Studies On Differentially Expressed Genes Of White Egg2of The Silkworm,Bombyx Mori

The white egg2mutant (w-2) of the silkworm, Bombyx mori, is suitable as a screening marker for screening of transgenic silkworm because of its features, such as egg and eye color, regular hereditary. However, the biochemical processe and molecular mechanism of the w-2mutant still remains unclear so far. In this study, the multidisciplinary approach combined with cDNA microarray based on SSH library, silkworm genome microarray, expression sequencing, and Real-time quantitative PCR, was employed to explore the differentially expressed genes in wild type strain Jingsong A and its near-isogenic line w-2mutant Jingsong A white, the current study provide useful data for further studying of the molecular mechanism of the white egg2mutant.1. The differentially expressed genes identified by SSH and cDNA microarray analysisBased on construction of suppression subtractive hybridization cDNA library from wild type strain Jingsong A and its near-isogenic line w-2mutant,300clones were randomly selected for further analysis of cDNA microarray, and finally11differentially expressed genes were identified.J241exhibited the most different expression level between wild type Jingsong A and mutant Jingsong A white among these11genes. The expression difference between wild type and mutant reached5752.60and7951.40folds at24hour point and48hour point after normally incubated respectively. In the early development stage of egg, J241was found to be abundantly expressed in wild type, but no expression was observed in w-2mutant.. The result of RACE shows that J241is specific sequence splicing only in wild type Jingsong A.After microinjection of double-stranded RNA (dsRNA) of J241into the wild type eggs, no inhibitory effect was observed on the ommochrome pigment formation, indicating that J241is not the core gene on the course of ommochrome biosynthesis.2. The differentially expressed genes identified by silkworm genome microarray At the early stage of embryonic development of wild type Jingsong A and mutant Jingsong A white,163and186differentially expressed genes were identified by23K silkworm genome microarray at24hour and48hour respectively. The maximal up-regulation in mutant was33.00folds higher, and maximal down-regulation was32.47folds lower compared to wild type at24hour point, whereas the maximal up-regulation reached14.52folds, and maximal down-regulation22.83folds at48hour point.26genes were identified to differentially express between wild type and w-2mutant, representing8%of total differentially expressed genes, among them, there are17genes with known function including8metabolic enzymes,3intercellular or intracellular transport-related proteins, and3regulatory and receptor proteins.Analysis of GO and metabolic pathway has demonstrated that differential expressions of genes involve in cellular components, biological processes, and molecular functions, including involvement of eight metabolic pathways at24hour point and23metabolic pathways at48hour point respectively.The further validation using real-time quantitative RT-PCR indicated that the degree of concordance between data generated by the two methods was high, and the maximal differential expression reached826folds between wild type and w-2mutant.3. The differentially expressed genes identified by expression profile sequencingSolexa sequencing, a high-throughput expression profile sequencing technology, was employed to explore the differentially expressed genes during certain development stage in egg of silkworm. In this experiment, we got3493817clean tags and112384distinct clean tags from the time course mixture sample of w-2mutant, and2497077clean tags and95089distinct clean tags from wild type respectively. Saturation analysis showed that the sequence data of this experiment was a true reflection of the expressed genes in these two wild type and mutant samples.All these distinct tags of wild type were mapped to13501silkworm genes, and the matching ratio is up to35.30%. These distinct tags of w-2mutant were mapped to14342genes, and the matching ratio is up to39.46%, suggesting that the data of sole sequence have been fully representative of gene expression profile in wild type and mutant.In this study,859differentially expressed genes were identified by sole sequencing between wild type and white egg2mutant.595genes of them are up-regulated, and264genes are down-regulated in w-2mutant. Interestingly, there are93genes specifically expressed only in wild type or w-2mutant. It is most likely that among these differentially expressed genes, some are involved in the regulation of w-2mutant phenotype, and worth to be further investigated.379differentially expressed genes are annotated to161metabolic pathways, including in89genes in metabolic pathway,19genes in ribosomal channels,16genes in purine metabolism,13genes in oxidative phosphorylation, and11genes in pyrimidine metabolism.There are five differentially expressed genes involved in tryptophan pathway, which metabolites are the source of eye pigment synthesis. Among them, dopa decarboxylase (EC:4.1.1.28) gene and L-tryptophanyl-tRNA synthetase gene (EC:6.1.1.2) expressed in w-2mutant are likely required to maintain the in vivo dynamic balance of the tryptophan metabolism.The results obtained from the expression profile sequencing analysis are perfectly consistent with data of real-time quantitative RT-PCR. The maximal differential expression level is up to16158times tested by real time quantitative RT-PCR between wild type and w-2mutant.The present study has harvested tons of information on genes differentially expressed in wild type or w-2mutant, and will be helpful to elucidate the molecular mechanism of the white egg2mutant.