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|Title:||TOWARDS THE MINIMAL SYMBIOTIC GENOME OF SINORHIZOBIUM MELILOTI|
|Keywords:||Symbiotic nitrogen fixation;Minimal genome;Histochemical staining of nodules;Sinorhizobium meliloti|
|Abstract:||Sinorhizobium meliloti is a model bacterium for the study of symbiotic nitrogen fixation (SNF). It infects the roots of alfalfa as well as some other legumes and differentiates into N2-fixing bacteroids within the plant cells of specialized nodule organs. To understand genes essential for SNF and, in the longer term, to facilitate the manipulation of this SNF process for agricultural purposes, it is highly desirable to construct the minimal genome for SNF in this organism. S. meliloti harbors two replicons required for SNF, a 1.7-Mb chromid (pSymB) and a 1.4-Mb megaplasmid (pSymA). A previous deletion analysis revealed that only four gene regions, accounting for <12% of the total sequences of pSymA and pSymB that, were essential for SNF. In the first part of the thesis, I report the cloning of these two pSymA SNF-essential regions on a plasmid (pTH3255) in Escherichia coli, and the integration of this plasmid into the genome of a ∆pSymA S. meliloti derivative strain (the strain was named as RmP4291 after integration). Plant root dry weight and nitrogenase-catalyzed acetylene reduction assays were carried out on RmP4291 with four host plants, including Medicago sativa, Medicago truncatula, Melilotus alba and Melilotus officinalis. Nodule kinetic assays were also performed on RmP4291 and RmP110(wt). The results showed that the SNF-essential regions from pSymA were sufficient to restore the symbiotic capabilities to the ∆pSymA derivative strain with all the host plants tested, except a significant reduction (~40%) in SNF by RmP4291 was noticed on M. officinalis compared to that by wildtype S. meliloti. A higher alfalfa nodulation efficiency of RmP4291 compared to that of wildtype RmP110 was also discovered. In the second part of the thesis, a histochemical staining method for S. meliloti nodules was developed by integrating the marker genes gusA (β-glucuronidase) and celB (β-glucosidase) into the S. meliloti genome. This staining method was found to be useful in the study of nodule competitiveness. A nodule competition assay was carried out between RmP4291 and RmP110 using the new staining method. RmP4291 was found to be significantly reduced in nodulation competitiveness compared to wildtype S. meliloti. The development of the histochemical staining method for S. meliloti nodules will accelerate the identification of genes required for nodule competitiveness in the organism, which will be of crucial importance to the construction of the minimal genome strains with high SNF efficiency.|
|Appears in Collections:||Open Access Dissertations and Theses|
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