[Cloning and expression of metallothionein gene of Bombyx mori]

Yeast MTI gene from vector pCMI-1 was used as a probe. It appeared strong hybridization signals when the total DNA of Bombyx mori Huishu eggs hybridizes with the probe. The 1-6 kb DNA fragments were isolated from the EcoR I digested total DNA of Bombyx mori Huishu eggs and ligated with M13- vector digested by restriction EcoR I. The ligation mixtures were used to transform E. coli DH5 alpha. blue/White colonies selection was used to identify colonies with insert. Approximately 4000 white colonies were selected, so the part genomic library of Bombyx mori was constructed. Three positive colonies were gained from the genomic library by southern blotting analysis, designated T1 (pZHC-1), T5 (pZHC-5), T7 (pZHC-7). Digesting the recombinant plasmid pZHC-5 with 12 restriction enzymes, the results suggested that the inserted fragment was about 1.2 kb and there was only a Hind III site. The experiment of resistant to CuSO4 proved that the DH5 alpha cells contain recombinant plasmids were more resistance than the recepient DH5 alpha cells. According to these results, the inserted fragment possibly contains the gene encoding Metallothionein of Bombyx mori. The sequence analysis of the inserted fragment and its high-expressions in E. coli are in progress.