| Insect juvenile hormone and moulting hormone regulate their growth and metamorphosis by the titer’s alterations at different developmental stages. But these two hormones are regulated by neuropeptide of the endocrine system. The moulting hormone is a kind of steroid hormones synthesized and released by the prothoracic gland, and the brain neuropeptide PTTH is important for the moulting hormone’s synthesis. It was found that, apart from the PTTH, the insect brain produces many other neuropeptides such as CCAP、AST-B and FMRFamide to regulate their ecdysis and metamorphosis. So the brain is the control centre of insects’growth and development and plays a critical role in insects’post embryo development. Although researches about neuropeptides have made great progress on several insects like fruit flies, Drosophila, for the silkworm, Bombyx mori, it is still a new area. The silkworm, a typical member of Lepidoptera, is the only one that has completed genomic sequencing. The homologous gene may have different functions in various species, so it’s necessary to study the silkworm’s brain neuropeptide genes based on the genomic sequencing. First of all, how many neuropeptide genes does the silkworm have? And how many mature peptides are expressed and produced? Secondly, how does the brain tissue, the main organ to produce neuropeptides, change in the form during the development? What expression pattern do the metamorphosis-related brain neuropeptide genes have? What kind of cells express neuropeptide and the peptide-like genes? What kind of development-regulating function do these genes have? All these questions need to be answered. The researches on the brain tissue-related genes, and especially on neuropeptide and the peptide-like genes at the metamorphosis stage, can not only help to find out the factors which control silkworm’s metamorphosis and development to provide information on lengthening or spontaneous terminating the pupal period, but also can help us to know more about Lepidoptera’s metamorphosis development and the controlling mechanism, which is meaningful to find a new pathway to pest control and develop new biological environment-protecting pesticides.In this study, using the silkworm’s whole genome sequences and the microarray data of the brain tissues, the bioinformatics analyses have been conducted on the silkworm’s development expression-related genes, especially neuropeptide genes, based on the current researches on the model organism, fruit flies. In addition, RT-PCR, hybridization in situ, hormone induction (In vivo and In vitro) and RNA interference were used to study the expression profiles and functions of AST-C and NPLP4 genes. The main findings are as follows:1. Neuropeptide genes screening and mature peptides prediction in the silkworm, Bombyx moriNeuropeptides play an important role of regulation for the silkworm, Bombyx mori. To fully understand the regulation of neuropeptides in the growth of silkworm, Bombyx mori and to get more neuropeptide genes in B. mori, the online program tblastn of BLAST and the search program of OPENOFFICE software were used to screen genes encoding putative neuropeptide precursors in the databases of silkworm genome and theoretical protein based on the homology and the structure of the neuropeptides conserved in other insects and invertebrates. Several online programs were used to analyze the structures of the genes and the theoretical proteins to predict the mature peptides. As results,31 neuropeptide gene families (excluding bombyxin family) with 37 gene subfamilies were identified. A total of 44 neuropeptide genes and 193 mature peptides were predicted. Among them,73 neuropeptides are amidated at the C-terminal, 6 neuropeptides are cyclized at the N- terminal, and 9 neuropeptides are sulfated. The findings suggested that almost all neuropeptide precursors of insects, except for vasopressin-like peptide, were found in the silkworm, Bombyx mori. The interspecies differences were revealed in amino acid sequences in most of the mature neuropeptides in the evolutionary process, but the high conservation of the motif with family traits showed that the growing regulating pathways to different kinds of insects might be conservative. Although most of the mature peptides share the similarity because of their belonging to the same family, compared with those of other insects, the structure of mature peptides of Proctolin, CCAP and CAPA-PK are extended. The results suggested that in the course of evolution, some neuropeptides’structure changed and performed various features for their own species. This study laid the foundation for future researches on neuropeptides’functions, and it provided useful information for neuropeptides’regulation on development of the silkworm, Bombyx mori, especially the development and