嗜碱性兼性自养莫拉氏菌M-z1.5-二磷酸核酮糖羧化酶基因的克隆

Ribulose-1,5-bisphosphate Carboxylase/oxygenase Genes Cloning of an Alkalophilic C-1 CompoundAssimilating Moraxella sp.M-z

以莫拉氏菌(Moraxella sp).M-z 1.5-二磷酸核酮糖羧化酶(RubisCO)大小亚基N-末端氨基酸序列和真养产碱菌(Alcaligenes eutrophus)RubisCO大亚基靠C-末端保守区域DNA序列为依据,合成3个引物,以莫拉氏菌M-z染色体DNA为模板通过PCR扩增制备了二种探针,Southern杂交分析表明上述探针与莫拉氏菌M-z染色体DNA的两个ApaⅠ酶切片断(5kb、4kb)呈阳性杂交反应。认为完整的RubisCO基因包含在这两个片段中。分别将含目的基因的5kb,4kb DNA片段克隆至pUC118和pBluescript KS^+,构成质粒pP-CL511和pBS4,并绘制了这二个质粒限制性核酸酶酶谱。

Moraxella sp. M-z is an alkalophiiic C-l compound assimilating bacterium. For isolating its RubisCO genes, two primes were synthesized according to the N-termi-nal amino acids sequences of LSU and SSU of its RubisCO, the third prime was determined from the conservative region of RubisCO's LSU gene of Alcaligenes eutrophus. Two probes corresponding to the LSU gene and LSU gene with the initial part of SSU gene of Rubis CO were prepared via PCR. Southern hybridization showed the total RubisCO genes were harboured in two DNA fragments (5kb and 4kb) derived from the chromosomal DNA of strain M-z with Apa I digestion. The 5kb DNA segment which believed to bear the LSU gene of RubisCO and its upstream genes and the 4kb DNA segment encoding the SSU gene of RubisCO and its downstream genes were cloned into plasmid pUC118 and pBluescript I KS+, respectively, to yield plasmids pPCL511 and pBS4. Restriction endonuclease map of the cloned target fragments were constructed using pPCL511 and pBS4. It is the first report of cloning the RubilCO genes from an alkalophiiic C-1 compound assimilating bacterium.