期刊
PLANT PHYSIOLOGY
卷 177, 期 2, 页码 465-475出版社
AMER SOC PLANT BIOLOGISTS
DOI: 10.1104/pp.17.01618
关键词
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资金
- CEA IRTELIS PhD grant
- Initiative d'Excellence program (DYNAMO) [ANR-11-LABEX-0011-01]
- Agence Nationale de la Recherche ChloroPaths [ANR-14-CE05-0041-01]
- CNRS INSIS PhotoModes [72749]
Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b(6)f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.
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