regulation at the pupal stage.2. Microarray-based gene expression profiles of silkworm brainsMolecular genetic studies of Bombyx mori have led to profound advances in our understanding of the regulation of development. Bombyx mori brain, as a main endocrine organ, plays important regulatory roles in various biological processes.With the help of scanning electron microscope we can clearly observed the brain tissue’s change in form at various developmental stages. The microarray technology made it possible to analyze the expression profiles of the silkworm’s brain tissue genes. In this study we observed the brain tissue’s changes in form at seven stages including V5, V7, W, P1, P3, P5 and P7 and based on the genome wide chips the gene expression profiles of silkworm’s brain tissue were investigated. The result showed that the 5th instar larva’s brain tissue is round, and at the final stage of larva, the stage of metamorphosis, it changed acutely in form. At the pupal stage, colored spots are deposited on the surfaces of brain tissue and the form is more irregular. All these suggested that the brain tissue is not only the metamorphosis development control center but also its form was regulated by 20-hydroxyecdysone (20E).The findings of microarray data showed a total of 4,550 genes were transcribed in at least one selected stage. Of these, clustering algorithms separated the expressed genes into stably expressed genes and variably expressed genes. The results of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis of stably expressed genes showed that the ribosomal and oxidative phosphorylation pathways were principal pathways. Secondly, four clusters of genes with significantly different expression patterns were observed in the 1,175 variably expressed genes. And the genes of cluster I and cluster III were highly expressed at V7 and P5 respectively with the largest number. This is in accordance with the great changes in form at the stage of V7 and P5. So it is presumed that in these two clusters some genes are involved in the form-change of the brain tissue. Thirdly, compared with the homogenous sequence,6 silkworm predicted neuropeptide-like precursor 4 genes were identified, which mainly distributed on the 11th chromosome of nscaf 3031, and their theoretical protein C terminals usually have three highly conservative amine acids, YYX(X usually refers to L,V, I,G).The analysis of microarray data showed thirty-two neuropeptide genes, six NPLP4 genes, and 117 cuticular protein genes were expressed in the silkworm’s brain tissue at the selected developmental stages. Through this study we had a more profound understanding to the changes in form of the silkworm’s brain tissue during the development process. And the analyses to the expressed genes of the brain tissue will provide important information for the further function researches on the related genes of silkworm’s brain tissue, especially neuropeptides and peptide-like substance.3. The research on expressions and functions of the silkworm’s putative C-type allatostatin (AST-C)Allatostatins (AST), a battery of neuropeptides, play important roles in insects. They are categorized in three types according to the structure including A-type allatostatin (AST-A), B-type allatostatin (AST-B) and C-type allatostatin (AST-C). Aforementioned silkworm’s brain tissue microarray data showed that only AST-C gene was found to be expressed at the stages we investigated. And the AST-C gene was highly expressed at the prophase of metamorphosis, but its expression level descended at the pupal stage. It has the same developmental expression pattern with that of PTTH. All these data suggested the putative AST-C may play the same role in the silkworm’s metamorphosis developmental regulation as PTTH. In order to confirm its developmental regulation pattern and functions, we further studied the AST-C gene in the silkworm.We compared the homology of AST-C amino acid sequence in different species. And the results showed that in non-conservative area, the amino acid sequences differ largely, but the amino acid sequences in AST-C mature peptide are very conservative, which show the conservatism in the function. Based on the microarray data, using RT-PCR technique, the space-time expression characteristics of AST-C and PTTH genes were compared and further analyzed in this study. The results showed that AST-C and PTTH gene were both expressed in the silkworm’s brain and prothoracic gland at V7. And AST-C was expressed in midgut. It presumed that AST-C is involved in the developmental regulation of the midgut before the metamorphosis. The expression characteristics of silkworm’s brain tissue and CNS at various stages showed the two genes resemble in expression patterns, that is, both were highly expressed before pupation, descended at the