| In the long-term natural evolution process,the high adaptability of insects to changeable environment is closely related to their metamorphosis development.There are many kinds of insect metamorphosis,but all of them are undergoing the death of old organs and the reconstruction of new organs.As an important component of the female reproductive system,the ovary undergoes remodeling during the larval-pupal transition process,which makes the ovarian membrane degenerated and ovarioles rapidly development to ensure the normal development and maturation of eggs in the ovary.In addition,in the process of the replacement of each instar of insects,a few organs will undergo renewal and remodeling.For example,the original structure of trachea will be removed during the molting process for forming a larger gas exchange network to accommodate the increased oxygen demand that accompanies individual growth.Matrix metalloproteinases(MMPs)are important endopeptidases involved in tissue and organ remodeling,mainly through the degradation of extracellular matrix(ECM),which plays a very important role in the balance of ECM degradation during tissue and organ remodeling and development.Therefore,it is of great biological significance to explore the function and mechanism of MMPs in insect organ remodeling and development,so as to enrich and improve entomological research.Meanwhile,the study of insect organ metamorphosis development has potential value for new biological control strategies of insect pests.As a model of lepidoptera,Bombyx mori is a completely metamorphosis insect,undergoing four stages in its life: egg,larva,pupa and adult.Previously study showed that the deletion of MMPs in B.mori would lead to developmental abnormalities,such as abnormal tracheal branch development of larva,abnormal mating and oviposition process of adults,etc.It is suggested that MMPs play an important role in the metamorphosis development of B.mori.In this study,the role of Bm MMPs in organ development of B.mori was analyzed based on the different phenotypes presented by Bm MMPs transgenic knockout lines.It was confirmed that Bm MMP1 gene plays an important role in trachea development of B.mori,and the mechanism of Bm MMP2 gene regulating ovarian development of B.mori was explored.It is expected to provide new clues for the regulation of silkworm ovary development and theoretical reference for the analysis of insect ovary development mechanism.The main research results and conclusions are as follows:1.Bm MMP1 gene is involved in the trachea development of Bombyx moriPreviously study found that the Bm MMP1-KO transgenic silkworm strains began to die in large numbers on the third day of the fifth instar and could not complete the generation,and anatomy revealed abnormal development of trachea.In this study,quantitative real-time PCR(q RT-PCR)was used to analyze the expression of Bm MMP1 gene during tracheal development of B.mori,and it was found that Bm MMP1 gene was up-regulated during tracheal remodeling.Furthermore,the effects of Bm MMP1 gene on tracheogenesis and development had been analyzed.Firstly,the tracheogenesis of Bm MMP1-KO strain in embryonic stage was observed by in vitro culture of silkworm embryos and real-time microscopic observation,and the results showed that the deletion of Bm MMP1 gene did not affect the tracheogenesis in embryonic stage.Secondly,the tracheal development of silkworm larvae from the first instar to the fifth instar was observed,and the results showed that the number of tracheal branches decreased significantly at the third instar after the deletion of Bm MMP1 gene.Lastly,the tracheal taenidium spacing,tracheal cell spacing and proliferation ability were detected and analyzed by microscopic measurement,tissue immunofluorescence and5-Bromo-2’-deoxyuridine(Brd U)labeling,and the results showed that the taenidium spacing of trachea could not be increased with individual development,and the tracheal cells were crowded with the intercellular space reduced,and the cell proliferation ability was significantly weakened after Bm MMP1 gene was knocked out.All the results indicate that Bm MMP1 gene is essential for tracheal development and tracheal cell growth of B.mori.2.Bm MMP2 gene regulates ovarian remodeling in Bombyx moripreviously study found that the Bm MMP2-KO strain and Bm MMP3-KO strain had