Identification And Characterization Analysis Of Manganese Utilization Genes MDG6 And NRAMP6 In Arabidopsis Thaliana

Manganese is an essential trace element for plant growth and development.Manganese is directly involved in many important metabolic processes in plants.Therefore,it is of great theoretical and practical significance to study deeply the mechanism of absorption,transport and utilization of manganese by plants.It has been reported that the NRAMP family has a wide range of metal transport activities in plants,can transport a variety of metal elements,the NRAMP family has six family genes,in Arabidopsis,it has been reported that NRAMP 1 is a high-affinity manganese transporter,NRAMP2 is a manganese transporter located in TGN,which is mainly responsible for root elongation under manganese deficiency.NRAMP3 and NRAMP4 also play an important role in intracellular manganese homeostasis.NRAMP6 is involved in the transport of Cd.In addition,in order to further study the mechanism of Mn utilization,we established a EMS mutagenesis library based on nramp1 mutant,screened Mn deficient mutants under Mn deficiency conditions,and obtained a Mn dependent mutant named mdg6nramp1(mn-dependent defective growth 6).In addition,the transport mechanism of NRAMP6 to Mn is not clear at present.Therefore,it is of great significance to explore the mechanism of Mn utilization by NRAMP6.This study focused on the phenotypic and functional analysis of NRAMP6 and MDG6 genes.The main findings are as follows:(1)The growth of nramp1 mutant was inhibited under Mn deficiency condition,but the nramp6 single mutant had no obvious phenotype under Mn deficiency condition.Therefore,nramp1 nramp6 double mutant was obtained by constructing a hybrid population.Under Mn deficiency condition,compared with the control,the root elongation of nramp1 nramp6 was inhibited and the root hair was shortened obviously,and the growth of nramp1 nramp6 was significantly inhibited by Mn deficiency treatment in water culture.Nramp1 nramp6 was specifically sensitive to Mn deficiency,but not to other divalent metals such as iron deficiency and zinc deficiency,and was not sensitive to other bivalent metals such as iron deficiency and zinc deficiency.Under the condition of water culture,the content of metal ions was determined by manganese deficiency and sufficient treatment.The results showed that compared with nramp1,the content of manganese in the new leaves of nramp1 nramp6 decreased significantly,but the content of manganese in roots was over-accumulated.In order to verify this conjecture,the manganese content in the injured fluid of xylem was detected,and it was found that the manganese content in the injured fluid of nramp1nramp6 was significantly lower than that of the nramp1 control.In order to verify this conjecture,the manganese content in the injured fluid of nramp1 nramp6 was obviously decreased as compared with that of the nramp1 control.This further suggested that NRAMP6 may be involved in the transport of manganese from root to shoot in vivo.After setting up grafting experiment of nramp1 and nramp1 nramp6 to exchange shoot and root,the content of Mn was determined by water culture and Mn deficiency treatment.The results showed that there was no difference in Mn content between nramp1 and nramp1 nramp6 in shoot and root as compared with nramp1 above ground and root.This suggested that NRAMP6 may be involved in the regulation of the signaling pathway,which in turn controls the transport of manganese.In order to verify the expression pattern of NRAMP6 in plant,it was found that NRAMP6 was expressed in root cap,root vascular bundle and hypocotyls through NRAMP6 promoter and genomic fusion GUS as well as in microtubule and hypocotyls of shoot.This suggests that NRAMP6 may be involved in the transport of manganese through vascular tissue.The specific localization of NRAMP6 in Arabidopsis protoplasts driven by 35s strong promoter NRAMP6-GFP was investigated.The results showed that NRAMP6 could co-locate with endoplasmic reticulum-specific marker.The overexpressed NRAMP6 plants grew better in Mn deficiency than that in the control,and could significantly enhance the tolerance of plants to low Mn.(2)Screening EMS mutagens under the condition of Mn deficiency,and screening a Mn-sensitive mutant.The root length of the mutant was significantly inhibited under Mn-deficient condition.Mdg6 nramp1 was sensitive to low manganese and insensitive to other divalent metals such as iron deficiency and zinc deficiency,and was not sensitive to other bivalent metals such as iron deficiency and zinc deficiency,and mdg6nramp1 was sensitive to low manganese.The gene was cloned by genome-wide sequencing combined with development markers and named MDG6,which encodes a COPII transporter family SEC23,which is mainly involved in the transport of proteins from endoplasmic reticulum to Gaul matrix.SEC23 mainly functions in the form of SEC23/SEC24 complex.In order to explore the difference of element content in the mutant under Mn deficiency,more Mn was accumulated in mdg6 and mdg6nramp1 double root under Mn deficiency,whether in new leaf,mature leaf or root,compared with the nramp1,and under the Mn deficiency condition,more Mn was accumulated in the new leaf,mature leaf or root,and there was no significant difference in the content of elements between the mutant and the mdg6nramp1 control.The content of manganese accumulated in the single line was more than that in the double process.Under the condition of Mn deficiency,the content of Zn in mdg6 and mdg6 nramp1 decreased significantly in root,old and new leaves,suggesting that SEC23 may be involved in the localization of Mn and Zn transporter under Mn deficiency.35s strong promoter drives MDG6-GFP,to transform Arabidopsis thaliana protoplasts into Arabidopsis thaliana protoplasts and the specific marker SEC23D,can overlap completely,indicating that MDG6 is located in ERES(endoplasmic reticulum exit site)and cytoplasm.The expression pattern of MDG6 was further analyzed by quantitative analysis of qRT-PCR.It was found that Mn deficiency resulted in a significant decrease in the expression of MDG6 both in root and shoot under wild-type conditions.This indicated that Mn deficiency inhibited the mRNA level of MDG6.In conclusion,SEC23,was cloned by forward genetic method and hybridized to obtain nramp1nramp6,NRAMP6 involved in manganese transport process.It is important for plant growth and development that the functional analysis of SEC23 needs to be further analyzed.