Functional Analysis Of A HMA5 Gene During Symbiotic Nodulation In Medicago Truncatula

Heavy metal transporter P-type ATPase?HMA?has been confirmed to be involved in the transport of heavy metals in plants,and HMA5 is mainly involved in the transport of copper.In the model plants Arabidopsis thaliana,Oryza sativa and Cucumis sativus,a large number of studies have been conducted on HMA5.However,studies on HMA5 in leguminous plants were rarely reported.A HMA5 gene associated with copper stress and symbiotic nodulation in Medicago lupulina was isolated in our lab.In order to further study the role of such genes in the symbiotic nodulation of leguminous plants,this study screened the genome of Medicago truncatula,which the whole genome was sequenced,looking for homologous genes of HMA5 and exploring the relationship with symbiotic nodulation by expression pattern analysis.On this basis,the MTR₈g079250 gene was used as an object to perform subcellular localization,tissue expression,RNA interference and overexpression,and preliminary identificate the gene's function in symbiotic nodulation.1.Subcellular localization of MTR₈g079250The onion epidermal subcellular localization vector of MTR₈g079250 was constructed,and the onion epidermal cells were transformed by particle bombardment.The MTR₈g079250::GFP fusion protein was mainly expressed in the cytomembrane and nucleus of onion epidermal cells under laser confocal microscope.The tobacco leaf subcellular localization vector of MTR₈g079250 was constructed,and transformed into Nicotiana benthamiana leaves through Agrobacterium tumefaciens.The results showed that the target gene was mainly localized on the cytomembrane and nucleus.2.Tissue expression of MTR₈g079250The PMTR₈g079250::GUS fusion expression vector was constructed and transferred into the roots of Medicago truncatula through the method of rhizogenes-mediated transformation.After culture,GUS staining was observed.The results showed that the target gene was mainly expressed in the meristem,infection zone and cortex of nodules and expressed in the middle column and root crown of roots.3.Analysis of growth and nodulation in transgenic plants with RNA interference and overexpression of MTR₈g079250The RNA interference vector and overexpression vector were constructed and transformed into roots of Medicago truncatula through the method of rhizogenes-mediated transformation,and the transgenic chimeric plants were formed.The transformed plants were analyzed from the infection event,the physiological phenotype,the microstructure of nodules and spatio-temporal expression analysis of related genes.There was no significant difference in the above mentioned aspects of RNA interference transgenic plants with the control plants,indicating that target gene RNA interference may have no significant effect on the symbiotic nitrogen fixation of Rhizobium.The number of infection thread in cortex and nodule primordia were significantly reduced in overexpression transgenic plants.The the fresh weight and the number of root nodules of the overexpressed transgenic plants were significantly reduced;The transmission electron microscope showed that the number of bacteroids in infected cells was decreased,the shape was irregular,and the peribacteriod space was increased,showing signs of aging;qRT-PCR analysis showed that the expression level of nodule-specified marker genes NIN,DMI1 and ENOD11 were down-regulated in different degrees,and the expression level of leghenmoglobin gene was also down-regulated.The expression level of two other homologue gene of HMA5,MTR₅g010420 and MTR₈g012390 were less changed.The homologue gene of HMA7/RNA1,MTR₂g035840 was significantly upregulated,while the expression level of MTR₄g094695 was not significantly changed.The expression level of CCH homologue gene was not significantly changed.In summary,it is speculated that overexpression of MTR₈g079250 inhibits symbiotic nodulation and affects plant growth.