桑蚕金属硫蛋白基因在大肠杆菌中的克隆和表达

CLONING AND EXPRESSION OF METALLOTHIONEIN GENE OF BOMBYX MORI

用BamHⅠ和SacⅠ双酶切质粒 pCM1 1 ,获得酵母的MTI基因片段 ,用非放射性地高辛标记作为探针。提取桑蚕肥苏蚕卵的总DNA ,分别用EcoRⅠ、BamHⅠ和HindⅢ酶切 ,与MTI探针进行Southern杂交 ,出现较强的杂交信号。然后用EcoRⅠ完全酶切桑吞的总DNA ,电洗脱法回收 1～ 6kb的染色体片断 ,与EcoRⅠ酶切的M1 3- 载体以 3∶1比例连接 ,转化受体菌DH5α。筛选到 40 0 0多个白色转化子 ,与探针MTI进行Southern杂交筛选阳性转化子 ,选择到有强杂交信号的三个转化子 [编号为T1 ( pZHC 1 )、T5( pZHC 5)、T7( pZHC 7) ]。用 1 2种限制性内切酶对 pZHC 5重组质粒进行酶切分析表明插入片段约 1 2kb ,在基因内有一个HindⅢ位点。抗性测定表明受体菌DH5α在含有 5 0mmol/LCuSO4的培养基上生长 ,在含有 5 2mmol/LCuSO4的培养基上不生长 ,而转化子确能在含有 5 2mmol/LCuSO4以上的培养基上生长。上述研究结果表明 1 2kb左右的插入片段含有桑吞的金属硫蛋白基因。

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 1～6kb DNA fragments were isolated from the \%Eco\%RⅠ digested total DNA of \%Bombyx mori\% Huishu eggs and ligated with M13\+- vector digested by restriction \%Eco\%RⅠ. The ligation mixtures were used to transform \%E.coli\% DH5α. 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 recombinat plasmid pZHC\|5 with 12 restriction enzymes, the results suggested that the inserted fragment was about 1.2kb and there was only a \%Hin\%dⅢ site. The experiment of resistant to CuSO\-4 proved that the DH5α cells contain recombiant plasmids were more resistance than the recepient DH5α cells. Acording 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.