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The Isolation And Characterization Of A Galactosly Transferase Mutant Of Rhizobium Sp. Strain Tal1145 That Nodulates Leucaena Leucocephala

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Title: The Isolation And Characterization Of A Galactosly Transferase Mutant Of Rhizobium Sp. Strain Tal1145 That Nodulates Leucaena Leucocephala
Authors: Higashide, Wendy
Advisor: Borthakur, Dulal
Issue Date: 15 Jan 2014
Publisher: University of Hawaii at Manoa
Abstract: The root nodule bacterium Rhizobium produces both lipopolysaccharides (LPS) and exopolysaccharides (EPS), which are required for the effective nodulation of legumes and nitrogen fixation inside the nodules. Rhizobium sp. strain TAL1145 forms nitrogen-fixing nodules with tree legumes such as Leucaena, Gliricidia, and Acacia. Previously, Parveen et al. (1996) cloned and characterized a gene for LPS synthesis (lps) located on a 3.8-kb fragment within the TAL1145 chromosome. Sequence analysis of this 3.8-kb fragment showed the presence of an UDP-glucose epimerase gene (uge) and a galactosyl transferase gene (gat) upstream and a mannosyl transferase gene (mat) downstream of the lps gene. The objectives of this project were to: 1) isolate gat, mat, and uge mutants of TAL1145 and 2) determine the role of these genes in symbiosis with Leucaena. A gat mutant of TAL1145 was constructed by site directed transposon mutagenesis. The position of the transposon Tn3Hogus that disrupted the gat gene in the mutant was determined by sequencing a PCR-amplified DNA fragment flanking the Tn3Hogus insertion in the mutant. The Tn3Hogus insertion was located 149 nucleotides downstream from the putative start site (ATG) of the gat gene. The mutant WH97 appeared to be indistinguishable in colony morphology and the amount of EPS production for the wild type strain TAL1145. However, WH97 showed a slower growth rate than TAL1145 on yeast extract mannitol (YEM) medium. The mutant was used to inoculate Leucaena to determine the symbiotic phenotype. WH97 formed nodules on Leucaena, but the nodules were smaller than those formed by TAL1145. The growth and the nodulation defects of WH97 could be complemented by the cloned 3.8-kb fragment containing the wild type gat gene in plasmid pUHR252.
Pages/Duration: vii, 52 pages
URI/DOI: http://hdl.handle.net/10125/32028
Rights: All UHM Honors Projects are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:Honors Projects for Biology



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