normal growth in larval stage,and normally become an adult after the metamorphosis in the pupal stage.However,the mating and oviposition difficulties occurred in the adult stage,suggesting that the development of their reproductive systems were affected.Compared with Bm MMP3-KO strain,Bm MMP2-KO strain had more significant phenotypic differences in female reproductive system,especially in ovarioles and eggs.In order to further study the development of ovarioles and eggs in the ovary of B.mori,anatomic analysis was conducted on the ovarian remodeling process of Bm MMP2-KO strain.The results showed that knockout of Bm MMP2 gene resulted in the ovarian membrane degradation and ovarioles development abnormally,and both ovaries could not be reshaped to form four independent ovarioles,while they were wrapped together and easily broken with completely losing their original elasticity and toughness.Subsequently,transmission electron microscopy(TEM)showed that the epithelial cells of the ovariole were reduced after the deletion of Bm MMP2 gene,and the edge was not clear.At the same time,5-Ethynyl-2’-deoxyuridine(Ed U)labeling results showed that the proliferation ability of ovariole epithelial cells was significantly reduced and apoptosis occurred.Furthermore,the cryotomy-tissue staining experiment was used to analyze the development of ovary of Bm MMP2-KO strain before remodeling,and the results showed that there was no significant difference through compared with the control.These results indicate that the deletion of Bm MMP2 gene hinders the remodeling of silkworm ovary.In order to further determine the regulation of Bm MMP2 gene on the remodeling process of silkworm ovary,the Bm MMP2 with a Ds Red label overexpressed transgenic line(Bm MMP2-OE)was constructed,and the overexpression of Bm MMP2 gene was successfully detected without affecting the gene expression at the insertion site.In this study,Bm MMP2-OE strain was used to detect Bm MMP2 in situ expression in the female reproductive system of B.mori,and the results showed that Ds Red label fluorescence was mainly located in the ovarian membrane and ovariole.Furthermore,the quantitative analysis of the expression of Bm MMP2 in female reproductive system by q RT-PCR and Western Blotting(WB)showed that Bm MMP2 was indeed expressed in the ovariole,and the expression level of Bm MMP2 was higher in the ovarian membrane during ovarian remodeling.In addition,q RT-PCR was used to analyze the expression characteristics of Bm MMP2 gene in the whole development process of silkworm ovary from the larval fourth instar to the adult.It was found that the expression level of Bm MMP2 gene was the highest on the second day of silking stage during the process of ovarian remodeling.These results indicate that Bm MMP2 gene regulates the remodeling of silkworm ovary by involving in the degradation of ovarian membrane and the development of ovariole.3.Effects of Bm MMP2 gene on egg development of Bombyx moriThe developmental process of eggs is happening in the ovary,and the ovariole provides nutritional transportation and morphological support for them.In order to explore the influence of Bm MMP2 gene on egg development,the eggs development of Bm MMP2-KO strain was analyzed by anatomical observation,egg number statistics and scanning electron microscope(SEM)observation.It was found that the number of eggs decreased significantly,and most of the eggs were not fully developed,but a few eggs still developed to normal size and formed egg shell and pore structure after knocked out Bm MMP2 gene.Furthermore,parthenogenesis experiments were carried out on these approximately normal-sized eggs,and it was found that they could develop.Meanwhile,SDS-PAGE and WB experiment showed that the synthesis,transport and absorption of vitellogenin(Vg)could be carried out normally.In addition,the expression of Bm MMP2 in the female reproductive system of B.mori showed low expression in the eggs.These results suggest that Bm MMP2 gene is mainly involved in the regulation of ovarian remodeling,and controlling the degradation of ovarian membrane and the development of ovariole to provide space for egg development,but not determining the maturation off eggs.4.Bm MMP2 regulates silkworm ovarian development by degrading type ⅠcollagenIn order to explore the mechanism of Bm MMP2 gene regulating