Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I.
Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I.
复制标题
莱茵衣藻的定向叶绿体转化：编码铁硫蛋白的 psaC 基因的插入失活使光系统 I 不稳定。
作者：
Yuichiro Takahashi;M. Goldschmidt;Su Yin Bianca Maria Soen;L. Franzen;J. Rochaix
编码绿色藻类莱茵衣藻光系统I铁硫蛋白（PSI）的叶绿体基因psaC已被克隆并表征。推导的氨基酸序列与高等植物和蓝藻的氨基酸序列高度相关。使用粒子枪，野生型C。用携带psaC基因的质粒转化reinhardtii细胞，所述psaC基因被设计用于在叶绿体中表达壮观霉素/链霉素抗性的aadA基因盒破坏。在含有乙酸盐作为还原碳源和壮观霉素的平板上选择的转化体不能在缺乏乙酸盐的基本培养基上生长，并且PSI活性不足。对转化子细胞总DNA的Southern印迹分析表明，野生型psaC基因已通过同源重组被中断的psaC基因所取代。虽然不再检测到psaC基因的真实转录物，但aadA在转化体中产生少量转录物。生化分析表明，无论是PSI反应中心亚基还是属于PSI的7个小亚基都不能稳定地积累在转化体的类囊体膜上。细胞蛋白的脉冲追踪标记显示PSI反应中心亚基正常合成，但在转化体中迅速翻转。我们的结论是，psaC基因编码的铁硫结合蛋白是光化学活性和PSI稳定组装的重要组成部分。目前的研究表明，任何编码光合机构成分的叶绿体基因都可以在C。reinhardtii使用所述的策略。
The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse‐chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described.