Mutational effect of the “- 35” element of sorghumpsbA gene promoter
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Mutations of the first position T and the third position G in TTGACA, the “- 35” element of sorghumpsbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATTACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants and the wild type sorghumpsbA gene promoter was tested in a spinach chloroplast protein extract system. Gel retardation assay of the wild type showed a strong protein-binding band. On the other hand, the protein-binding band of the mutant resulting from single base mutation, ATGACA or GTGACA, showed reduced intensity, while that of the mutant resulting from double base mutation, ATTACA, showed increased intensity. It is thus shown that the “- 35” element plays an important role in controlling the binding betweenpsbA gene promoter and the specific chloroplast proteins; mutation of a single base may exert a substantial influence on the binding affinity.
Keywordssorghum (Sorghum valgare P.) psbA gene 5′-noncoding region site-specific mutagenesls expression control
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