ovarian development,label-free proteomics was used to analyze the expression of pre-pupal ovarian protein in Bm MMP2-KO strain and wild-type Dazao strain.Firstly,the expression differences of ECM related proteins were analyzed,and Collagen alpha-1(I)with the most significant difference was screened,which was significantly up-regulated after knocked out Bm MMP2 gene.QRT-PCR results showed that Collagen alpha-1(I)gene expression trend was consistent with Bm MMP2 gene during ovarian development,which was up-regulated on the second day of silking stage and eighth day of pupal stage;and the expression of Collagen alpha-1(I)gene in different parts of the ovary during remodeling process was also consistent with Bm MMP2,which was highly expressed in the ovarian membrane.Meanwhile,in situ enzyme assay result showed that type I collagen was degraded in the position of ovarioles and ovarian membrane in wild type Dazao strain,but there was no obvious degradation in the ovary of Bm MMP2-KO strain.In addition,the immunohistochemical results of ovariole at adult stage showed that the fibrocytes in ovariole of wild-type Dazao strain were uniformly full and collagen was orderly distributed around cells,while,the fibrocytes were significantly reduced and elongated,and collagen accumulation was abnormal in the Bm MMP2-KO strain.These results indicate that collagen degradation in the ovarian membrane and ovariole of silkworm after Bm MMP2 gene knockout is hindered,and collagen-Ⅰ can be the substrate of Bm MMP2.5.Bm ILP and Bm MMP2 genes co-regulate ovarian development of Bombyx moriAccording to the reports that MMPs can induced by Relaxin,a member of the insulin family,to prevent collagen accumulation in the heart,lungs and other organs,which causes tissue fibrosis.There are many insulin-like family members in B.mori and the insulin-like peptide(Bm ILP)as the member of it was screened out form four proteins with the most significant differences,which including ATP Synthase subunits,Serine/threonine-protein kinase Aurora-2,insulin-like peptide and an unknown protein through the proteomic differential protein screening in this study.The expression pattern of the fifth instar larva of B.mori showed that Bm ILP gene was highly expressed in the ovary.The expression during ovarian development showed that Bm ILP gene was gradually up-regulated after ovarian remodeling,but its expression level was significantly down-regulated after ovarian remodeling after the deletion of Bm MMP2 gene.The expression of adult reproductive system,Bm ILP and Bm MMP2 gene were highly expressed in the ovariole.In addition,after interference with Bm ILP gene,the ovariole of B.mori showed phenotype similar to that of Bm MMP2-KO strain.In order to explore the regulatory relationship between Bm ILP and Bm MMP2 gene,the experiments including Bm ILP protein was added in vitro and in vivo and gene overexpression and interference experiments were conducted in Bm NS cells were carried out.The results showed that Bm ILP gene regulates the expression of Bm MMP2 gene.Therefore,it is speculated that Bm ILP gene may be involved in silkworm ovarian development by inducing the up-regulation of Bm MMP2 gene to degrade collagen-I.In order to investigate the causes of significant downregulation of Bm ILP gene after Bm MMP2 gene knockout in silkworm ovary remodeling,The expression of key proteins in insulin-related signaling pathways was detected by WB,which also enriched in the KEGG(Kyoto Encyclopedia of Genes and Genomes),and the results showed that knockout of Bm MMP2 gene led to increased phosphorylation of AKT(Protein kinase B)in silkworm ovary after remodeling.Additionally,to verify whether Bm MMP2 gene is regulated by PI3K/AKT(phosphoinositide 3-kinase/Akt)signaling pathway,the pathway inhibitor LY294002 was used for individual injection and cell addition experiments.And the results showed that the expression of Bm MMP2 gene was inhibited in both ovary and ovarian cell line Bm NS.These results indicate that Bm ILP gene is crucial for silkworm ovarian development.It is speculated that Bm MMP2 gene may be regulated through PI3K/AKT signaling pathway by Bm ILP gene.While,the abnormal phosphorylation of AKT after the deletion of Bm MMP2 gene is speculated to be the cause of the down-regulation of Bm ILP gene after ovarian remodeling. |
PDF Full Text Request