Functional Research On Heavy Metal Transport P-type Atpase Gene HMA7 In Symbiotic Nodulation Of Medicago Truncatula

Heavy metal P-type ATPases have been confirmed to be involved in heavy metal transport in plants.HMA7?also known as RAN1?transports copper ions to ethylene receptors,ethylene binds to receptor in the presence of copper ions,thus initiating the expression of ethylene pathway.Studies have shown that ethylene inhibits symbiotic nodulation,but the function of HMA7 in symbiotic nodulation is unknown.In order to investigate the effect of heavy metal transport P-type ATPase7?HMA7?on symbiotic nodulation,we obtain two HMA7 candidate genes by screening the genome of Medicago truncatula.Based on the analysis of its expression pattern,subcellular and tissue localization of MTR₂g035840 was performed.we carried out RNA interference of two target genes and overexpression of MTR₂g035840 to analyze its function in nodule formation.1.Analysis of expression patterns of two target genes.Detection of expression of two target genes under symbiotic conditions shows increase of MTR₂g035840 and MTR₄g094695 expression in late symbiotic period,presumably it plays a role in late nodule maturation.Tissue expression analysis showed that MTR₂g035840 was mainly expressed in root nodules and MTR₄g094695 was mainly expressed in roots.Under different copper concentrations,compared with controls,The expression of MTR₂g035840in 50 mg kg^-1 and 200mg kg^-1 decreased in the early and late stages of symbiosis.MTR₄g094695 is mainly affected by 200mg kg^-1 copper,it shows different changes in different periods of inoculation.It is speculated that they play different roles in the absorption or transport of copper ions.2.Location analysis of MTR₂g035840.MTR₂g035840 subcellular localization vector for tobacco leaves was constructed,transient transformation system of tobacco cells was mediated by Agrobacterium tumefaciens.The gene of interest with the Golgi?GmMAN1?and endoplasmic reticulum?AtCHS?marker genes was co-transformed.Co-localization of GmMAN1 and AtCHS with target gene under laser scanning confocal microscopy was observed.The results showed target gene is mainly located on endoplasmic reticulum.MTR₂g035840 Promoter::GUS fusion expression vector was constructed.Transformed into roots by Agrobacterium.After growing,The results of GUS staining showed the gene was expressed in the nodule meristem and nitrogen fixation zone of the nodule and in the whole root tissue.3.Construction of interference and overexpression vectors of target genes and analysis of transgenic plants phenotype and nodulation.MTR2g035840 and MTR₄g094695 RNA interference vectors and overexpression vectors of MTR₂g035840 were contructed,then its was transferred into the roots of Medicago truncatula through the method of rhizogenes-mediated transformation,formed transgenic chimeric plants.The infection events of rhizobia,the morphological and physiological indexes of plants,the space-time expression of related genes and the microstructure of rhizobia were analyzed respectively.The results showed no significant difference in the growth of RNAi-40 and RNAi-95 transformed plants compared to the control group.Analysis of infection events showed that the number of nodule primordial was significantly decreased in RNAi-40,but there was no significant change in the number of root hair curlings,root hair infection lines,and cortical infection lines.No significant changes in RNAi-95 infection events.The results of transmission electron microscopy images showed that there was no significant change in nodule cells in the RNAi-40 and RNAi-95 bacteroids contracted,Some symbionts fused.Overexpression of MTR₂g035840 significantly inhibited the invasion and nodulation of rhizobia.The observation of infection events showed a significant reduction in the number of cortical infection lines and nodule primordia.The number of nodules at the 28 day post inoculation was also significantly reduced,transmission Electron Microscopy?TEM?results showed that the density of bacteroids in single infected cells in the overexpression group was reduced,bacteroid forms are also irregular.