Abstract：A cysteine proteinase inhibitor from silkworm (Bombyx mori), named Bombyx cysteine proteinase inhibitor (BCPI), demonstrated the inhibitor activity for Bombyx cysteine proteinase (BCP) and cathespin L in vitro. By proteomics tools, BCPI was also identified from silk gland of silkworm, which showed the abundant expressional level in the silk gland of Ras1CA system of transgenic silkworm. In order to explore the expression pattern and its regulation in silkworm in vivo, in this study, we cloned the gene of bcpi and investigated the spatial and temporal expression at transcriptional level and translational level. The result showed that bcpi had a length of 315 bp, encoding a protein of 105 amino acid with a signal peptide of 19 amino acid at the N-terminal and a domain of inhibitor I29 at the C-terminal. Though BCPI was a protein from silkworm with low molecular weight, it had the high similarity with N-terminal domain of the cysteine proteinase from the baculovirus of lepidopteran insect. By reverse transcription PCR (RT-PCR) and quantitative Real-time PCR (qRT-PCR), the gene demonstrated a strong expression in the haemocyte of silkworm, and had a weak expression in head and epidermis in the 3rd day of 5th instar, respectively. In addition, differentially expressional level of bcpi were analyzed during the developmental stages from 1st day of 4th instar to 10th day of pupa stage by qRT-PCR. An up-regulated expression of bcpi was found in the pre-pupal stage and late pupa stage before eclosion. The expression pattern of bcpi suggested it perhaps was involved in development and metamorphosis of silkworm. By the Western blot, the high level of BCPI was accumulated in the hemolymph of silkworm. The strongest expressional level of BCPI in hemolymph appeared in the wander stage and the stage before eclosion. The result showed the BCPI functions in the hemolymph of silkworm and regulated the activity of Bombyx cysteine proteinase in different developmental stage. In order to investigate whether expression of bcpi were affected by insect hormone, the 20-hydroxyecdysone were applied to silkworm at the 2nd day of 5th instar larval. The result showed that the up-regulated expression were induced in the silkworm by 20-hydroxyecdysone by qRT-PCR. In order to further analyzed bcpi promoter responsed to ecdysone, dual luciferase reporter assay system were applied to study the bcpi promoter with the treatment of 20-hydroxyecdysone. However, the activity of luciferase was down-regulated in the BmE cell with the treatment of 20-hydroxyecdysone when the upstream 1 500 bp region of promoter was cloned and analyzed by dual-luciferase reporter assay system. The down-regulation of bcpi promotor was also validated by gradient concentration of 20-hydroxyecdysone. We precluded that there were complicated mechanism in the regulation of expression of bcpi in silkworm. The region except upstream 1 500 bp region of bcpi may be responsible for enhancement the expression with 20-hydroxyecdysone. In conclusion, expression pattern of bcpi in different tissues and developmental stages were analyzed in this work and the relationship between expression of bcpi and 20-hydroxyecdysone was discussed. The results offer us more clues to study the expression and function of bcpi.
