A Deletion Mutant Generating by Replacement Construct in Sorbitol Dehydrogenase of Ralstonia solanacearum Bacterial Wilt Strain

Authors

  • Duangkhae Kanjanasopa Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140, Thailand.
  • Srimek Chowpongpang Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
  • Orawan Chatchawankanphanich National Center for Genetic Engineering and Biotechnology (BIOTEC), Kasetsart University, Khamphaengsaen, Nakhon Pathom 73140, Thailand.
  • Wichai Kositratana Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140, Thailand.
  • Niphone Thaveechai Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.

Keywords:

Ralstonia solanacearum, deletion mutants, overlapping PCR, sorbitol dehydrogenase gene

Abstract

A method for making precise mutations in the Ralstonia solanacearum genome at frequencies high enough to allow direct identification of mutants by PCR or other screening methods rather than by selection was developed. This method utilized a novel pBR322-based gene replacement vector that acted as a donor plasmid carrying the desired mutation into the target cell. Single-crossover occurred resulting in efficient replacement of the wild type allele on the chromosome by the modified sequence. The precision of this method was demonstrated by making deletion mutants in sorbitol dehydrogenase gene of R. solanacearum. Sorbitol dehydrogenase (polS) of R. solanacearum biovar 3 race 1 was cloned and further mutated by site direct mutagenesis by overlapping PCR to eliminate NADP binding and active sites. The deleted polS construct (ΔpolS) was introduced to R. solanacearum by triparental mating. Homologous recombination mutants were screened based on antibiotic resistance and
biochemical properties.

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Published

2006-08-30

How to Cite

Duangkhae Kanjanasopa, Srimek Chowpongpang, Orawan Chatchawankanphanich, Wichai Kositratana, and Niphone Thaveechai. 2006. “A Deletion Mutant Generating by Replacement Construct in Sorbitol Dehydrogenase of Ralstonia Solanacearum Bacterial Wilt Strain”. Agriculture and Natural Resources 40 (4). Bangkok, Thailand:902-13. https://li01.tci-thaijo.org/index.php/anres/article/view/243831.

Issue

Vol. 40 No. 4 (2006): July-August

Section

Research Article

License

online 2452-316X print 2468-1458/Copyright © 2022. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/),
production and hosting by Kasetsart University of Research and Development Institute on behalf of Kasetsart University.