Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides
| Autor: | B L, Randolph-Anderson, R, Sato, A M, Johnson, E H, Harris, C R, Hauser, K, Oeda, F, Ishige, S, Nishio, N W, Gillham, J E, Boynton |
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| Rok vydání: | 1998 |
| Předmět: |
Oxidoreductases Acting on CH-CH Group Donors
DNA Complementary Transcription Genetic DNA Mutational Analysis Molecular Sequence Data Drug Resistance Transformation Genetic Animals Protoporphyrinogen Oxidase Amino Acid Sequence Cloning Molecular Genomic Library Base Sequence Sequence Homology Amino Acid Herbicides Nucleic Acid Hybridization DNA Exons Sequence Analysis DNA Blotting Northern Cosmids Clone Cells Blotting Southern Genes Mutation RNA Oxidoreductases Chlamydomonas reinhardtii |
| Zdroj: | Plant molecular biology. 38(5) |
| ISSN: | 0167-4412 |
| Popis: | In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the A rabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G--A change at 291 in the first putative exon, resulting in a Val--Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G--A change at the equivalent position (5751) within exon 10. |
| Databáze: | OpenAIRE |
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