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KMID : 0381120110330060635
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Genes and Genomics
2011 Volume.33 No. 6 p.635 ~ p.643
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A powerful ubiquitous activity of Bombyx mori heat shock protein 70 promoter
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Goo Tae-Won
Kim Sung-Wan
Kim Yong-Bo
Kim Seong-Ryul
Park Seung-Won
Kang Seok-Woo
Kwon O.-Yu
Yun Eun-Young
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Abstract
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For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This clone, named bHsp70 was ubiquitously expressed in all tissues and developmental stage of 5th instar B. mori larvae, and stimulated by thermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (?1003/+147) in the 5′-flanking region of bHsp70 gene, which has about 264 fold more intensive promoter activity than BmA3 promoter. Transcription activity of bHsp70 promoter under heat shock condition (42°C, 4 hr) was increased over 2 fold than normal condition. In bHsp70 promoter, there are an ER stress response element (ERSE) and a heat shock element (HSE). Accordingly, we suppose that the bHsp70 promoter may be used for conditional expression of heterogonous proteins under thermal or ER stress. Moreover, the bHsp70 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHsp70 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.
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KEYWORD
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Bombyx mori, Heat shock protein 70, Luciferase assay, Promoter
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