Effects Of Nutritional Signaling Pathways On Storage Proteins In Silkworm,Bombyx Mori

Bombyx mori is a Lepidoptera model organism and is a completely metamorphic insect.The silkworm’s life has to go through four stages:egg,larva,pupa and adult.The larval stage of the silkworm is divided into five instars,from the first to the fifth instar.In the fifth instar,the last stage of the larva,the silkworm will eat a lot of mulberries and ingest a lot of nutrients and grow rapidly.There is a type of protein in the silkworm body,which is expressed in high amounts in the fat body of the fifth instar,secreted into the blood,and quickly absorbed by the fat body during the wondering.This type of protein is called the storage protein.The storage protein of the female silkworm accounts for 60%of the total fat body protein from the larva-pupa to adults.They can participate in the formation of the silkworm epidermis,provide an important source of amino acids for the reproductive and development of adults,and provide nutrients during the development of eggs,While also participating in the immune response.The expression of storage protein has obvious characteristics of tissue,period,sex specificity,and high expression.Its transcriptional regulation process is very complicated,and previous related reports are rare.This study intends to explore the molecular regulation mechanism of the high-level expression of the silkworm storage protein at the fifth instar and to provide references for elucidating the research on the expression regulation mechanism of this kind of insect storage protein and the function of the storage protein.The main research results obtained are as follows:1.Starvation treatment down-regulates the expression of BmSPsTo determine whether the high expression of BmSPs at the fifth instar is related to the large amounts of nutrients ingested by the silkworm,this study first used starvation to treat the fifth-instar female silkworm individuals and found that the expressions of BmSPs at the m RNA level and protein level are decreased sharply.The expression of insulin receptor(In R),an important factor in the nutrition-related insulin signaling pathway,and the transcription factor Bm Fox O downstream of the insulin signaling pathway was significantly up-regulated.2.Insulin nutrition signaling pathway is involved in regulating the expression of BmSPsIndividuals injected insulin and Akt inhibitorand(Akti)PI3K inhibitor(PI3Ki)of the insulin signaling pathway.Molecular testing found that insulin induction significantly up-regulated the expression of BmSPs at both the m RNA and protein levels;after Akti injection,the expression of BmSPs tended to decrease.After PI3Ki injection,the expression of BmSPs was significantly down-regulated at the m RNA level and protein level.By culturing the fat bodies of female silkworms on the third day of the fifth instar in vitro and adding trehalose and PI3Ki,the test found that trehalose was significantly up-regulated The expression of BmSPs,and PI3Ki significantly down-regulates the expression of BmSPs.Insulin and Akti and PI3Ki were added to silkworm embryonic cells(Bm E cells),the test found that insulin significantly up-regulated the expressions of BmSPs,The expression of BmSPs was significantly down-regulated after Akti and PI3Ki treatment.Therefore,individual experiments,tissue culture,and cell experiments have consistently proved that BmSPs are affected by factors of the insulin signaling pathway.3.Bm Fox O,a transcription factor downstream of the insulin signaling pathway,induces down-regulation of BmSPs expressionTo further prove that BmSPs are affected by the insulin signaling pathway,the relationship between Bm Fox O and BmSPs was studied in depth.The expression of Bm Fox O was significantly down-regulated after individual injection of insulin,while the expression of Bm Fox O was significantly up-regulated after PI3Ki injection;by constructing a Bm Fox O cell-level overexpression vector,after successfully overexpressing Bm Fox O in Bm E cells,the expression of BmSPs was reduced significantly.The Bm Fox O potential binding element(Bm Fox O-like CRE)prediction was performed on the 3 000 bp upstream promoter of BmSP1,and it was found that there are four Bm Fox O-like CRE on the BmSP1 promoter.EMSA experimental results show that Bm Fox O can be directly combined with the second Bm Fox O-like CRE element of BmSP1.The chromatin immunoprecipitation experiment(Ch IP)was used to analyze the binding in vivo at the cellular level.The results of the Ch IP experiment showed that the chromatin precipitated by the Flag tag antibody fused to Bm Fox O can be amplified to the second Bm Fox O specific sequence of the