华癸根瘤菌urtB基因突变株的构建与共生固氮表型分析

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

【目的】尿素ABC转运体透性酶亚基编码基因urtB可能参与尿素代谢及支链氨基酸转运;本文旨在获得实验证据阐明urtB基因对华癸根瘤菌结瘤和固氮的影响,为深入研究其功能机制提供一定的科学依据。【方法】利用生物信息学分析urtB基因的结构特征及生物学功能,通过荧光定量检测urtB基因在自生和共生条件下的时空表达特征和启动子原位表达技术检测urtB基因组织表达特征,采用插入突变构建urtB突变株,通过植物盆栽并结合添加氮素处理,检测与分析突变体的共生固氮表型变化。【结果】分析表明urtB基因对于氮素转运非常重要,在共生条件下的表达水平比自生培养条件下显著上调表达;在成熟根瘤的固氮区中大量表达;正确构建和筛选获得了根瘤菌urtB突变株;接种urtB突变株与野生型菌株7653R相比较,突变体根瘤发育异常;植株地上部分生物量和根瘤固氮酶活性显著降低;添加氮素可恢复其共生缺陷表型。【结论】华癸中慢生根瘤菌urtB基因可能通过影响根瘤中氮转运或同化,进而在根瘤发育与共生固氮中发挥重要作用。

[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 7653 R was screened and obtained. Compared with the wild-type strain 7653 R, 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.