蒺藜苜蓿重金属转运蛋白HMA5基因在共生结瘤中的功能分析

Functional analysis of heavy metal transporter HMA5 gene in symbiotic nodulation of Medicago truncatula

重金属转运ATP酶(heavy metal transporting ATPase,HMA)主要参与植物体内重金属的转运.前期研究发现,HMA5基因与苜蓿铜胁迫及共生结瘤相关.为探明豆科植物根瘤中的铜离子吸收、转运及平衡机制,本研究以模式豆科植物蒺藜苜蓿(Medicago truncatula,Mt)为材料,对其HMA5同源基因MTR_8g079250在共生结瘤中的功能进行了初步研究.亚细胞定位分析表明,MTR_8g079250主要定位于细胞膜和细胞核上;GFP::MTR_8g079250融合表达分析显示,MTR_8g079250主要在根瘤的分生区、侵染区、皮层和根的中柱、根冠表达,RNAi沉默目的基因后对共生固氮表型没有产生明显影响.然而,目的基因过表达植株的鲜重和单株根瘤数目较野生型明显降低,侵染线和根瘤原基也显著减少;透射电子显微镜分析表明,根瘤细胞中含有少量的类菌体且形状不规则,环类菌体空间增大,呈现出早衰的迹象;qRT-PCR结果显示,结瘤标志性基因NIN、DMI1、ENOD11和豆血红蛋白基因的表达水平均有不同程度的下调,豆血红蛋白基因的表达水平也有所下调.这些结果表明,MTR_8g079250过表达对共生结瘤有抑制作用.

Leguminous plants are able to grow in a nitrogen-deprived environment because they have evolved a symbiotic relationship with rhizobia in the soil,leading to the formation of new organs,namely nodules.In root nodules,rhizobia reduce nitrogen to ammonia through biological nitrogen fixation and provide ammonia to the host legumes in exchange for carbon source and energy they need.In the symbiosis,legumes also provide the metal trace elements to rhizobia,including copper(Cu).As one of the essential elements for plant growth,Cu participates in multiple biological processes.However,excess Cu induces oxidative stress to the detriment of many physiological processes including symbiotic nitrogen fixation.Therefore,the copper content in plants must be maintained within a certain physiological range.The higher plants have evolved complex physiological mechanisms to maintain the copper homeostasis in cells.The heavy metal transporter,Ptype ATPase(HMA),is mainly involved in the transport of heavy metals including copper in plants.All members of the HMA family in the dicot model plant Arabidopsis have been identified and classified.Preliminary research in the laboratory found that HMA5 gene is related to copper stresses and the symbiotic nodulation of Medicago.In this study,the function of HMA5 homologous gene was investigated during symbiotic nodulation in Medicago truncatula(Mt),a model leguminous plant.A phylogenetic tree was constructed based on the currently classified HMA family genes in Medicago truncatula and the expression patterns of the genes were analyzed.A homologous gene MTR_8g079250 of HMA5 was selected for further study.Subcellular localization assays showed that MTR_8g079250 was localized to the cell membrane and nucleus.The fusion expression analysis of GFP::MTR_8g079250 showed that MTR_8g079250 was mainly expressed in the meristem,infection zone and cortex of the nodules,as well as in the stele and cap of the roots.Knockdown of MTR_8g079250 by RNA interference(RNAi)technology had no significant effect on the symbiotic nitrogen fixation phenotype.However,overexpression of MTR_8g079250 impaired the infection process and nodule development.Compared with the control,the fresh weight and nodule number per plant were dramatically reduced in MTR_8g079250-overexpression plants.The numbers of the infection threads and nodule primordia were also significantly decreased.Ultrastructure analyses showed that the nodule cells contained fewer bacteria in the overexpression plants.In particular,the remarkable deformation of bacteroids and enlargement of peribacteroid space occurred in the infected cells,suggesting premature aging of the nodules.qRT-PCR analyses showed that the expression levels of the nodulation marker genes,NIN,DMI1,ENOD11 and leghemoglobin gene were down-regulated to different extent.These results indicate that the overexpression of MTR_8g079250 inhibited the symbiotic nodulation.In summary,this study preliminarily assessed the function of HMA5 in symbiotic nodulation,laying a foundation for the subsequent exploration of the absorption,transport and equilibrium mechanisms of copper ions during root nodule symbiosis.