华癸中慢生根瘤菌7653R MCHK-3535基因在自生和与紫云英共生中的功能

Role of Mesorhizobium huakuii 7653R MCHK-3535 gene in the free living condition and symbiosis with Astragalus sinicus

【目的】探究Mesorhizobium huakuii 7653R MCHK-3535基因在共生固氮中的功能。【方法】构建MCHK-3535插入失活突变体、超表达和互补菌株,对其进行共生表型鉴定;并测定各菌株的生长曲线、运动性及生物膜形成;利用qRT-PCR方法和组织表达定位分析MCHK-3535在共生过程中的时空表达特征。【结果】与野生型7653R相比,突变体Δ3535达到稳定期的生物量增加,运动性下降,生物膜形成增加。此外,MCHK-3535基因的失活会显著提高紫云英固氮能力和植物地上部分鲜重,但不影响根瘤数量及重量;且互补菌株C3535能部分回补到野生型性状,超表达菌株OV3535均无显著性差异;qRT-PCR和启动子组织表达定位发现,MCHK-3535基因主要在根瘤的侵染区、过渡区及固氮区表达,并且表达持续整个固氮期。【结论】MCHK-3535基因在自生及与紫云英共生过程中发挥重要功能,参与根瘤的正常发育并负向调控固氮酶活性。

[Objective]To study the function of MCHK-3535 gene in symbiotic nitrogen fixation between Mesorhizobium huakuii 7653 R and Astragalus sinicus.[Methods]We constructed the deletion mutantΔ3535,overexpression strains OV3535 and complementary strains C3535,followed by identifying symbiotic phenotypes,detecting the growth curve,swimming motility and biofilm formation Besides,we used qRT-PCR and promoter-GUS fusion vector to detect the expression characteristics of MCHK-3535 in the symbiotic process.[Results]Compared with the wild type 7653 R,mutantΔ3535 grew faster,decreased motility,and increased biofilm formation,moreover,Δ3535 significantly increased the ability of nitrogen fixation and the fresh weight of plants,while the number and weight of root nodules were not affected,in addition,the complementary strain C3535 partially compensated to the wild-type phenotype,whereas OV3535 had no significant difference in these aspects.The results of qRT-PCR and the promoter-GUS reporter system showed that MCHK-3535 expressed in the infection zone,transitional zone and nitrogen fixation zone of the nodules,and the gene expressed lasted for the entire period of nitrogen fixation.[Conclusion]MCHK-3535 functions in the symbiotic nitrogen fixation process and may participates in the normal development of nodules and negatively regulates nitrogenase activity.