Studies Of The JNK Pathway And BmNep1Gene Depending On The Degeneration Model Of Silkworm Silk Glands

The silkworm, Bombyx mori, is a central model in Lepidopteran insects, andaffords an important influence on national industry. Its silk gland is the mostcharacteristic and valuable organ, and it degenerates by programmed cell death duringthe larval-pupal metamorphosis. This organ can be taken as a model of development andmetamorphosis to study signal transduction pathways, gene functions and so on insilkworm. Based on this model, we cloned several genes in the JNK pathway, andprimarily studied the functions of silkworm JNK pathway by RT-PCR, RACE,qRT-PCR, Western blotting, Biochemical fraction, Immunocytochemistry, and RNAi;we also cloned and characterized BmNep1, a novel gene in silkworm, studied itsregulation and function, and primarily analyzed its function in silkworm.1. Cloning and characterization of novel genes, BmNep1, BmJun and BmFos,in silkwormBased on DJun protein, DFos protein and deduced Nep1amino acid sequence ofDrosophila, this study cloned cDNA of BmJun, BmFos and BmNep1in silkworm. Thelargest ORF of BmJun has720bp, which encodes239amino acids with a molecularweight of²6.8kD. The largest ORF of BmFos has720bp, which encodes380aminoacids with a molecular weight of⁴1.6kD. Similar to Drosophila DJun and DFos, theC-terminal of both BmJun and BmFos has a typical BZIP domain. The largest ORF ofBmNep1has711bp, which encodes236amino acids with a molecular weight of²6.5kD. This gene has three exons and two introns. It is localized to chromosome15. Theanalysis on conserved domain suggested the deduced protein belongs to the EMG1（Nep1） superfamily, essential for40S ribosomal biogenesis. Both multiple sequencealignment and the phylogenic tree demonstrated that BmNep1is a highly conservedprotein in various species. 2. The JNK pathway was involved in the degeneration of silkworm silkglandsIn Drosophila, DJun and DFos genes are involved in the degeneration of salivaryglands. The functions of these genes in the JNK pathway have not been studied insilkworm. Depending on the polyclonal antibody of BmJun, it was localized to thenucleus of BmN cells, which was consistent with its role as a transcriptional factor.During the degradation of silk glands, BmJun expression was significantly up-regulatedat the pre-pupal stage, which was up-regulated³28.7-fold to that at day7of the fifthinstar; and BmFos expression also dramatically increased at the pre-pupal stage, whichwas up-regulated¹40.8-fold to that at day7of the fifth instar. Moreover, both BmJunand BmFos were notably expressed in different parts of the silkworm silk glands.During this physiological process, the transcriptional level of BmJNK was significantlyup-regulated. It was first found BmJNK had2splice variants at least.3. The JNK pathway involved the regulation of adult eye and progeny eggdevelopmentIn Drosophila, DJun involves the regulation of adult eye and egg development. Weused mixed siRNA to disturb BmJun gene expression at the wandering stage （or at theearly spinning stage）. qRT-PCR results showed that the transcriptional level of BmJungene was down-regulated by mixed siRNA. RNAi of BmJun gene resulted in abnormaladult eyes in the pupal stage, and led to the decline of progeny eggs. Compared tonormal eggs, the eggs after RNAi were small and plicated. These results revealed thatthe functions of the JNK pathway in silkworm were similar to Drosophila in somerespects.The study firstly characterized the physiological functions of the JNK pathway insilkworm, which provided a clue for further studies on silkworm signal pathways.4. BmNep1gene was implicated in silkworm tissue degeneration during themetamorphosisIn yeast, Nep1involves40S ribosome biogenesis. And mutation of human Nep1results in BCS disorder. There are no reports about this protein in insects at present. During the5^th instar when silk glands produce mass of silk proteins, and during thelarval-pupal metamorphosis when silk glands degenerate by programmed cell death,expression pattern of BmNep1was different from other genes, BmP0, BmP1and BmP2,which were involved in ribosome biogenesis. During the larval-pupal metamorphosis ofsilkworm, the transcript level of BmNep1was highly induced as the degeneration of silkglands. During the degeneration of middle and posterior silk glands, BmNep1expression was induced. At the wandering stage, BmNep1protein could not be detected,and at the spinning stage, BmNep1protein level was up-regulated. During themetamorphosis, the transcriptional level of BmNep1was obviously up-regulated inmidguts from the spinning stage, which was different from BmP1gene. At thewandering stage, BmNep1protein could not be detected, and at the spinning stage,BmNep1protein level was up-regulated. These results suggested that BmNep1geneinvolved the degeneration of silkworm tissues during the larval-pupal metamorphosis.RNAi of BmNep1gene resulted in abnormal silkworm metamorphosis.5. Moulting hormone regulated BmNep1expressionThe transcriptional level of BmNep1and its protein level were in accordance withchanges of BmE74A,20E primary-response gene in silkworm. In other word, when themoulting hormonal level was up-regulated, BmNep1expression increased. UsingBiochemical fraction and Immunocytochemistry, BmNep1protein was localized to thenucleus of BmN cells. By BmN cells exposed to20E with a concentration of2and4μmol/L, both the transcript level of BmNep1and its nuclear protein level wereup-regulated, demonstrated that the moulting hormone regulated BmNep1expression insilkworm.In our study, it was first found that Nep1gene was regulated by an endocrinehormone, and involved the degeneration of silkworm tissues. It might regulate a processof programmed cell death. These results provided a new basis for further functionalcharacterization of Nep1, which might contribute to deeply understand development andmetamorphosis of insects as well as tissue degradation, and provide a clue for themolecular mechanism of neurodegenerative diseases due to Nep1mutation.