The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the fragment within the agp DNA was deleted and replaced by an erythromycin resistance cassette to generate plasmid pUCAE, which was used to transform the Synechocystis sp. PCC 6803 wild-type strain and a mutant with resistance to erythromycin was obtained. PCR analysis of the genomic DNA from the resulting mutant indicated that the appropriate deletion and insertion indeed had occurred. The cell growth and Chla, glycogen content in the mutant showed difference from those in the wild-type strain. The obtained biomass as well as the Chla content in the mutant strain was higher than that of the wild-type strain, which suggested that the photosynthesis efficiency in the agp- strain was higher than that in the wild-type strain. No glycogen was found in the mutant, providing evidence for the correction of the mutant in physiological level.
蓝细菌6803 agp 基因的分子克隆和缺失突变株的构建
吴桂芳1,2 沈忠耀2 吴庆余1* 赵南明1
（1. 清华大学生物科学与技术系，北京100084；2. 清华大学化工系生物化工研究所，北京100084）
摘要：PCR扩增了蓝细菌集胞藻6803（Synechocystis sp. PCC6803）的agp基因（编码ADP-葡萄糖焦磷酸羧化酶），进一步以pUC118为载体将其克隆到大肠杆菌中，构建了pUCA质粒。通过DNA体外重组，以红霉素抗性基因部分取代agp基因片段，构建了既含agp基因上游及下游序列、又携带选择性标记——红霉素抗性的pUCAE质粒。该质粒转化野生型集胞藻6803细胞，获得了能在含红霉素的培养基上正常生长的agp基因缺失突变株。对该突变株基因组DNA进行PCR扩增，验证了其基因结构的正确性。突变株细胞生长速度较野生型细胞快，胞内的叶绿素含量比野生型细胞高，表明该突变株具有较高的光合效率。在突变株中未检测到糖原的存在，进一步从生理水平上验证了突变株构建的正确性。
关键词： 蓝细菌；集胞藻6803 ; agp 基因克隆；缺失突变；糖原合成；光合作用
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