蓝细菌ORF469的分子克隆和缺失突变工程株的构建

PCR扩增了蓝细菌Synechocystis sp．PCC 6803的ORF469(编码469个氨基酸的开放阅读框),进一步以pUC118为载体将其克隆到E．Coli中，构建了pOQ2质粒。通过DNA体外重组，以红霉素抗性基因取代部分克隆化ORF469片段，又构建丁缺失ORF469片段(保留部分上游和下游序列)的pOQ22质粒。用pOQ22质粒转化Synechocystis sp．PCC 6803野生株细胞，获ORF489缺失突变工程株，它在红霉素抗性培养基上生长正常。对缺失突变工程株DNA的PCR和Southern blot分析证明，Synechocystis sp．PCC 6803的ORF469已被删除。色素测定结果揭示Synechocystis sp．PCC 6803中ORF469表达产物控制细胞内不依赖光的叶绿素生物合成。

Molecular Cloning and Deletion-mutant Construction of ORF469 in Cyanobacterium (Synechocystis sp. ) PCC 6803

ORF469 (an open reading frame encoded 469 amino acid residues) in Syne-chocystis sp. PCC 6803 was amplified by PCR. The PCR-amplified ORF469 fragment was cloned in pUC118 to generate the pOQ2 plasmid. Part of fragment within this ORF469 was deleted and replaced by an erythromycin resistance cassette to generate the pOQ22 plasmid. This pOQ22 plasmid was used to transform the Synechocystis sp. PCC 6803 wild type strain and the mutant resulting from deletion of ORF469 was obtained. The mutant cells could grow normally in the culture medium with erythromycin. PCR and Southern blot analysis of genomic DNA form the mutant indicated that part of ORF469 fragment had been deleted and replaced by erythromycin resistant cassette. The analysis of pigments showed that the expression product of ORF469 in Synechocystis sp. PCC 6803 was involved in light-independent biosynthesis of chlorophyll.