Elucidation of the symbiotic mechanism through the point of osmoregulation of root nodule bacteria.

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1.Construction of glycinebetaine production strain (GB strain) : GB synthetic gene (codA) from Arthrobacter globiformis was introduced into pTE3 expression vector and GB strains of leguminous bacteria were constructed. Choline oxidase activities of Bradyrhizobium japonicum and Mesorhizobium loti GB strains were 0.25 and 0.35 units/mg protein, respectively. Intracellular GB content (μg/mg dry wt of cell) of BjGB strain was 0.56.2.Nodulation test : Nodulation of BjGB strain started earlier than that of the parent strain. Besides, the number and weight of nodules, nitrogen fixation activity (ARA), and plant weight were increased to around 1.3-5 times that of the parent. The nodulation ability seemed to be superior in lower temperature (20℃/10℃) (number and weight of nodules, ARA, and plant weight against parent (%) : 145.0, 152.4, 222.6, and 107.9, respectively).3.Measurement of GB content : GB contents in nodules formed with BjGB strain were around 43.5μg/gFW (no GB was detected in the parent). According to the marker enzyme assay of NF methods, GB of around 87% produced by BjGB (bacteroids) was released to plant cytosol and some of them had a possibility to be incorporated into plant organelles such as mitochondria and vacuoles.4.Global expression analyses of Bj USDA110 genes : Results obtained from global expression analyses of Bj USDA110 genes which respond to soybean seed extracts (SSE) showed that clones located within symbiosis island such as nod and type III (ttss) were conspicuously up-regulated and these clones occupied around 45% of up-regulated clones in the whole genome. The expression profile was similar to that of the induction by genistein. Besides, some clones out of symbiosis which was related to osmoregulation (ABC transporter, etc.) were also up-regulated. However, genes for nitrogen fixation (nif, fix etc.) were not expressed.