Regulation Of BmFoxO On The Metabolism And Growth Of Silkworm,Bombyx Mori

The silkworm,Bombyx mori,is a member of Lepidoptera for holometamorphosis insects.Growth and metabolism are mainly regulated by three hormones in silkworm,which contain the juvenile hormone,ecdysone and bombyxin.Silkworm converted the nutrients they stored or obtained from the outside into the necessary energy for growth through catabolism and anabolism in their life cycle.Once the balance between these two metabolisms was destroyed,the individual’s growth and development would be affected.Metabolism is the most basic system in individual development,learning the metabolism in different situations,which is of great significance to the study of growth and development in silkworm.The fat body of silkworm is the main organ that equivalent to the liver and adipocyte of mammalian which responsible for storing nutrients and participating in energy metabolism.In addition,fat body is the target tissue of hormone such as insulin.The insect insulin signaling pathway,is similar to that of mammals,plays a major role in the metabolism of protein,carbohydrates and lipid,individual reproduction,life span and growth.FoxO,an essential transcription factor downstream of the insulin signaling pathway,is widely exists in eukaryotes from yeast to mammals.Activated FoxO stimulates the expression of target genes,sequentially,induces apoptosis,inhibits the cell cycle,improves immunity and promotes gluconeogenesis and lipolysis in individuals.The current research shows that knocking out the BmFoxO results in smaller individuals.Moreover,20 E can induce the activity of BmFoxO and then promote the decomposition of fat body in the silkworm.However,the function of BmFoxO in the growth and metabolism of silkworm is not yet clear.In view of this,basic features of BmFoxO were inquired firstly,and then preliminary explored the function of BmFoxO in nutrition metabolism through starvation treatment of silkworm and overexpression of BmFoxO in BmE cells.Finally,explore the effects of BmFoxO on growth and metabolism using the transgenic overexpression technology.The main results are as follows: 1.Analysis of basic characteristics of BmFoxOThe expression patterns of BmFoxO in each tissue of the 3rd day of 5th instar larvae were analyzed by RT-qPCR.The results showed that BmFoxO was expressed in various tissues and highly expressed in the midgut,fat body and hemolymph.Fat body expression profile showed that the expression level was higher at newly molt of 5th instar larvae and pupae stage than other stages.To understand the effects of insulin,ecdysone,and juvenile hormones on BmFoxO during growth and development of the silkworm,same hormonal treatment(Akt inhibitor,20 E,JHA)at BmE cells and vitro of the fat body.It was noted that the expression of BmFoxO was significantly increased after treating with the Akt inhibitor and 20 E,whereas reduced via disposal of JHA.Overexpression of BmFoxO in BmE cells promoted the process of apoptosis whereas inhibited cell proliferation.Further,the expression of genes involved in apoptosis such as caspase1,caspase4 and ice were elevated analysis by RT-qPCR.The expression of 4EBP,which inhibited the translation initiation was increased,while myc that regulated biosynthesis of ribosome and promoted cell growth was decreased.It was proved that overexpression of BmFoxO in BmE cells inhibited the cell growth.2.Effect of BmFoxO on nutrition metabolism in silkwormIn order to comprehend the role of BmFoxO in nutrition metabolism,the silkworm began to be starved on the 2nd day of 5th instar.Indexes of lipid and trehalose in the fat body and hemolymph were observed at different time during starvation.Data showed that the content of glycogen and triglyceride in the fat body were decreased continuously during the period of starvation,and the concentration of trehalose in the hemolymph increased during the starvation of 48 h-72 h.Expression levels of genes of insulin pathway such as BmFoxO,InR and Akt and those linked to lipid metabolism enhanced remarkably during starvation stress determined by RT-qPCR,which accelerated lipolysis.It was confirmed that the genes related to glucose and lipid metabolism and insulin pathway mentioned above had the similar expression trend after overexpressing BmFoxO in BmE cells compared with that of starvation stress.These results postulated that BmFoxO plays an important role in the nutritional metabolism of silkworm.3.Generation and detection of transgenic silkworm overexpressed BmFoxOFor the purpose of analyzing the effect of BmFoxO on the growth and metabolism of silkworm by stably changing the expression of BmFoxO in the silkworm.A transgenic vector of overexpressing BmFoxO systemically with A4 promoter was constructed and microinjected into silkworm eggs.The red fluorescent protein expressed in eyes of silkworm was selected termed Over-FoxO.Test of insertion site suggested that BmFoxO was inserted on nscaf 2529 of chromosome 5 without disturbing the expression of other adjacent genes in Over-FoxO.The expression of BmFoxO in the lavare on 5th instar and various tissues of 3rd day of 5th was verified by RT-qPCR and RT-PCR.The larvae,pupae and moths of Over-FoxO were smaller and the body weight was considerably reduced.Therefore,we analyzed the expression of genes that regulate growth and metabolism in insulin and TOR pathways in Over-FoxO.We determined that the expression of InR and Akt was increased.On the contrary,the Tor1 and Tor2 were down regulated.Moreover,the expression of 4EBP was induced in Over-FoxO and thus inhibited the initiation of translation.The transcription level of myc in the fat body was increased whereas decreased in muscle.Eventually,cell growth was inhibited might be due to changes in 4EBP and myc.Expression of genes concerned with lipid decomposition(bmm,al-1,al-2,akhr,gk)and synthesis of glucose and trehalose(g6p,tres,tre1,tre2)and glycogenolysis(gp)were increased in the fat body of Over-FoxO silkworm.The results listed above indicate that overexpression of BmFoxO could inhibite the expression of genes related to synthesis of proteins and ribosomes in the silkworm,which inhibited the growth of the silkworm.Besides,the gluconeogenesis and lipolysis were promoted in the silkworm.4.Metabolomic analysis of hemolymph in overexpressing BmFoxO transgenic SilkwormDue to significant changes in the expression of genes related to carbohydrate and lipid metabolism in the fat body of Over-FoxO silkworm,indexes detection was carried out with the fat body and hemolymph of Over-FoxO as well as the control silkworm of wild type on 3rd day of 5th instar.It was informed that the concentration of glucose and trehalose raised significantly and the content of triglyceride decreased in the hemolymph in Over-FoxO silkworm.The content of glycogen in the fat body of Over-FoxO also declined.Based on the principle that Oil Red O stained triglyceride in red,the fat body isolated from Over-FoxO was discovered staining less than wild type individuals.A total of 29 different metabolites were examined in the hemolymph on 3rd day of 5th instar larvae of Over-FoxO and wild type silkworms by GC-MS.The principal metabolites were carbohydrate and intermediate metabolites,fatty acids and acids,and most of them were increased in Over-FoxO silkworm.Metabolic pathway analysis showed that the different metabolites mainly existed in glucose and lipid metabolism.Diverse metabolites focused on galactose metabolism,ubiquinone and terpenoid biosynthesis,phenylalanine tyrosine tryptophan biosynthesis,vitamin C metabolism was tested by the MetPA,which manifested that energy metabolism has changed in Over-FoxO silkworm.In a word,the content of lipid was decreased and the concentration of glucose was enhanced in the OverFoxO silkworm.It suggested that the metabolic balance of silkworm was destroyed,which affected the growth of silkworm.