Identification, Expression And Function Analysis Of Specific Genes In The Silkworm, Bombyx Mori

Insects are the most abundance animals in the world, which play important roles in the ecological system, biodiversity, agricultural, and human health concerns. The diversity of insects is a result for the adaptation of the variational environment in the evolutionary process. There must be an essential contact between the species diversity and physiological functions, in other words, the genetic materials determine the biological characteristics of species. As more and more genome projects of species were completed, studying on the functional of specific genes could provide crucial clues to understand the differentiation, ethology characteristic, and morphology of the species in their evolutionary process.The silkworm (Bombyx mori) is a typical model organism for Lepidoptera, and is also an important economic insect domesticated by human for more than 5,000 years. The completed genome sequence of the silkworm and other species will help us study on the species-specific genes in the silkworm and also in Lepodoptera. Species-specific genes are protein-coding regions with no recognizable homolog in distantly related species, which are also called orphan genes or ORFans. These genes play important roles to each species in the developmental and evolutionary processes, which might operate several special functions in their growth, adaptation and special metabolism pathway. In this work, we identified the species-specific genes in the silkworm, analyzed the expression pattern on the basis of the silkworm genome microarray, and explored biological function of thirteen candidate genes by RNAi. The following are the main results:1. Identification and sequence analysis of species-specific genes in the silkworm Based on the genome sequences from B. mori, Drosophila melanogaster, Anopheles sinensis, Aedes albopictus, Apis mellifera, Tribolium castaneum, Caenorhabditis elegans, Homo sapiens, and Gallus gallus, and all the encoding sequences from Butterflybase, we identified the species-specific genes in six insect genomes and found 2,051 species-specific genes in the silkworm, which account for 14% in all genomes. Using all sequences which already are publicized in butterflybase, we also identified 340 Lepidoptera-specific genes. There also 878,2611,1078,1383,4142 species-specific genes are indetified in A. mellifera, D. melanogaster, A. sinensis, A. albopictus, and T. castaneum, respectively. They account for 7.9%,12%,8.2%,8.2%, and 24.8% in all genes of each insect, respectively.911 and 227 genes have the expression evidence from the silkworm EST and Lepidoptera, respectively. And 190 genes have the functional annotations. A large part of these genes with annotations are identified from gene families and were classified into seven functional groups. About 1710 genes are single copy; other genes belong to 171 gene families, such as antibacterial peptides and paralytic peptide-binding proteins.2. Genome-wide analysis of species-specific genes expressional profiles by microarrayUsing the silkworm oligonucleotide microarray containing 22,987 probes,1,535 species-specific genes of silkworm (including Lepidoptera-specific genes) has the probes. Survey the microarray data of several tissues in the day-3 fifth instar larvae, the results showed that 567 genes were expressed in at least one tissue, and 268 genes were tissue-specific expression genes, other 63 genes had the tissue co-expression characteristics. There are 468 genes differentially expressed in male and female embryos by microarray analysis,200 genes were detected expression in early embryo stage. We analyzed the expression of species-specific genes during larval molting, the results showed that 623 genes were expressed periodically.16 genes were specifically expressed during the molting stage which might play an important role in molting. Other 14 genes were just expressed in the feeding stage. We surveyed the expression patterns of the silkworm specific genes in the metamorphosis stage, the conclusion showed that the expressional signal of 640 genes were greater than 400, and most of these genes have the similar expressional profiles in male and female, still part of genes have significant patterns, this result indicate that these genes may be related to arise and development of sperm or germ. We also analyzed the induced expression of specific genes,451 genes were differential expressed in day-3 fifth instar silkworm larvae after infected by four microorganism. 3. Functional analysis of candidate genes by RNAiAccording to those genes which have the evidence of EST, higher expression in pupae stage, and single copy as candidate conditions, we chose 13 genes in all species-specific genes as our target. We microinjected the in vitro synthesized dsRNA into prepupae, there are 6 genes had the significant phenotype in 48h after injection, BGIBMGA000702 gene got the absolute phenotype, the body of the pupae varied to shorter than the control and the body fluid was not so many as the control in the 72h after injected dsRNA. The results indicated that BGIBMGA000702 gene might be effecting the development of pupae through some special pathway. And the results of qRT-PCR and western blotting also showed that the expression level of this gene was declined after RNAi. RT-PCR was carried out to validate the microarray data, the results showed that this gene was expressed in the beginning of spinning, and had a higher expression in male pupae.4. Prokaryotic expression, polyclonal antibody preparation, and western blotting analysis of BGIBMGA000702Comparative and chromosomal location analysis of genomic sequence found that BIBMGA000702 gene is located on Chromosome 1, and it is a single exon gene. The CDS of BGIBMGA000702 was cloned in this study, including a 588bp ORF, which was predicted encode a protein of 195 AA with molecular weight of 23kDa and isoelectric point of 9.64. The cDNA of BGIBMGA000702 was ligated into modified p28 plasmid to construct prokaryotic expression vector, which was named 000702/p28. A 25kDa recombinant protein was obtained. The molecular weights were a little higher than the original protein because of adding a 6×His-tag. Protein composition identification showed that the recombinant protein was expressed in inclusion form.With the methods of affinity chromatograph and electroelution, we purified recombinant protein 000702/p28, and then immunized male rabbits with the recombinant protein to prepare polyclonal antibodies. Western blotting results showed that the expression of BGIBMGA000702 was knocked down by RNAi. The whole developmental expression pattern of this gene were also detected, and the results showed that this gene was not expressing in the larva stage, but had higher expression level in oosperm and pupal stage.According to our results, we identified the species-specific genes in six insects and Lepidopetera, and also analyzed the spatial-temporal expression patterns of specific genes in the silkworm (including species-specific genes of Lepidopetera). Identification of species-specific genes in the silkworm will provide clues to study biological functions of these genes.