Construction and analysis of symbiotic phenotypes of urtB mutant in Mesorhizobium huakuii 7653R

Abstract:

[Objective] Urea ABC-transporter permease subunit UrtB may be involved in urea metabolism and branched-chain amino acid transport. This study aimed to obtain experimental evidence to clarify the effect of urtB gene on the nodulation and nitrogen fixation of Mesorhizobium huakuii and provide scientific basis for further study of its functional mechanism.[Methods] The structure characteristics and biological functions of UrtB protein were analyzed by bioinformatics, the expression pattern of urtB gene under free-living growth and symbiosis conditions were detected by fluorescent quantitative real-time PCR, and in situ expression of urtB gene in nodule tissue during nodulation process were detected by promoter fusion with β-glucuronidase activity. The urtB gene mutant was constructed by site-directed insertion, pot plant experiments and in combination with nitrogen addition treatment were carried out to identify symbiotic phenotypes and restoration.[Results] It showed that the urtB gene was anticipated to be involved in nitrogen transport of rhizobia. The expression level of urtB gene under symbiotic conditions was significantly up-regulated compared to that under free-living conditions, and urtB gene was highly expressed in the nitrogen-fixing zone of the mature nodules; an urtB mutant of 7653R was screened and obtained. Compared with the wild-type strain 7653R, the 7653R△urtB mutant inoculated with host plant resulted in an abnormal development of the nodule, and the above ground biomass of plants and nodule nitrogenase activity were significantly decreased. Adding nitrogen in the pot experiment could restore its symbiotic defective phenotype.[Conclusion] The urtB gene of Mesorhizobium huakuii may affect nitrogen transport or assimilation in root nodules, and therefore it plays an important role in root nodule development and symbiotic nitrogen fixation.