Construction of transgenic silkworm spinning antibacterial silk with fluorescence
- 514 Downloads
A targeting vector consisting of a fusion gene of the green fluorescent protein (GFP) gene gfp and the antimicrobial peptide cecropin gene cec flanked by pieces of the 5′ and 3′ sequences of the fibroin L chain gene fib-L of the silkworm (Bombyx mori) and a negative selection DsRed marker gene driven by the baculovirus immediate early gene 1 (i.e.-1) promoter, was used to target the silkworm genome in order to explore the possibility of improving the performance of silk. A transgenic silkworm with a green fluorescent cocoon was obtained and PCR analysis of its genome confirmed that the target genes had been integrated into the silkworm genome correctly. Furthermore, in the posterior silk glands of the G6 generation transformation silkworm, a band representing the fusion protein Fib-L-GFP-Cec with a molecular mass of 68.7 kDa was detected by western blotting with an antibody against GFP. An investigation of the number of bacteria attached to a cocoon showed the transgenic silkworm cocoon possessed antibacterial properties. These results suggested the performance of silk can be improved by modifying the fibroin gene.
KeywordsBombyx mori Gene targeting Green fluorescent protein Fibroin light chain Cecropin
We gratefully acknowledge financial support by the National Basic Research Program of China (973 Program, 2012CB114600), the Specialized Research Fund for the Doctoral Program of Higher Education (20113201130002) and a Project funded by the Priority Academic Program of Development of Jiangsu Higher Education Institutions.
- 5.Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18(1):81–84PubMedCrossRefGoogle Scholar
- 12.Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84PubMedCrossRefGoogle Scholar
- 18.Zhang F, Zhao Y, Lu CD (1999) Fluorescent transgenic silkworm. Acta Biochim Biophys Sin (Shanghai) 31:119–123Google Scholar
- 19.Zhao Y, Chen X, Peng W, Dong L, Huang JT, Lu CD (2001) Use of homologous recombination to change heart heavy chain gene of silk fibroin protein. J Biochem Biophys 33(1):112–116Google Scholar
- 23.Guo XY, Zhou ZY, Feng LC, Wang L, Lu C, Xiang ZH (2001) The sperm mediated method is used to import the exogenous gene into silkworm. Prog Biochem Biophys 28(3):423–425Google Scholar
- 24.Cao G, Xue R, Shen W, He Z (2006) The research of hGM-CSF gene transgenic silkworm based on piggyBac. Sci Seric 32(3):324–327Google Scholar
- 28.Liu C, Zhao P, Cheng T, Cha X, Xia Q, Xiang Z (2005) A new transcription pattern analysis of silkworm Fhx/P25 gene. Prog Biochem Biophy 32(8):740–746Google Scholar
- 29.Wang S, Lu C (2006) Fibroin heavy chain promoter cloned fragment leakage expression in silkworm vivo and cultured insect cells. Sericulture 4:491–493Google Scholar
- 31.Pan X, Cao G, Xue R, Gong C (2009) Fibroin heavy chain promoter-driven DsRed instantaneous secretory expression. Biol Eng 25(5):761–766Google Scholar