Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides |
| |
Authors: | Randolph-Anderson Barbara L Sato Ryo Johnson Anita M Harris Elizabeth H Hauser Charles R Oeda Kenji Ishige Fumiharu Nishio Shoichi Gillham Nicholas W Boynton John E |
| |
Institution: | (1) Developmental, Cell and Molecular Biology Group, Departments of Botany and Zoology, Duke University, Box 91000, Durham, NC 27708-1000, USA;(2) Sumitomo Chemical Company Limited, 2-1,4-Chome, Takatsukasa, Takarazuka-Shi, Hygogo-Ken, 665-8555, Japan |
| |
Abstract: | 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 Arabidopsis 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. |
| |
Keywords: | Chlamydomonas complementation herbicide resistance indexed cosmid library nuclear transformation protoporphyrinogen oxidase |
本文献已被 SpringerLink 等数据库收录! |
|