Influence Mechanism About The Posterior Silk Gland-specific Expressed Silk Fibroin Heavy Chain-like Protein Gene Hpl On The Silk Gland Development In The Silkworm, Bombyx Mori

Silkworm,Bombyx mori(B.mori),as the distinct Chinese characteristics insects in animal husbandry,is the essential invertebrate model and a model of Lepidopteran insects.The silk gland in B.mori is a highly specialized organ that specifically synthesizes and secretes silk protein.The excellent regulation mechanism of protein synthesis and secretion in the silk gland of B.mori is a fascinating and complicated research topic,which has attracted the attention of the industry.We utilized a laboratory-created TBH mutant resource for posterior that specifically expresses an Fib-h like protein-coding sequence Hpl,focusing on the protein synthesis ability of fibroin cells in the early age larvae of TBH mutant and the developmental changes and related regulatory mechanisms of fibroin cells during the embryonic stage,further explain the commonly occurring silk gland development and the mechanism of functional effects of fibroin transgenic silkworms represented by TBH.The results are as follows:(1)Synthesis and secretion of cocoon protein is influenced by expression of the Hpl gene in the larval posterior silk glandHpl is specifically expressed in the posterior silk gland(PSG)tissue of the N4 silkworm,which is normally secreted by transgenic technology.The mature larvae of TBH mutant could not cocoon normally.Although exogenous protein gene(Hpl)expressed at a extremely low level in PSG cells throughout the whole 5th instar larval stage when fibroin is vigorously synthesized,the transcription levels of endogenous fibroin protein gene BmFib-H,BmFib-L,and BmP25 were significantly up-regulated compared with wild type(WT)in both of the early and middle of 5th instar stages,except BmFib-H gene at wandering stage and BmP25 at 5L3d;on the other hand,the transcription level of fibroinase gene BmBcp and its inhibitor gene BmBcpi were significantly up-regulated.It showed that the level of fibroin synthesis in TBH mutant posterior silk gland cells was up-regulated simultaneously triggered a significant increase in the level of protein degradation.(2)Cytohistogenesis and development of silk gland are influenced by expression of the Hpl gene in the posterior silk glandIn the mutant TBH silkworm,the middle silk gland(MSG)develops normally throughout the larval stage,but the PSG develops abnormally and its length was significantly shorter than that of WT,and a large number of nodules and distortions appeared.The results of green fluorescent probe DiO and DAPI staining showed that the PSG nuclei of TBH mutant in the late 5th instar were dense in the swollen nodule,sparse in the bending depression,extremely uneven in nuclear branches,and squeezing at the expansion and distortion resulted in the rupture of the cell membrane.From the 3rd to 5th instar larvae,the changes were aggravated with the progress of the nuclear division and the aggravation of the nuclear branching of PSG cells.The silk gland of B.mori started at the middle stage and completed at the late stage of embryonic development.The number of silk gland cells does not increase in the larval stage.In the newly hatched larvae,there was no significant difference in the nuclear morphology of silk gland between TBH and WT,but the number of PSG cells was significantly less than that of WT.From the 2nd instar,the arrangement of PSG cells was disordered and the morphology began to be irregular.The results showed that the expression of Hpl gene in PSG significantly affected the development of early silk gland cells.In TBH silkworm embryos,although no trace amount of exogenous Hpl mRNA was detected,the transcription levels of BmFib-H and BmFib-L genes specifically expressed in posterior silk gland were significantly changed compared with WT embryos in silk gland cytohistogenesis phase(W3),silk gland cell mitosis proliferation phase(J3),and silk gland forming phase(J5).In addition,the transcription levels of the cell cycle genes BmCyclinB and BmCyclin3 were significantly up-regulated compared with WT during the three investigation periods.Transcriptome analysis showed that PSG specifically expressed Hpl gene,inhibited the gene expression of glucose metabolism regulation,weakened the gene transcription levels of Wnt pathway responsible for signal transduction,and affected the expression of genes related to cell differentiation,cell cycle,protein transport and secretion.Conclusion:The results showed that the developmental mechanism of PSG malformation in TBH mutant,is related to the abnormal transcription level of silk genes from the beginning of embryogenesis to the entire larval stage,the increasing of cell cycle regulation pressure,and the weakened signal caused by down-regulation of Wnt signaling pathway and carbohydrate metabolism pathway gene transcription levels.It is directly manifested by the slower proliferation of PSG cells and the decrease of cell number in embryonic stage,the slower proliferation of mitosis in PSG cell nucleus and the abnormal distribution of nuclei in larval stage.