pupal stage, and ascended at the later pupal stage. Results of in situ hybridization showed that the AST-C gene was expressed in a pair of neurosecretory cells at the lateral of the brain tissue, locating at the same place as that of PTTH gene. All these suggested that both the AST-C and PTTH genes, with the same expression pattern, regulated the development of metamorphosis. The function study revealed that the pupation of the silkworm’s larval was delayed by RNA interference of the AST-C gene. The findings of the in vitro assay of the tobacco hornworm indicated that AST-C mature peptides restrained the synthesis of juvenile hormone (JH) at corpora allata (CA). All these data suggested that in order to save the space of biological tissue, the silkworm regulated the moulting hormone enhancing factors and the juvenile hormone inhibiting factors with the same cells expression. So we assumed the patterns of developmental regulation as follows:the silkworm’s brain tissue expressed various neuropeptides through the same secretory cell, and using the same expression pattern to promote the syntheses of moulting hormones and inhibit the syntheses of juvenile hormones, the silkworm’s development of metamorphosis was then regulated.4. The research on expressions and functions of the silkworm’s putative neuropeptide-like precursor 4 geneThe neuropeptide-like precursor genes were firstly identified and named in the peptidomics of the central nervous system in Drosophila. Above mentioned researches showed a battery of presumed NPLP4 genes were expressed in the silkworm’s brain tissue at the specific period investigated. To know more about developmental expression-related genes of the silkworm’s brain tissue and to find out more function-unknown genes, we further studied the function of the NPLP4 genes.The results of RT-PCR showed that the NPLP4 genes were expressed distinctly in the brain tissue and central nervous system (excluding the brain) from the 3rd day of the 5th instar to the 7th day of the pupation. The NPLP4C gene was only highly expressed in the brain tissue and central nervous system at the 7th day of the pupation, but the rest of the NPLP4 genes were faintly expressed in the brain tissue at the 5th day of the 5th instar, and then their expression increased at the end of the 5th instar and the 3rd day of the pupation. The NPLP4A、NPLP4E and NPLP4F genes largely increased in the central nervous system at the stage of metamorphosis, but the expression level of NPLP4E increased at the 3rd day of the pupation.All these findings are in accordance to the titer peak of 20E at the end of larva stage and later papal stage of silkworm.20E and JH were used in the induction in vitro of the silkworm’s brain tissue at the 5th day of the 5th instar. The result showed that apart from the NPLP4C,20E induced the expression of the NPLP4 genes. But JH inhibited the expression of the NPLP4A, NPLP4E and NPLP4F. Different genes show different concentration and dose-response relationship to the induction of 20E.20E were used in the induction in vivo of the silkworm’s brain tissue at the 5th day of the 5th instar. The result showed that compared to the control group all the silkworms injected with various doses turned into the pupa in advance. Excluding the NPLP4C gene, the NPLP4 genes injected with various doses of 20E at different time showed the some inductive effects. All this suggested that different NPLP4 genes have different expression pattern response to 20E and JH. The results of the whole mount hybridization of the silkworm’s brain tissue at the 7th day of the 5th instar showed that NPLP4A and NPLP4E expressed in the intermediate neurosecretory cells at the pars intercerebralis, but NPLP4F expressed in the area of mushroom neuropil of the brain. The various distribution and expression patterns of NPLP4 genes in the silkworm’s brain suggested that they may be regulated from different regulating pathway. The RNA interference of the NPLP4E genes resulted in the developmental defect of imago’s wings. It suggested that the NPLP4E genes play important role in the formation of the imago’s issue. And this may be the reason that the expression level of NPLP4E genes increased in the brain and CNS at the 3rd day of the pupation. The RNAi to NPLP4A (16.67% of treated lavae) led to the defect of ecdysis from larva to pupa, suggesting that NPLP4A probably plays a role in the silkworm’s ecdysis. Moreover, RNA interference of the NPLP4F genes delayed the development from larva to pupa, suggesting that the NPLP4F gene took part in the larva’s metamorphosis. All researches on NPLP4 genes showed that different NPLP4 genes have different functions, and there is a long way for us to know the molecule mechanism to educe their specific functions. |
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