like CRE element of BmSP1 indicates that Bm Fox O can directly bind to the second Bm Fox O-like CRE element of BmSP1 at the cellular level.Then the enzyme activity analysis of the BmSP1promoter at the cellular level found that when there is a second Bm Fox O-like CRE element,Bm Fox O can significantly reduce the activity of the BmSP1 promoter;but truncating here,Bm Fox O cannot significantly change the activity of the BmSP1promoter.The above results indicate that the insulin nutrition signaling pathway down-regulates the expression of Bm Fox O and then up-regulates the expressions of BmSPs.4.Amino acids affect the expression of BmSPs through the TOR signaling pathwayIn addition to carbohydrates,protein is also the main component of mulberry leaves.Therefore,we speculate that the amino acid signaling pathway is involved in the regulation of the expressions of BmSPs.Twenty mixed amino acids were used to inject female silkworms on the second day of the fifth instar.The experimental results showed that individual injection of amino acids can significantly up-regulate the expressions of BmSPs;the main nutritional signaling pathway that amino acids can activate is the TOR signaling pathway.After injection of rapamycin(Rapa)an inhibitor of the signaling pathway,the expressions of BmSPs were significantly down-regulated at the m RNA level and protein level.Using amino acids and Rapa to culture the fat bodies of female silkworms on the third day of the fifth instar in vitro,it was found that the addition of amino acids significantly up-regulated the expressions of BmSPs,while Rapa significantly down-regulated the expressions of BmSPs.GATA transcription factors are the key transcription factors in the TOR signaling pathway.Six subtypes of GATA transcription factors have been successfully overexpressed in Bm E cells.The test results found that only Bm GATAβ4 can significantly up-regulate the expressions of BmSPs.The expression of Bm GATAβ4 was detected on the material cultured in vitro and found that the addition of amino acids can significantly up-regulate the expression of Bm GATAβ4,while Rapa inhibits the expression of Bm GATAβ4.The double-stranded interference of Bm GATAβ4 at the cellular level showed that the expressions of BmSPs were significantly down-regulated with the interference of Bm GATAβ4;the expressions of BmSPs were also significantly down-regulated by Bm GATAβ4 intervention in female silkworm individuals on the second day of the fifth instar.The dual fluorescence reporter system further proves that the promoter activity of BmSP1 can be up-regulated by Bm GATAβ4.The above results indicate that the nitrogen source substance-amino acids can affect the expression of Bm GATAβ4 of the TOR nutrition signal pathway,and then affect the expression of BmSPs.5.Bm GATAβ4 and sex-regulated transcription factor doublesex synergistically affect the expressions of BmSPsThere are obvious gender differences in the expressions of BmSPs.To explore its mechanism,first,we cloned 6 doublesex transcription factors with transcriptional activity,including 4 female cleavage forms(Bmdsx F~1,Bmdsx F~2,Bmdsx F~3,Bmdsx F~5)and 2 male cleavage forms(Bmdsx M~1 and Bmdsx M~3),and a cell-level overexpression vector was constructed.Various doublesex transcription factors were successfully overexpressed in Bm E cells and found that except for the effects of Bmdsx F~3 and Bmdsx F~5 on the expressions of BmSPs,the other doublesex had no significant effect on the expressions of BmSPs.Predictive analysis of doublesex possible binding elements on the promoters of BmSPs revealed that the promoters of BmSP1,BmSP2,and BmSP3all have three possible doublesex binding elements,and there are GATA transcriptions near the two possible doublesex binding elements on the promoters of BmSPs.It is speculated that there may be interactions between doublesex and GATA transcription factors.To prove this point,doublesex and Bm GATAβ4 were co-transfected into Bm E cells.The co-transfection of six transcriptionally active doublesex and Bm GATAβ4could all inhibit the upregulation of Bm GATAβ4 on BmSP1,with Bmdsx M~1 and Bmdsx M~3 inhibiting the most significant;As far as the expression changes of BmSP2and BmSP3 are concerned,the co-expression of two male splicing forms of doublesex and Bm GATAβ4 can further promote the up-regulation of Bm GATAβ4 on the expression of BmSP2 and BmSP3.Co-immunoprecipitation experiments further proved the interaction between Bm GATAβ4 and Bmdsx M~1.The above results preliminarily indicate that the different transcription forms of doublesex and Bm GATAβ4 may interact with each other and have different regulatory effects on different types of storage proteins.