Construction Of SSH CDNA Library And Investigations Of The Ctlp Gene In Sex-limited Yellow Cocoon Silkworm, Bombyx Mori

Bombyx mori is the most well studied Lepidoptera model organism, and its nature colored cocoon is the excellent genetic resource for production and application. At present, the research on molecular mechanism of colored cocoon is proceeding at home and abroad. The cocoon color is regulated by multiple genes, and its genetic mechanisms are still not clear, how to study cocoon coloration mechanism is a difficult problem puzzling scientists, nowadays to explore and identify novel cocoon color-regulated genes is considered to be an effective way.The sex-limited strain Ys is reared and preserved in our laboratory, of which female cocoon color is yellow and male is white, so it is a perfect material for researching on differentially expressed genes between yellow and white cocoons. Based on this, suppression subtractive hybridization (SSH) was employed to construct cDNA library of specially expressed genes in female Ys strain. Chymotrypsin-like proteinase (Ctlp) gene, differentially expressed between female silkworm and male in the SSH cDNA library, was cloned and identified by RACE, RT-PCR and bioinformatics methods. Meanwhile, the tissue expression pattern of Ctlp gene on day 3 of the fifth instar larvae was examined by RT-PCR and Real-Time PCR. In addition, the Ctlp protein was tested to be expressed in prokaryotic expression system.1 Construction of SSH cDNA library of specially expressed genes in female sex-limited Ys strain of silkworm, Bombyx moriA subtracted cDNA library of specially expressed genes in female sex-limited Ys strain in the silkworm, Bombyx mori was constructed. About 94 randomly-selected clones containing inserts were sequenced. Tentative annotations were performed by BLASTX and results were manually validated. 5 ESTs remained unknown, whereas 9 ESTs were assigned to a defined annotation, including the gene encoding NAPDH oxidoreductase, eukaryotic translation initiation factor 5A, Heat shock cognate protein, Serine protease precursor, chorion locus sequence encompassing gene, beta-tubulin (Tub2) and three ribosomal RNA or ribosomal protein. Web Gene Ontology (WEGO) analysis revealed that these annotated genes were defined as cell components, molecular function and biological process, involved in energy metabolism, cell structure, binding activity and catalytic activity.2 Cloning and Identification of chymotrypsin-like proteinase gene in the silkworm, Bombyx moriThe EST highly expressed in the midgut of female silkworm was cloned. The full-length cDNA sequence of cloned gene is 976 bp, of which the longest ORF coding 279 amino acids is 840 bp. Protein property analysis shows that the protein encoded by this gene is a secreted protein. Molecular weight of the deduced amino acid sequence is 29 780.10 (or 29 781.00) and isoelectric point is 8.75 (or 9.07). There are two transmembrane helices from inside to outside at its amino acid residues from 1 to 20 region and from 50 to 100 region. SOPMA analysis implicats that this protein contains about 18.64% -helix, 26.88% extended strand, 7.89%β-turn and 46.59% random coil. N-glycosylation site, Protein kinase C phosphorylation site, N-myristoylation site, Serine proteases histidine active site, Serine proteases serine active site, Carbamoyl-phosphate synthase subdomain signature 2 are predicted in the protein sequence. Protein functional domain (SMART) predicts the region of the protein from 38 to 274 amino acids is trypsin-like serine protease domain. Molecular evolution analysis indicates that the gene encodes Chymotrypsin, and homologous with Lepidoptera insects such as H.virescens, S.exigua and H.armigera, whereas far away from vertebrate, which is consistent with traditional theory about evolutional relationship. Therefore, chymotrypsin-like proteinase gene (Ctlp gene) was named.3 Ctlp gene expression profile in the silkworm, Bombyx moriEST profile shows that Ctlp gene specially expresses in the midgut of silkworm larves. Microarray expression analysis reveals that Ctlp gene is significantly expressed different in eighteen tissues and organs in DaZao strain. Ctlp gene is specially expressed in the midgut.RT-PCR and Real-Time PCR analysis showed that Ctlp gene was expressed both in the midgut and gonad on day 3 of the fifth instar larvae in Ys strain, while almost not expressed in the silkgland and fatbody. It was notable that the expression in the midgut of female was the highest among the tissues and organs, much higer than that of male.In addition,it had lower expression in the gonad.4 Prokaryotic expression analysis of Ctlp geneProkaryotic expression in E. coli Rosetta 2 (DE3) could be identified by SDS-PAGE and Western bolt, but the band is less distinct, maybe due to the inclusion bodies do not formed. For the further research we need to optimize the expression conditions.