Study On The Regulation Mechanism Of Silk Gland Factor 1(SGF1)Nuclear Localization In Bombyx Mori

The silkworm Bombyx mori is economically important insect due to its silk synthesis ability and used as a model organism in metamorphosis and developmental biology studies known for lepidopetra representative.The posterior silk gland(PSG)is the longest part which can synthesis fibroin is the effective component of silk and prior to the quality and yield of fibroin directly.Illustrating the detailed regulatory mechanism of fibroin synthesis will provide important theoritic refference for the future to molecular breeding in silkworm.SGF1,a member of FoxA transcription factor family is the first discovered transcription factor involved in the synthesis of fibroin in silkworm and plays an important role in development process of silk gland.Our collaborate lab has found that the phosphorylation level of SGF1 peptide(EQEAAS*PTSALQR)increased in PSG of RaslCA transgenic silkworm.We assumed that it may responsible for high silk yield by transgenic silkworm,However it is remained to be clarified whether phosphorylation of SGF1 influence nuclear localization to enhance the transcriptional regulation of fibroin synthesis related genes and regulation mechanism of Rasl downstream Raf/MAPK and/or PI3K/AKT signaling pathway.during this process.We used bioinformatic software to analysis SGF1 and predicted the phosphorylation site and regulated kinase of SGF1 phosphorylation peptide(EQEAAS*PTSALQR).Then we verified SGF1 whole length and phosphorylation antibody in vitro and detected SGF1 developmental profile in PSG,The preliminary data indicated that the SGF1 nuclear localization is positive related with the expression of fibroin related gene by immuno-staining and western blot.The PI3K/AKT or Raf/MAPK signaling inhibitors treatment in vivo aimed to figure out which pathway play the important role in regulating SGF1 nuclear localization.In the end,we explored if the influence of inhibitors to SGF1 and its homologous gene FoxAl nuclear localization through regulating SGF1 phosphorylation in silkworm and mammalian cell lines.The details are as follows:1.Verification of SGF1 and P-SGF1 antibody and SGFl development profile analysis in PSG.Based on known SGF1 full length sequence and phospho-peptide sequence information,we prepared whole length and phosphorylated SGF1 antibody.We constructed two vectors pIEx4-SGF1-V5 and pIEx4-SGF1S91A-V5 and transfected them into BmN cell individually to verify SGF1 and P-SGF1 antibody by Western Blot.Then we analyzed the development profile of SGF1 in the PSG.The transcription level of SGF1 increased gradually from Day 4 of the fourth instar(4L4D)to Day 3 of the fifth instar(5L3D)and then decreased gradually and the protein level of SGF1 increase gradually from Day 4 of the fourth instar(4L4D)to Day 7 of the fifth instar(5L7D)and then decrease dramatically.2.Nuclear localization of SGF1 and relationship with fibroin expressionBy immunostaining of SGFlin PSG we found that SGF1 nuclear localization was commonly found during feeding stage.SGF1 could be observed in nuclear until the Day 1 of pre-pupation(PPl)and translocated from nuclear at Day 2 of pre-pupation(PP2).The localization switch of SGF1 from assembling in nucleus to dispersing in cytoplasm happened from PP1 to PP2.Finally,we observed that the precise time of this localization switch time is W36h.By immuno-staining experiment and Western Blot,we compared the protein expression and localization of SGF1 in both Ras1CA transgenic and wildtype silkworm.The results showed enhanced SGF1 nuclear locolization and protein level in PSG of Ras1CA transgenic silkworm than in wildtype.3.Study on regulation of SGF1 nuclear localization by Raf/MAPK or PI3K/AKT signaling pathways in vivo.In order to study the regulation of SGF1 nuclear localization,we used the inhibitors(PLX and PD60)of Raf/MAPK signaling pathway and inhibitors(Wort、Rapa and LY294)of PI3K/AKT/TORC1 signaling pathway to treat silkworm at different development stage.The results showed that.after inhibitors treatmentsin vivo,the expression and nuclear localization of SGF1 decreased by immuno-staining experiment and Western Blot.So we presumed that this two signaling pathways can regulate the function of SGF1 nuclear localization.4.Study on regulation of SGF1 nuclear localization by Raf/MAPK or PI3K/AKT signaling pathways in vitro.Inhibitor treatment and point mutation experiments have been proved that Raf/MAPK or PI3K/AKT regulating SGF1 Ser91 site to enhance its nuclear localization in vitro.To clarify whether this regulatory mechanism is evolutionary conserved,we constructed wildtype and mutated plasmids of SGF1 homologous gene in mammal,FoxAl.By immuno-staining and Western Blot,we detected the phosphorylated site and signaling pathways regulation to FoxA1.The results showed that phosphorylation of FoxA1 has nothing to do with nuclear localization,however its nuclear locolization is regulated by Raf/MAPK signaling.In conclusion,the nuclear localization of SGF1 can be regulated by Raf/MAPK and PI3K/AKT/TORC1 signaling pathways and positively correlated to the fibroin transcription.