Serial Analysis Of Gene Expression In Silkworm, Bombyx Mori

The domesticated silkworm, Bombyx mori, is one of the most important economic insects in China, India, and many other developing countries owing to its propagation on a large scale and its utilization in silk production. Silkworm is one member of the holometabolous class of insects, which possesses four distinguished developmental stages, i.e., egg, larva, pupa, and adult. It is an ideal model for studying metamorphosis. Genetically, silkworm is one of the best-studied lepidopteran insects because large number of mutants has been identified. Lepidoptera also include highly destructive agricultural pests that cause widespread economic damage to crops and trees. Consequently, information on silkworm gene expression adds to our understanding of silkworm metamorphosis and provides novel candidate targets for pest control.Serial analysis of gene expression (SAGE) is one of the most versatile methods for genome-level studies of gene expression. The SAGE technology samples short sequence tags (14-bases long) from mRNA transcripts found in the population of interest. Each tag contains sequence information that can identify, in basic local alignment search tool (BLAST) analysis, the transcript from which an individual tag has been derived. The frequency of each tag in the SAGE library is an accurate estimate of the abundance of its corresponding mRNA transcript. Numerous groups have successfully used this technique and have described the SAGE protocol in detail. SAGE analysis has been used to study gene expression in a wide range of organisms, including yeast, Arabidopsis thaliana, rice, mouse, and human. SAGE is particularly well suited to the different gene expression analysis of organisms whose genomes are not completely sequenced, since it does not require a hybridization probe for each transcript and provides quantitative information on gene expression that is not readily obtainable using other methods. Also SAGE is suited to do chromosome annotation and find novel genes. Our researches including the following aspects:Firstly,SAGE was used to examine the profile of expressed genes during embryonic development in the domesticated silkworm, B. mori, after irradiation with cobalt-60. A comparison of the SAGE sequence tags derived from irradiated embryos with those from normal embryos revealed 673 differentially expressed genes (p < 0.001 and at least 3 folds change). Of these, 292 genes were highly expressed in normal embryos and 381 genes were highly expressed in irradiated embryos. These results provide valuable information for understanding the mechanisms of radiation-induced changes in gene expression. In addition, we found that the generation of longer cDNA fragments from SAGE tags is an efficient way to identify genes, thereby facilitating the analysis of large numbers of unknown genes.Secondly,we used SAGE to derive profiles of expressed genes during the developmental life cycle of the silkworm, and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool (BLAST) on the EST database, 35% of the tags matched known silkworm expressed sequence tags (ESTs). SAGE demonstrated that a number of the genes were up- or down- regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags (GLGI) constituted the most efficient method for gene identification, which facilitated the analysis of a large number of unknown genes.Thirdly,basing on the different expression SAGE tag: TGTCGTCCTT, we cloned the full-length cDNA sequence of silkworm bursicon gene and then studied the role of the gene function in wing expansion. Its gene expression at different developmental stages has been investigated in the silkworm, B. mori. Bursicon gene was expressed at low levels in larvae, high levels in pupae, and low levels again in adults. Then, we injected the double-stranded bursicon RNA into B. mori pupae to test RNA interference. The level of bursicon mRNA was reduced significantly in pupae, and a deficit in wing expansion was observed in adults. In addition, the differential display reverse transcription polymerase chain reaction (DD-RT-PCR) was used to reveal differences in the expression of transcripts in response to the inhibition of bursicon. In conclusion, bursicon plays a key role in the stereotyped behavioral program involved in wing expansion.Fourthly, in 2004, the draft genome (-428.7 Mb) of the domesticated silkworm, B. mori has been sequenced by the whole-genome shotgun method and should provide important insights into the functional genomic research. However, this genomic data was short of detailed gene annotation, even the chromosome location. Basing on the published SSR linkage map, 475 scaffolds have been located in the 28 chromosomes. Combined with the SAGE tags, these scaffolds also have been annotated. There were 1444 transcripts have been found in the totally 15 Mb genome and the number of unique gene is 909. From the data, we also get the gene expression level in the silkworm development stages about the located genes.