The Investigation Of Molecular Mechanism Underlying Silk Production And The Modification Of Silk Property In Silkworm,Bombyx Mori

Silkworms have been domesticated to produce silk for five thousands years.In recent years,with the improvement of genome sequencing,silkworm has become an important model of insects with considerable economic value and especially important scientific research value.Silk fribroin is a good biological material which not only can be used to make clothing quilts,jewelry and some high-end consumer goods,but also can be used as medical sutures and other medical supplies.Similar to these complex traits,such as high blood pressure,diabetes,obesity in human,rice yield,soybean oil concentration,and dairy cow milk production in animal and crops,silk protein synthesis and secretion is also a typical complex traits determined by multiple genes.It is a tough work to determine the genetic mechanism of a complex trait because these phenotypes are often caused by interactions between the environment and the genes.The inherent conditions of silkworms,such as short life cycle,high density of breeding,high safety and easy control of feeding indoor,make it an ideal model for studying the molecular mechanism of complex traits.At the same time,as a biological material used in the medical career needs,the transformation of silk property is also an imminent and very meaningful work.In this study,a series of research were done with these scientific issues,the main results are as follows:1.Analysis of the molecular mechanism underlying the trait of silk production in silkworm based on quantitative proteomics and transcriptomicsTo investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain,we used both SDS-PAGE-based and gel-freebased proteomic techniques and transcriptomic sequencing technique.The results showed that,in the third day of fifth instar when the silk protein synthesis and secretion reached to a peak,in the ZB strain,numerous ribosomal proteins and transcripts were down-regulated,along with the transcripts of translational related elongation factors and genes of important components of fibroin,the proteasome pathway was significantly enhanced,and the energy-metabolismrelated pathways,oxidative phosphorylation,glycolysis/gluconeogenesis,and citrate cycle were also enhanced.These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis,the degradation of proteins,and the inefficiency of the energy exploiting.The results laid the foundation for the future cultivation of high-yielding varieties of silkworm.2.Analysis of molecular mechanism underlying silk yield based on iTRAQ proteomics and RNA-sequencing in silkworm,Bombyx moriUsing isobaric tags for relative and absolute quantification(iTRAQ)quantitative shotgun proteomics and RNA-sequencing-based transcriptomics,139 proteins and 630 transcripts were identified as novel in the ZB strain compared with the Lan10 strain,indicating that these results significantly expand the coverage of proteins and transcripts of the posterior silk glands in the silkworm.Of the 89 differently changed proteins,23 were increased,and 66 were decreased.Of the 788 transcripts,779 were upregulated,and 9 were downregulated.These results confirm that decreased energy utilization/protein translation and enhanced protein degradation are the key factors in lower silk production.Moreover,this study provides novel insight into the molecular changes that may result in lower silk production,namely,a combination of impaired transcription activity,missed protein folding/transport,and lowered yields of the main components of fibroin,along with weakened growth/development of the posterior silk gland.3.Studies on characterization of transgenic silkworm yielded biomaterials with calcium-binding activitySilk fibers have many inherent properties that are suitable for their use in biomaterials.In this study,the silk fibroin was genetically modified by including a Ca-binding sequence,[(AGSGAG)6ASEYDYDDDSDDDDEWD]2 from shell nacreous matrix protein and silk fibrion.It can be produced as fibers by transgenic silkworm.The Ca-binding activity and mineralization of the transgenic silk fibroin were examined in vitro.The results showed that this transgenic silk fibroin had relatively higher Ca-binding activity than unmodified silk fibroin.The increased Ca-binding activity could promote the usage of silk fibroin as a biomaterial in the pharmaceutical industry.This study shows the possibility of using silk fibroin as a mineralization accelerating medical material by generating genetically modified transgenic silkworm.