The Phenotypic And Mechanism Analysis Of A Posterior Silk Gland Mutant In Silkworm, Bombyx Mori

The most characteristic value of silkworms that is widely used by humans is their ability to produce silk protein fibers efficiently. The silk gland (SG) of silkworm is a highly specialized organ that specifically synthesizes and secrets silk proteins, which is the main target of human domestication and silkworm selection. Increasing the efficiency of protein synthesis and secretion in silk glands and improving the components of protein fibers synthesized by silk glands are major issues in sericulture industry that attract attention around the world. As the transgenic silkworm technology matures, effective use of silk glands’ superior protein synthesis ability to produce exogenous proteins has become a hot subject in bioreactor research area. The studies of silkworms as model animals with Chinese characteristics have also attracted much attention in agricultural field of insect model application and biomedical field of invertebrate alternatives. This paper investigated a transgenic silkworm mutant phenotype of posterior silk glands and its mutation mechanism. The main results are as follows:1. Constructing Silkworm posterior silk gland developmental mutant TBH by transgenic technologyA total length of 3057 bp Fib-H like coding sequence Hp1(Fib-H-like gene) was designed and transferred to the silkworm genome by Piggybac transposon. Mutant TBH (hp1/hp1) that specifically expressed Hpl in the posterior silk gland (PSG) was screened. Successful expression of exogenous gene Hp1 in PSG was confirmed by measuring mRNA and the protein. The anterior silk gland (ASG) and the middle silk gland (MSG) of TBH developed normally at larval stage, while its PSG was significantly shorter than that of the wild type, the diameter of PSG became larger, its light transmittance was reduced and the number of folds decreased, a large number of "beadlike" curly nodules appeared and the arrangement of nuclei became disorganized from dendritic branching shape of the wild type. The amount of silk spun by TBH mature larvae was reduced, thus the proportion of naked pupae and semi-naked pupae in the pupation process was significantly increased.2. Reducing the expression of endogenous fibroin genes increased the amount of exogenous recombinant protein expression in the posterior silk glandIn TBH individuals, the level of transcription and protein synthesis and secretion of endogenous genes Fib-L and P25 did not decrease, while mRNA level of Fib-H reduced by 71.1% and Fib-H proportion in secreted fibroin proteins dropped to 43.74% from 91.20% of the wild type. Although mRNA level of exogenous Hpl was only 0.73‰ and 0.74‰ of that of Fib-H and Fib-L respectively, HPL protein accounted for 18.85% of fibroin and 15.46% of the total amount of secreted proteins, thus exogenous macromolecular protein HPL was very efficiently translated and secreted. Analysis of DEGs was further carried out in the PSG cells and 891 DEGs were detected. Not only did 239 genes related to protein processing in endoplasmic reticulum significantly differentially expressed, but the expression of 37 genes associated with stress and apoptosis, as well as neural responses was also significantly affected in the PSG cells of TBH. These results indicate that when Fib-H transcription was down regulated in TBH system, the production of exogenous recombinant protein was significantly improved, thus providing an important reference and idea of optimization for the production of other recombinant proteins in the silk glands.3. The developmental abnormalities in the posterior silk gland affected the process of programmed cell death (PCD) and normal expression of 30K protein during the process of silk gland degenerationDuring the process of silk gland degeneration of larval-pupal metamorphosis, HE staining and PCD-related test of silk glands of transgenic mutant TBH showed that the degeneration process of TBH silk glands was significantly slower than that of the wild type. The determination of 30K protein levels in PSD showed that there were no significant difference of its level between the 5th instar larvae TBH and wild type silkworms. However, during the process of silk gland degeneration, the level of 30K protein synthesis was improved in the wild type silkworms after silk protein synthesis was slowed down, while the transcription level of 30K protein gene and 30K protein levels in TBH silkworm silk gland were significantly lower than those in the wild type. We speculate that the repression of normal expression of Fib-H and other silk proteins resulted in developmental malformations of the (posterior) silk gland, and further changed the PCD and silk gland function of synthesizing physiological proteins during the process of silk gland degeneration, affected a series of physiological functions including reproductive development that 30K protein was involved in, for instance, leading to reduced number of eggs laid by the silkworms.4. A model of hyperproteinemia/hyperaminoacidemia was preliminarily established with TBH as a materialJust as the effect of hyperlipemia and hyperglycemia on the human bodies, hyperproteinemia or hyperaminoacidemia also poses serious threat to human health. However so far, animal disease model of hyperproteinemia/hyperaminoacidemia has not been reported. In this article, concentrations of proteins and free amino acids in the hemolymph of the transgenic mutant TBH individuals during the pupa stage rose sharply, and a high protein/amino acid environment in circulatory blood existed for 6d-7d, which induced the death of TBH silkworm individuals. This is an example that a model of hyperproteinemia/hyperaminoacidemia had a long window phase accounting for over 10% of its life cycle, which has a great significance in studying the pathogenesis of hyperproteinemia/hyperaminoacidemia in animal tissues and developing related therapeutic drugs. Furthermore, during this phase the animals (silkworms) are inactive and don’t eat, so they are a closed biological system that can significantly improve the accuracy of relevant studies.