Altering Fibroin Heavy Chain Gene of Silkworm Bombyx mori by Homologous Recombination.
A gene unit, which encoded fibroin-like peptide, was synthesized and constructed. The unit was multimerized to about 2 400 bp using BamHI and BglII at each end of the unit, then was fused with gfp reporter gene. The fusion gene, flanked by the 5'and 3'sequence of the fibroin heavy chain gene of silkworm Bombyx mori, was transferred into the eggs of silkworm by electroporation. After the silkworms developed and spinned silk, 73 out of about 5 400 cocoons were brighter than normal ones under UV light. The protein extracted from the brighter cocoon could react with the GFP polyclonal antibodies. Genomic DNA from these silkworms and their progenies were analyzed. The integration of gfp gene into genomic DNA of silkworm and the occurrence of expected homologous recombination event had been proved by Southern hybridization. It was shown that gfp-fibroin like fusion gene had integrated into the genomic DNA of silkworm by homologous recombination and the phenotype of "brighter cocoon" could be used to select transgenic silkworms.
Zhao
Yun
Y
Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China. lucd@sunm.shcnc.ac.cn
Chen
Xiu
X
Peng
Wei-Ping
WP
Dong
Liang
L
Huang
Jun-Ting
JT
Lu
Chang-De
CD