Functional Studies Of C-Jun Gene And Nucleolar Essential Protein 1 Gene In Silkworm, Bombyx Mori

c-Jun N-terminal kinase(JNK) signaling pathway plays an important role in cell proliferation, transformation, differentiation and apoptosis. Nucleolar essential protein 1(Nep1) gene is functionally conserved in many species. It has been demonstrated that Nep1 in yeast is essential for the biogenesis of the 40 S ribosomal subunit. Mutation of Nep1 in humans causes the autosomal- recessive disorder Bowen-Conradi syndrome. The silkworm Bombyx mori is an important economic insect and a central Lepidoptera model. The research about Jun and Nep1 is unclear. The BmJun, BmFos and BmNep1 genes were cloned preliminary by our laboratory, but their biological functions were largely unknown. We used RT-PCR, q RT-PCR, IP, Western blotting, immunohistochemistry and LC-MS/MS to study the relationship between BmJun protein and BmFos protein in JNK signaling pathway and their regulation function of reproductive development in silkworms Bombyx mori. We studied the expression pattern of BmNep1 protein which was induced by ecdysone hormone during larva-pupa metamorphosis, and explored the process of its participation in ribosome biogenesis. The main results are as follows: 1 The JNK pathway was involved in the regulation of reproductive development by regulating the synthesis and storage of BmVgc-Jun protein not only can form homodimer but also heterodimer AP-1 with Fos protein to play a regulatory role of transcription factor in mammals. Drosphila DJun and DFos protein only form heterodimer. According to the sequences of BmJun and BmFos gene cloned by our lab priliminary, we constructed the recombinant plasmids of pCMV-BmJun-EGFP and pCMV-BmFos-FLAG by using pCMV-EGFP and pCMV-FLAG plasmids and expressed in HEK293 T cell line. Total protein was extracted for IP experiment. The results showed that BmJun interacted with BmFos protein to form BmAP-1, and it was speculated to play a regulatory role.Our previous interference BmJun showed the development of progeny egg was abnormal. The synthesis of vitellogenin is in fat body during larva-pupa metamorphosis. Based on this physiological basis, we investigated the expression patterns of BmJun, BmFos and BmVg genes during larva-pupa metamorphosis. The expression pattern of BmVg was consistent with BmJun/BmFos gene, which were significantly upregulated in the pre-pupa stage in the fat body. Further investigation confirmed that the BmJun protein level was significantly increased at pre-pupa stage in fat body, and the expression pattern of BmJun gene and protein in ovary was consiste nt with that in the fat body. The expression of BmVg gene was significantly down-regulated by interfering BmJun gene at wondering stage. We used JNK inhibitor SP600125 to treat with silkworm, the results showed that the expression of BmVg protein decreased in both fat body and ovary in female silkworm of experimental group, the GSI was significantly decreased at pupa stage, the percentages of abnormal eggs and t he adult who layed abnormal eggs were increased, and there was a dose effect. These results showed that BmJun interacted with BmFos to form BmAP-1 dimer and participate in the regulation of BmVg gene synthesis in silkworm fat body. They were involved in the regulation of ovary and egg development. 2 BmNep1 protein which was regulated by ecdysone was involved in ribosome biogenesis in Bombyx moriIn yeast, Nep1 protein involves in 40 S ribosome biogenesis. The mouse homologue Nep1 can restore functional defects caused by a mutation in yeast Nep1. And mutation of human Nep1 causes BCS disease. There are no reports on the function that Nep1 involves in the ribosome biogenesis in insects. We found that the expression pattern of BmN ep1 gene was consistent with BmE74 A, a 20 E primary-response gene, during the degeneration of silk gland in our previous study. And the expression of BmNep1 in BmN cell line was induced by 20 E. We investigated the expression profiles of BmNep1 gene and protein in tissues at 5t h instar of Bombyx mori, and found that BmNep1 gene was highly expressed in the silk gland. Further investigation the expression of BmNep1 protein during 4t h instar larvae molt and larva pupa metamorphosis in silk gland, it was increased with the increase of ecdysone titer. BmNep1 was up-regulated during 5L3 to pre-pupa stage, immunoprecipitated proteins who interact with BmN ep1 proteins, LC-MS/MS results showed that almost all of the identified proteins associated with the ribosome biogenesis. Further investigation in ovary showed that both of BmNep1 gene and protein are induced by ecdysone hormone. These results indicated that the regulation of ecdysone to BmNep1 was non-tissue-specific, and BmNep1 protein might be involved in the ribosome biogenesis.