Cloning Of Foxo Gene And The Functional Investigation In Programmed Cell Death Of The Bombyx Mori Silk Gland

FOXO is pivotal in physiological processes such as growth, development, CellDifferentiation, Metabolic Apoptosis, Signal Transduction and so forth. In mammals,signal transduction pathways of Insulin-like Growth Factors like IGF/PI3K/FOXO arerelated to Cell Division Cycle, Cell Death, Stress, Detoxification and some other cellprocesses.Metamorphosis from larvae to pupae in Bombyx mori is combined with fiercetissue renewal, including the rapid degeneration and vanishing of specific tissues likesilkgland. It hasn’t been reported whether Bombyx FOXO was engaged in regulatingcell differentiation and apoptosis of specific-status tissues. Pinpointing the ProgrammedCell Death initiated or induced from larvae to pupae, the author cloned the completesequence of Foxo gene and investigated the expression profiles of it. Methods ofhormone treatment, starvation and food restrain were conducted to observe alterations inFoxo expression level of silkgland tissue in individual and BmN cells and any othergene upstream or downstream the signal transduction pathways, as well as variation ofFOXO protein under certain circumstances, hoping to provide reference to theregulation mechanism of PCD genesis during Bombyx metamorphosis. The results areas follows:1. Cloning and verification of Bombyx Foxo geneThe complete mRNA sequence of Foxo gene was cloned by RACE and verified bythe obtained Foxo segments in our laboratory. The gene was3326bp, and the longestORF was1539bp. It was localized to nscaf2890of Chromosome9with9extrons and8introns. It encoded512amino acids. The theoretical molecular weigh of seduced proteinwas55679.76with the isoelectric point of6.08. Two trans-membrane helixes forminner to outside and one from outside to inner were observed in the amino acid402-434 area. The secondary structure predicted that α helix of FOXO approximately took by33.01%, extension chain by about7.81%, β corner by3.21%and random rollingstructure took by about56.05%. Analysis of functional domain （conservative area）indicated that amino acids92-181of FOXO protein were the specific Winged helix/Forkhead domain of FOXO family. The front and middle jaws of FOXO protein indifferent species were highly conservative. Cluster analysis demonstrated that BombyxFOXO, which belongs to insect FOXO proteins, was far from vertebrates but closest toLepidoptera ones in evolution. This result accorded to the traditional evolution history.Therefore, the newly cloned gene was named Bombyx Foxo gene then （GenBankaccession number: JQ081294）2. The spatial and temporal expression profiles of Bombyx Foxo geneIn the expression profile of tissues, Foxo was expressed in all tissues on day3ofthe5th instar. However, there were differences between tissues in Foxo expressionlevels. Specifically, Foxo expression in ovary, head, fatbody and hemolymph was highwhile low in testis and silk gland, which was agreed in chip expression profile fromSILKDB.In the profile of development stages, Foxo expression was low on day3and day4of embryonic stage after incubation, and it was also low from the2nd instar topre-pupation. Foxo expression was high only in periods like1st newly molted insatr,pupation stage and moth stage. On the day3of pupation, the Foxo expression infatbody of male was significantly higher than that of female, highlighting the giantdisparity between the sexes. The expression was high in the fatbody of feathered femalemoths, but still it was lower than that of male moths.3. The relation between FOXO and PCD induced by molting hormoneFoxo, EcR, E74A, InR, PTEN, Ywhaz （14-3-3zeta）, Tor1, Atg8, Saposin-like and soforth in silkgland and BmN cells were investigated for expression levels in order todemonstrate the influence of molting hormone on Foxo.Based on the expression levels of EcR and E74A in the silkgland of normal5thinsatr larvae, it could be speculated that variation in the titer of molting hormone was agreed with hemolymph, namely the titer of molting hormone at the prior period of the5th instar sustained low and went up on day6of the5th instar. It zoomed at maturationstage, and so did the value of FOXO protein. It was reported that20E would restrain thephosphorylation