| Insulin is a kind of protein hormone induced by endogenous and exogenous compounds such as glucose and secreted by pancreas isletβcells. It can effcct on reproductive and aging through adjusting glucose, fat and protein metabolism. Insulin signal transduction is conservative on evolution, and many species aging is involving in it. So it is possible to study on complex life phenomenon in lower eukaryote.In this paper, using information mining in the silkworm genome database, RT-PCR, RACE, RNA interference techniques and so on, we the cloned genes of Ir and Foxo, analyzed the gene expression patterns and preliminary identification of Ir and Foxo genes, Meanwhile, we also investigated the expression level of insulin and insulin-Like growth factor Signal Pathway homologous genes Akt,Ir,Foxo-s,Pdk,Irs,Ir-lp,Sirt2,Foxo,Pi3k60 and Pi3k110 in diet restriction and starvation Bombyx mori.1 The information mining of Ir and Foxo genes in silkworm sequences databaseUsing the IR and FOXO homologous protein sequences in Drosophila melanogaster, Aedes aegypti and other species as query sequence respectively, by blasting in the silkworm genome database and EST database from NCBI, and SILKDB, we acquire six potential sequences as coding part of the FOXO in silkworm genome, and three homologous genes of Ir. One of the three is Ir gene in silkworm, another is epidermal growth factor gene which has not being cloned, the third is the IR homologous protein which lacking the membrane kinase part (named IR-LP). We found five potential sequence fragments as coding part of epidermal growth factor protein, and two potential sequence fragments as coding sequence part of IR homologous protein which is unknown function by tblastn. 2 cDNA cloning and analysis of Ir-lp, Foxo and Rtpk in the silkworm, Bombyx moriAccording to the clues we found based on genome database,we cloned the genes of Ir-lp and Foxo and a similar code sequence only has kinase region, which named Rtpk by experiment method. The biggest ORF of silkworm Foxo is 1641bp, codes 546 amino acids, with molecular weight of 59574.45 D, and has a longer 5'UTR and a shorter 3'UTR, there is a typical structure domain of protein FOXO at the region between the 92 and 181 amino acids, and it's a member of transcription factors family. The homologization blast suggested that the protein coded by the gene is homologous with the human FOXO3 and FOXO1,fruit fly FOXOA and FOXOE, And nematodes daf-16, high homologous with the fruit fly, and there more the difference between human and nematodes.The longest ORF of cloned Ir-lp(insulin receptor-like protein gene,Ir-lp)of Bombyx mori is 1812 bp. The gene codes 604 amino acids, has a receptor L-arm and fibronectin 3. The result of 3'RACE suggested that, it no kinase domain like the insulin receptor, consistent with the result of gene mining prediction, the first 400 amino acid residues homologous to human and Drosophila proteins are highly conserved, further explain the cloned gene is the homologous gene of the IR family of silkworm.During the process of searching the homologous code sequence of the IR in silkworm genome, we find a sequence that has the highest homology with the Ir's kinase region only include the 400 amino acid residues of the kinase region. The result of cDNA fragment clone suggested that, the coding protein belongs to protein tyrosine kinase (PTK) family, is a kind of receptor tyrosine-protein kinase. Its kinase sequence has a high homologous with human, fruit fly, nematode this kinase sequence.3 Expression patterns of Ir and Foxo genesExpression levels of Ir gene were almost the same among 8 examined tissues on day 3 of the fifth instar larvae, but relatively low in blood, while expression levels of Ir-lp gene were significantly different among 8 examined tissues. It was the highest in head, then haemolymph and Malpighian tubule, and the lowest in fat body. Ir-lp gene, a insulin receptor analogue gene, was highly expressed in embryos incubated for 3 days, however, expression level decreased on day 4 of embryos, then there was high peak expression in the newly exuviated silkworm of the first, second and third instar larvae. In the newly exuviated silkworm of the fifth instar larvae, Ir-lp expression level decreased in fat body, then it fluctuated on day 7 of the fifth instar larvae and at the matured larval stage, gradually up-regulated during the pupal stage, and was highly expressed in pupae and adults, no matter female or male. It was suggested that maybe up-regulation of Ir-lp expression level during the pupal and adult stages was related with energy utilization efficiency, whereas up-regulation mechanism of other stages could not be well understood at present.Expression level of Foxo gene in blood was almost one time higher than other tissues, while Foxo-s sequence was expressed in every tissue, and expression level in fat body was twice of midgut. Foxo-s was almost not expressed on day 3 and 4 of embryos and in the newly exuviated silkworm of the first, second, third and fourth instar larvae, and highly expressed in fat body of the fifth instar prophase larvae, yet the level decreased as their development. It increased one day before the larvae changed into the pupae, then maintained at a high level during the pupal and adult stages. Foxo gene was lowly expressed in the most of our examined stages and relatively highly expressed during the first instar, pupal stage and moth stage.Expression levels of Rtpk gene significantly changed during our examined stages. Expression level was the highest on day 3 of incubated embryos, and then it was down-regulated. From the second instar larvae to the fifth instar larvae of day 7, expression level in fat body was low. The level was suddenly up-regulated at the matured larval stage, and then decreased during the pre-pupa stage, but it was relatively high during the pupal and moth stages, moreover, it was different between female and male.4 The effects of dsRNA treatment to genes of Ir and FoxoWe synthesized five dsRNA disruptors, and identified Ir600 and Foxo520 in them were two fragments which had obvious interference effects by screening, their effective concentrations are 1-20 nmol/L and 1-10 nmol/L, the effective time is 2-5d after interference treatment. In serum-free medium, the BmN cells of silkworm apoptosised and necrosised gradually. the interference group that contains 5 nmol/L Foxo520 has less Apoptotic cells than the negative control after interference treatment in 5 d, and its living cells also more; It suggested that Foxo gene might be promoting apoptosis of the cells which lack of serum induce.In Grace medium, the BmN cell of silkworm was treated with 5 nmol/L Ir600 dsRNA for 3-6d, The number of apoptotic cells increased significantly when compared with the control group in 5 d. It prompted that the down-regulation of Ir gene might be lead to cells apoptosis.5 The effect of dietary restriction and starvation on expression pattern of homologous genes in the bombyxin signal pathway of silkwormWe reproduced the Bombyx model of dietary restriction. And using this model, we investigated the influence of long-term and short-term hunger on gene expression of 10 genes from signaling system homologous in the different tissues at fifth instar larvae. Thus, in the influence of short-term hunger, apart from Foxo and Ir gene,the other genes were up-regulated expressed in gonad, midgut and integument, while more than 5 genes in 10 were down-regulated expressed in Malpighian tubule, head and blood. In the influence of long-term hunger, all genes are were up-regulated expressed in Malpighian tubule and were down-regulated expressed in sex gland and head. Most genes were up-regulated expressed in many tissues, obviously in midgut and Malpighian tubule and oppositely in sex gland.
|
PDF Full Text Request