworm（Bombyx mori）belongs to Lepidoptera moth family and is an important silk insect that has been domesticated to produce silk for more than 8500 years.Silk production is a traditional industry in China,its annual production of raw silk accounts for more than 78% of the total raw silk in the world.China is a major silk production country.Increasing the yield of silk and improving the quality of silk are the core issues in the development of silk industry.The traditional breeding and biological screening technology has been very mature,it greatly improves silkworm silk production,the main problem facing the development of silk industry is how to further improve silkworm silk,the use of molecular biology is the latest method to improve silk production.Recent studies mainly focus on the interaction between silk fibroin coding genes and transcription factors,the tissue-specific expression of silk protein genes,and the signal regulation mechanism of silk fibroin synthesis.In recent years,with the development of science and technology,the development of nano-biotechnology has made great strides and achieved significant results in various fields,feed additives developed with nanotechnology are widely used in animal husbandry Titanium dioxide nanoparticles（TiO₂ NPs）is a typical nanomaterial,based on its surface effect,volume effect and quantum size effect,macroscopic quantum tunneling effect,and so on,is widely applied in biological field.The biological effect of TiO₂ NPs is closely related to its concentration,high concentrations of TiO₂ NPs is toxic to biology,low concentration of TiO₂ NPs is used as additive can promotes the growth of livestock and protein synthesis It can be used to improve various traits of silkworm,improve the synthesis ability of silk fibroin and directly affect the yield of silkworm.The study on its signal regulation mechanism has not been reported yet.1.Gene Expression Profiles of Silkworm Silkworms after TiO₂ NPs TreatmentIn order to study the signal regulation mechanism of TiO₂ NPs affecting silk fibroin synthesis,this study used 5 mg/L TiO₂ NPs feed Bombyx mori.In this study,digital gene expression（DGE）tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO₂ NPs.In total,5,702,823 clean tags and 55,096 distinct tags were detected in TiO₂ NPs treated,6,150,719 clean tags,and 74,715 distinct tags were detected in control groups,respectively.Compared with the control,TiO₂ NPs treated silkworms showed 306 differentially expressed genes,including 137 upregulated genes and 169 downregulated genes.Among these differentially expressed genes,106 genes involved in silk protein synthesis,of which 97 were up-regulated and 9 down-regulated.Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes（KEGG）showed that 20 pathways were significantly enriched in TiO₂ NPs treated silkworms,and the metabolic pathway-related genes were the most significantly enriched.The credibility of DGE results were verified by q RT-PCR analysis of eight differentially expressed genes.The results showed that the results of DGE and q RT-PCR of 6 genes were consistent,and the results of DGE and q RT-PCR of 2 genes were different,indicating that DEG could provide a reliable reference for subsequent analysis.2.Effect of TiO₂ NPs on Akt/Tor signaling pathway in silk gland of silkwormAfter obtaining the overall gene expression profile of TiO₂ NPs,the process was found to be closely related to the Akt/Tor signaling pathway,and the differentially expressed genes involved in the key genes in the Akt/Tor signaling pathway,In order to investigate the effect of TiO₂ NPs on Akt/Tor signaling pathway in the silk gland of silkworms,we determined the changes in Akt/Tor signaling pathway gene and protein expression in the silk gland following treatments with TiO₂ NPs and Akt/Tor signaling pathway inhibitors（LY294002 and rapamycin）using quantitative RT-PCR and Western Blot analysis.We found that Akt,Tor1 and Tor2 gene expression in the silk gland were upregulated by 1.657,12.073,and 4.797 folds after 6 h in vitro culture after 72 h fed with mulberry that treated with TiO₂ NPs.Tor1 and Tor2 m RNA expression in the silk gland cultured in vitro with LY294002 for 6 h were 0.021,and 0.223 folds of those in control group.The m RNA expression of Tor1 and Tor2 in the silk gland treated with rapamycin for 6 h were 0.470 and 0.141 folds of those in the control group.The m RNA expression of Fib-L in TiO₂ NPs-treated group were increased at 6 h,12 h and 24 h,respectively,which was 1.446,1.301 and 1.351 folds higher than those in control group.The m RNA expression of Fib-L in LY294002 treated group was inhibited at 12 h,which was 0.664 times that of the control group.The m RNA expression of Fib-L in LY294002 treated group was inhibited at 12 h,which was 0.664 folds of those in control group.The m RNA expression of Fib-L in rapamycin treated group was inhibited at 6 h,which was 0.058 folds of those in control group.Western Blot results showed that TiO₂ NPs increased the phosphorylation of Akt and its downstream proteins;the phosphorylation of Akt and its downstream proteins LY294002;and the phosphorylation of Tor downstream proteins were inhibited by rapamycin.3.Silk protein synthesis transcription factor regulation of silk protein synthesisA variety of regulatory factors significantly regulate silk protein synthesis.In order to investigate the effect of regulators on silk fibroin synthesis,gene and protein expression levels of silk fibroin and related transcription factors were examined after treatment with Akt/Tor signaling inhibitors（LY294002 and rapamycin）.The experimental results showed that the m RNA expression levels of PI3 K,Fib-H and Fib-L,Bmsage,Bmdimm,Mbf2 and Bmftz-1 of silkworm silk gland were significantly decreased compared with the control after silkworm strain LY294002 was cultured in vitro,and as time went on,the protein expression of Fib-L and Bmsage significantly decreased at 6h compared with the control group.Bombyx mori silk fibroin rapamycin treatment in vitro,Tor,silk fibroin gene Fib-H and Fib-L,related regulatory factors Bmsage,Bmdimm and Mbf2 m RNA expression levels were significantly lower than the control,and with the passage of time,the protein expression of Fib-L and Bmsage decreased significantly at 6h compared with the control group.It shows that silkworm can regulate the expression of silk fibroin synthesis transcription factor through Akt/ Tor signaling pathway and regulate silk fibroin synthesis.This study also investigated the posterior silk gland protein concentrations of silkworms after 24 h of injection of LY294002 and rapamycin at the fifth instar 72 h,found that the protein concentration of LY294002 and rapamycin significantly decreased after injection.The results of this dissertation illustrate the signaling pathway that TiO₂ NPs promote the silk fibroin synthesis,it provides a new reference for silk fibroin signal transduction mechanism of silkworm silk fibroin synthesis.