嗜热硫杆菌启动子的克隆及定位

用DNA体外重组技术，将嗜热硫扦菌(Thiobacillus sp．)染色体DNA的Hind III片段插人启动子探测质粒pSDSI(Apr、Tc2)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240／μg／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插人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。

Cloning and orientation of a promoter of thermophilic Thiobacillus sp.]

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 degrees C and its optimum pH is 2.0-3.0. Using DNA recombinant technique, we inserted the HindIII fragments of the Thiobacillus sp. chromosomal DNA into the HindIII site of promoter-probe plasmid pSDSI (AprTcs, 5.65 kb). Transformants resistant to tetracycline were obtained on Tc plates (12 micrograms/ml). Of these, twenty transformants were able to grow on 120 micrograms/ml Tc plates, and two of them, designated pSDH7 and pSDH11, were able to grow on plates containing Tc at concentration up to 360 micrograms/ml. With HindIII, pSDH11 produced a 0.95kb fragment which had the function of promoter and a PstI site besides the 5.65 kb fragment of pSDSI. Southern blot hybridization showed that the 0.95 kb insert was from the Thiobacillus sp. chromosomal DNA. After restriction mapping, a 2.85 kb fragment of pSDH11 (which contained 0.7 kb of the inserted fragment) was removed with the aid of Pst1, and the remained fragment was used to construct a 3.75 kb plasmid (named pSDH114) which was resistant to a higher level of tetracycline (360 micrograms/ml) than for pBR322 (120 micrograms/ml). The remained 0.25 kb foreign fragment in pSDH114 still retained full function of the promoter contained in the original 0.95 kb. Thus we could orient the cloned promotor function fragment (0.25 kb) from Thiobacillus sp. in pSDH114.