环状芽孢杆菌C-2几丁酶基因的克隆

Molecular Cloning of a Chitinase Gene from Bacillus circulans C 2

环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ２总ＤＮＡ经ＰｓｔＩ部分酶切后分离２～１０ｋｂ的片段，插入质粒ｐＵＣ１９的ＰｓｔＩ位点，转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ），利用几丁质平板从约８０００个重组子中筛选到一个几丁酶基因阳性克隆（命名为ｐＣＨＴ１）。用１２种限制酶对重组质粒进行的酶切分析表明，重组质粒中的插入片段长３０ｋｂ，其中各有一个ＫｐｎＩ，ＳａｃＩ和ＳｓｐＩ位点。把该克隆片段反向插入ｐＵＣ１９的ＰｓｔＩ位点所得到的重组子同样具有几丁酶基因表达活性，说明此片段含有一个完整的几丁酶基因，其自身的启动子能被大肠杆菌转录系统所识别。Ｓｏｕｔｈｅｒｎ杂交证实了该片段来自于Ｂ．ｃｉｒｃｕｌａｎｓＣ２基因组，且以单拷贝形式存在，它不能与来自于其它７株几丁酶产生菌的总ＤＮＡ杂交。

The 2～10kb DNA fragments isolated from the PstI partially digested total DNA of Bacillus circulans C 2 was inserted into the PstI site of vector pUC19,and then the recombinant DNA was used to transform Escherichia coli. One chitinase gene containing clone (named pCHT1) was selected from about 8000 recombinants on chitin overlay plates.Analysis of pCHT1 cut with 12 restriction enzymes showed that the inserted fragment in this clone was about 3.0kb in size and had one site for each of the three restriction enzymes:KpnI,SacI and SspI.Cells harboring the recombinant plasmid (pCHT1 R) in which the insert had an inverted orientation also displayed chitinase activity,indicating that the cloned fragment from B.circulans C 2 contained an intact chitinase gene,and its own promoter could be recognized by E.coli transcriptional system.Southern hybridization analysis proved that the inserted fragment of pCHT1 was really from the genome of B.circulans C 2 and there was only one copy existed in the genome.The fragment could not hybridize with the total DNAs from other seven chitinase producing bacteria.