Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

被引：75

作者：

Gonzalez-Ballester, David ^{[1
,2
]}

Pootakham, Wirulda ^{[1
,3
]}

Mus, Florence ^{[1
,4
,5
]}

Yang, Wenqiang ^{[1
]}

Catalanotti, Claudia ^{[1
]}

Magneschi, Leonardo ^{[1
,6
]}

de Montaigu, Amaury ^{[2
,7
]}

Higuera, Jose J. ^{[2
]}

Prior, Matthew ^{[1
]}

Galvan, Aurora ^{[2
]}

Fernandez, Emilio ^{[2
]}

Grossman, Arthur R. ^{[1
]}

机构：

[1] Carnegie Inst Sci, Dept Plant Biol, Stanford, CA 94305 USA

[2] Univ Cordoba, Dept Bioquim & Biol Mol, E-14071 Cordoba, Spain

[3] Natl Sci & Technol Dev Agcy, Natl Ctr Genet Engn & Biotechnol BIOTEC, Pathum Thani 12120, Thailand

[4] Montana State Univ, Dept Chem & Biol Engn, Bozeman, MT 59171 USA

[5] Montana State Univ, Dept Microbiol, Bozeman, MT 59171 USA

[6] Scuola Super Sant Anna, PlantLab, I-56127 Pisa, Italy

[7] Max Planck Insitute Plant Breeding Res, Dept Plant Dev Biol, D-50829 Cologne, Germany

来源：

PLANT METHODS | 2011年 / 7卷

基金：

美国国家科学基金会;

关键词：

reverse genetics; insertional mutants; transformation; mutant library; mutant screening; paromomycin resistance; PCR-based screening; T-DNA; FUNCTIONAL GENOMICS; ARABIDOPSIS; MUTAGENESIS; REINHARDTII; PCR; PHOTOSYNTHESIS; IDENTIFICATION; REGIONS; SITES;

D O I：

10.1186/1746-4811-7-24

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

引用

页数：13

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