桑蚕金属硫蛋白基因在大肠杆菌中的克隆和表达

用\%Bam\%HⅠ和\%Sac\%Ⅰ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\%Eco\%RⅠ、\%Bam\%HⅠ和\%Hin\%dⅢ酶切，与MTI探针进行Southern杂交，出现较强的杂交信号。然后用\%Eco\%RⅠ完全酶切桑吞的总DNA，电洗脱法回收1～6kb的染色体片断，与\%Eco\%RⅠ酶切的M13-载体以3∶1比例连接，转化受体菌DH5α。筛选到4 000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12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\%Hin\%dⅢ位点。抗性测定表明受体菌DH5α在含有50mmol/L CuSO\-4的培养基上生长，在含有52mmol/L CuSO\-4的培养基上不生长，而转化子确能在含有52mmol/L CuSO\-4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。

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.