嗜热硫杆菌启动子的克隆及定位

CLONING AND ORIENTATION OF A PROMOTER OF THERMOPHILIC THIOBACILLUS SP.

用 DNA 体外重组技术,将嗜热硫杆菌(Thiobacillus sp.)染色体 DNA 的 Hind Ⅲ片段插入启动子探测质粒 pSDSI(Ap^r、Tc^s)的 Hind Ⅲ位点,在四环素平板上获得一批转化子。四环素抗性能力测定表明,有12个转化子可以在含240μg/ml 四环素的平板上生长,有2个可以在360μg/ml 的四环素平板上生长,对这二个抗性表达能力较高、分子量较小的质粒pSDH7和pSDH11作了限制性酶切图谱分析,并利用 PstI 位点,通过亚克隆将 pSDH11 插入片段上的启动子定位在0.25kb 片段内。分子杂交实验证明克隆的启动子活性片段确实来源于嗜热硫杆菌染色体 DNA。

Thiobacillus sp.is an obligate autotrophic thermophilic bacterium which was isolated from an acidic hot spring in Yunnan Province. Its optimum growth temperature is 45—50℃ and its optimum pH is 2.0—3.0. Using DNA recombinant technique,we inserted the HindⅢ fragments of the Thioba- cillus sp.chromosomal DNA into the HindⅢ site of promoter-probe plasmid pSDSI (Ap^rTc^(?), 5.65 kb).Transformants resistant to tetracyc- line were obtained on Tc plates (12μg/ml). Of these,twenty transformants were able to grow on 120μg/ml Tc plates,and two of them, designated pSDH7 and pSDH11,were able to grow on plates containing Tc at concentration up to 360μg/ml. With HindⅢ,pSDH11 produced a 0.95kb fragment which had the function of promoter and a PstⅠ site besides the 5.65kb fragment of pSDSI.Southern blot hybridization showed that the 0.95kb insert was from the Thiobacil- lus sp.chromosomal DNA.After restriction mapping,a 2.85kb fragment of pSDH11 (which contained 0.7 kb of the inserted frag- ment) was removed with the aid of PstⅠ,and the remained fragment was used to construct a 3.75kb plasmid (named pSDH114) which was resistant to a higher level of tetracycline (360/xg/ml) than for pBR322 (120μg/ml). The remained 0.25kb foreign fragment in pSDH114 still retained full function of the promoter contained in the original 0.95kb. Thus we could orient the cloned promotor function fragment (0.25kb) from Thiobacillus, sp.in pSDH114.