Study On The Function And Transcriptional Regulation Of Pupa-specific Vitellogenin Gene, In Silkworm, Bombyx Mori

Vitellogenin (Vg)/vitellin (Vn) is the main nutrition on the egg’s formation and embryonic development. The synthesis of this protein in the female fat body and sequestered by competent oocytes via receptor-mediated endocytosis. It formed into yolk granules for storage and usage. The synthesis of Vitellogenin is under the control of the hormones, Juvenile hormone and ecdysone in insect, estrogen and the analogous in oviporous vertebrates. Vitellogenin consistitutes80%of total egg proteins in the most of the insect. Being of the amout, Vg is considered as the most important nutrition of the embryonic development. However, except Vn, there are lots of other yolk proteins in silkworm and some other Lepidoptera insects. There are40%Vn,35%30kDa proteins and25%egg specific protein (ESP) in silkworm yolk protein.This study analyse the location of BmVg on the silkworm chromasome, the structure of the gene and putative protein. The evaluation of vitellogenin during the oviposesus anlimal was analysed. Then, the function of BmVg was analysed based on the gene transcription and the protein synthesis profile. Furtherly, the transcriptional regulation mechnism of Bm Vg by ecdysone was studyed. The maily results as followes:1. The bioinformatic analysis of BmVgBmVg gene is located on the nscaf2800of the27th chromosome of silkworm genome. All of the CDS sequences containes6346base pairs. This gene has7exons and6introns. The putative amino acid sequence contained1782amino acids. The putative protein has three conservative domains:LPD-N, DUF1943and VWD. The evlution analysis showes Azumapecten farreri Vitellogenin is the oldest one. And different order of the insect is get together. It means the evolution of Vitellogenin is followed with the evolution of species strictly in oviparous animals.2. The expression profile of BmVgThe synthesis of BmVg is maily started at the methphosis of larvae-pupa, quickly with a high level in the female. And the synthesis was sustained all of the silkworm pupation. However, the signal of the BmVg is hardly to be detected in male with the same condition;The expression profile of Bm Vg was named "a female specific gene". However, this kind of female specific expression is not absolute. On the specific experiment condition, we can also detecte the BmVg gene transcription and the protein synthesis. So we consider BmVg as "a female specific highly expression gene" for precise.3. The transcription of BmVg induced by ecdysone and the transplanted ovaryEcdysone induced BmVg transcription:the cultured female silkworm fat body was treated by ecdysone and Juvile. And found the transcription of BmVg was induced by ecdysone, rather than JH. Furtherly, the induction by ecdysone has time and dose dependence.The synthesis of BmVg induced by transplanted ovary:After transplanted, the ovaries developed in male silkworms and formed four complete oviducts with eggs. RT-PCR, qPCR and western blot showed BmVg transcription and the protein synthesis were induced in the acceptor male fat bodies and the content of BmVn was increased in eggs. It indicated that the secretion from ovary stimulates male’s fat body and made synthesis of the BmVg and transported into ovary for its development.We supposed that this kind of ovary secretion with female quality. And we treated the male fat body by17p-estroidol. And the result showed that17β-estroidol can induce BmVg synthesis. However, whether the ovary secretion is17β-estroidol and how to regulate BmVg synthesis needs more evidences.4. BmVg is irreplaceable to the egg formation and embrynic development of the silkwormRNAi showed the number and the color of eggs was affected by BmVg down regulated. The immunofluorescence histochemistry of eggs showed disformed and less number of the yolk granule was found in the dowregulated of BmVg. The results showed BmVg is still irreplaceable to the egg formation and embrynic development of silkworm, eventhough it content less. 5. Cloning and the regulation elements analyse of Bm Vg promoterThe BmVg promoter with1.5K base pair was cloned successful. In total,73cis-response elements were detected on the promoter. The Drosophila sex determination transcription factor doublesex motif has the ability to regulate BmVg transcription specifically in the female silkworm; DBRC-Z2and DE74are the elements of Drosophila ecdysoen steroid early response genes Broad-complex isoform Z2and E74; BEAF is a boundary element-associated factor and DREF is a DNA replication-related element factor. In addition, there are some homeodomain transcriptional factor elements in the BmVg promoter, including the Drosophila homeobox transcription factor with CUT domain, the POU domain, the Abd-B group, paired homeodomain factors etc.6. BmBrC-Z2transcriptional factor plays a critical role in Bm Vg transcription and egg formation.From the results of the BmVg promoter analyse, three BrC-Z2cis-response elements were predicted on the promoter. The CREs of-1100～-1082、-1088～-1070with forward direction are close to each other and the other one-186～-211with a reverse direction. We supposed that these three elements are the putative BmBrC-Z2CREs. The cell transfection assay showed that these BrC-Z2CREs are important to the BmVg promoter activity towards the ecdysone, especially the one near the transcriptional start site of the promoter. And transcriptional factor BmBrC-Z2can bind onto the BrC-Z2CREs specally; Then, the over expression of the BmBrC isoforms (Zl, Z2and Z4), showed only BmBrC-Z2can induce the activity of Bm Vg promoter; The qPCR and immunohistochemistry showed the trend synthesis of BmBrC-Z2and BmVg are corresponding during the metamorphosis of female silkworm. All of the results suggested ecdysone signal regulates BmVg transcription via BmBrC-Z2binding with three BrC-Z2CREs on the promoter.RNAi of BmBrC-Z2affected the egg formation and embrynic development of female silkworm. And the phenomenon is dure to the downregulation of BmVg.7. Homeodomain POU regulate BmVg transcription by binding with Broad complex in silkwormCell transfection assay and EMSA showed BmPOUM2transcriptonal factor binding with POU CRE (near the-186～-211BrC-Z2CRE) onto the promoter to participate BmVg transcriptional regualtion. qPCR and immunohistochemistry showed the transcriptional trend of BmPOUM2and BmVg are corresponding during the metamorphosis of female silkworm. And the BmPOUM2protein was concentrated in the nucleus of the fat body cell, BmVg in the cytoplasm. It means BmPOUM2as a transcriptional factor participates the regulation of BmVg. Over expression of BmPOUM2and BmBrC-Z2in the BmE-SWU1cell line, the BmVg promoter activity to ecdysone was higher than the control. GST-pull down and co-immunoprecipitation analysed that BmPOUM2and BmBrC-Z2can bind each other as a complex. These data showed BmPOUM2participates in BmVg transcriptional regulation by binding with BmBrC-Z2.Based on all the results of this study, we indicaded that the model of transcriptional regulation of BmVg by ecdysone is as follows:Edysone signal was first transmited to BmBrC-Z2transcriptional factor by ecdysone receptor complex (EcR/USP). And then, on the one hand, BmBrC-Z2can bind to the BrC-Z2CREs on the BmVg promoter. On the other hand, BmBrC-Z2can bind with BmPOUM2transcriptional factor as a complex, binding on the CRE of BrC-Z2/POU to participate in the transcriptional regulation of BmVg.