华癸根瘤菌外膜孔蛋白编码基因MCHK_1326突变株的构建与共生固氮表型分析

Construction and assignment of symbiotic phenotypes of outer membrane porin encoding gene MCHK_1326 in Mesorhizobium huakuii 7653R

【目的】Mesorhizobium huakuii 7653R的MCHK_1326基因编码一种外膜孔蛋白,可能参与根瘤菌侵染宿主植物以及结瘤固氮过程,本研究旨在探索该基因在共生固氮中的功能。【方法】生物信息学分析MCHK_1326蛋白的结构特征及生物学功能,启动子原位表达技术检测MCHK_1326共生时空表达特征,利用Cre-loxp系统构建MCHK_1326缺失突变株,考察其共生固氮表型及早期侵染事件,通过植物盆栽并额外添加无机氮源,检测突变株接种紫云英后的共生固氮表型变化。【结果】MCHK_1326基因在侵染早期如侵染线的延伸等过程中表达,在成熟根瘤的侵染区表达,与野生型相比,突变体△1326侵染线和根瘤原基数量显著减少;植株地上部分鲜重与固氮酶活性极显著降低,根瘤数量和根瘤重量显著降低;额外添加无机氮源能恢复其共生缺陷表型。【结论】MCHK_1326基因参与根瘤菌早期侵染和结瘤,在根瘤发育与共生固氮过程中发挥作用。

[Objective]The gene MCHK1326 from Mesorhizobium huakuii 7653R encoding an outer membrane porins may be involved in the process of rhizobial infection,nodulation and symbiotic nitrogen fixation.This study was aimed to explore the function of this gene in legume-rhizobium symbiosis.[Methods]Bioinformatics analysis used to study the structural characteristics and biological functions of MCHK1326 protein.The spatial and temporal expression characteristics of MCHK1326 gene was assayed by histochemical staining.The deletion mutant strain(7653R 1326 strain)was constructed by the Cre-loxp system.Early infection events and symbiotic phenotype of wild-type and mutant strain were observed.The symbiotic phenotype of the mutant strain was also measured under the conditions of adding nitrogen source.[Results]The gene MCHK1326 was highly expressed during the entire early phase of infection and in the infection zone of mature nodules,such as the elongation process of the infected thread.The infection threads and the number of nodules primordium by the mutant were significantly reduced;compared with the controls,the plants inoculated with 7653R△1326 strain displayed significant reduction in the fresh weight of the aerial plant,nitrogenase activity,and the amount and weight of nodules of 7653Δ1326 strain was significantly lower.The defected symbiotic phenotype of plants inoculated with 7653RΔ1326 strain could be restored to normal by adding inorganic nitrogen source.[Conclusion]The gene MCHK1326 participates in the early infection and nodulation processes and plays an important role in the development of nodule and symbiotic nitrogen fixation.