process, and in our study, InR and Ywhaz, which restrained the activityof FOXO, were relatively low at pre-pupation stage. At pupation stage, E74A andSaposin-like, related to apoptosis were rapidly up-regulated. Therefore,20E triggeredthe expression of FOXO, and the most efficient period it posed to cell apoptosis wasmaturation stage.The20E titer in the larvae went up after they were injected with exogenous20E onday4of the4th instar, and so were the Foxo transcription and translation levels.Injection to BmN cells with exogenous20E also up-regulated the expression of Foxo.Meanwhile, E74A, Atg8and Saposin-like were all up-regulated. The levels remainedhigh compared with the control group. Autophagy and apoptosis were observed in BmNcells by fluorescent staining. It could be inferred that FOXO did take part in the PCDprocess induced by molting hormone.4. The relation between FOXO and PCD induced by starvationThe expression levels of Foxo, InR, PTEN, Ywhaz （14-3-3zeta）, Atg8, Saposin-likeand some other genes in silkgland and BmN cells were investigated to demonstrate theinfluence of food restraining and starvation on Foxo.InR gene in the silkgland was up-regulated after the larvae were starved orrestrained by food. It had been reported that starvation would impair the titer ofbombyxin, probably by enhancing the sensitivity of silkgland to insulin signal. However,the result of sensitivity enhancing was more significant in starvation group than thefood-restrained group. Foxo expression levels in silkgland were both remarkablyup-regulated by the two treatments. Though sensitivity to insulin signal in silkgland wasenhanced, Ywhaz was down-regulated, inferring that the absence of nutrition factorcould up-regulated the expression of Foxo, and may enhance the activity of FOXO. Theless of the nutrition, the higher was the activity of FOXO. Thus the activated FOXOprobably took part in the degeneration of silkgland in silkworm. In cell experiments, starvation induced the autophagy and apoptosis of BmN cells.In this process, Foxo gene was up-regulated. PTEN, which restrains phosphorylationprocess, was tremendously up-regulated, though the up-regulation of InR gene wouldenhance the sensitivity of insulin signal, indicating that phosphorylation level wasgreatly restrained. Then the expression of Saposin-like was also tremendouslyup-regulated. Thus it could be predicted that the activity of FOXO was enhanced whenlarvae were in starvation, and FOXO was closely related to cell apoptosis byparticipating in PCD induced by starvation..5. The research of interfering Foxo geneAccording to the expression profile of Foxo at24h,48h and72h in the silkglandof the interference group and the negative control group, the interfering efficiency ofsiRNA₄90, siRNA₈21and siRNA₁161were confirmed, among which siRNA₈21stood out. According to the expression profile of Foxo, Atg8and Saposin-like in thesilkgland of the interference group and the negative control group, indicating thatFOXO and autophagy contact more closely, and the instantaneous increasing of FOXOexpression can induce autophagy. The results of silk gland cell nucleus staining and theanalysis of the genome DNA extracted from silk gland showed that interfering Foxogene delayed the process of apoptosis in silk gland cell. This confirmed that FOXO didtake part in the PCD process induced by molting hormone. However, no abnormalphenotype or pupation was observed in interfered Bombyx individuals. It could beinferred that interfering Foxo gene did not affect the fundamental biological functionplayed by Foxo gene.6. Autophagy and apoptosis at the early stage of cell death in Bombyx moriIn our research, the up-regulation of Atg8was prior to that of Saposin-like innormal individuals or individuals treated by molting hormone, indicating that autophagywas generated before apoptosis which accorded with the known morphology conclusion.Autophagy was observed prior to apoptosis when BmN cells were stained, meanwhile,the up-regulation of Atg8in starvation group was prior to Saposin-like. After interferingFoxo gene, we found that the high-expression period of Atg8gene delayed, and so did Saposin-like gene. It was predicted that in cells, the autophagy may restrain theapoptosis or putrescence, thus to protect cells.