Identification And Characterization Of NRAMP1 Matel Transporter In Chlamydomonas Reinhardtii

Heavy metal pollution has not been effectively controlled,and the bioremediation method is not only time-saving,energy-saving,environment-friendly,and low in cost,but also can recover heavy metals.It is an extremely valuable method for remediation of heavy metal pollution.Chlamydomonas reinhardtii has always been a model organism in the biological community and used as a model for the study of physiology,biochemistry,and molecular genetics in the dynamic balance and tolerance of cell-level transition metals.The identification of the function of C.reinhardtii transition metal ion transporters can lay the foundation for the further study of the distribution of metal ions in different cell compartments,the regulation of metal ion concentration,and the interrelationship between metal ions and other trace elements.It can also provide a large number of target genes that regulate the degree of metal enrichment,which may provide a solution for bioremediation of high-efficiency and low-cost polluted water bodies and soils.In addition,it can provide a theoretical basis for the biological production of C.reinhardtii.This article mainly analyzes the function of NRAMP1 transporter and predicts the mode of transport of Cd(?),Co(?),Zn(?)by the NRAMP family and the mode of transport of Zn(?)by the ZIP family.Firstly,a fragmented amplification and overlap PCR method were used to successfully amplify the NRAMP1 gene with a GC content of up to 70%.Secondly,using bioinformation analysis,it was concluded that Zn(?),Fe(?),Cd(?),Mn(?)as Os NRAMP7 protein and At NRAMP6 protein potential transporters are listed as candidates for the study of Cr NRAMP1 protein specific transport substrate.Furthermore,through the growth analysis of the wild-type CC4533 and NRAMP1 deletion mutant nramp1?,candidate metal ions that may be the specific transport substrate of the NRAMP1 transporter were screened out: Cd(?),Co(?),Zn(?).It is also known that Zn(?),Mn(?),and Fe(?)are necessary trace elements for the growth of C.reinhardtii,and only a very small amount of Zn(?)is required to maintain their normal growth.The sensitivity of Mn(?),high or low,will obviously inhibit the growth of C.reinhardtii.While the growth of C.reinhardtii is sensitive to the loss of Fe(?),it is very tolerant to the increase of Fe(?)concentration.Analysis of yeast transgenic strains and transgenic C.reinhardtii strains by metal ion growth stress,supplemented by analysis of the relative expression levels of the NRAMP family and the ZIP family,led to the following main conclusions:One of the NRAMP1 specific transport substrates is Zn(?),which may be localized on the cell membrane,and the direction of transport of Zn(?)is from the outside to the inside.Or it localized on the vacuole membrane,transporting Zn(?)from inside to outside.NRAMP1 transporter is associated with Co(?)and may interact with NRAMP4.NRAMP4 may play an important regulatory role in the detoxification mechanism of Co(?).The expression of NRAMP2 may not be regulated by Cd(?),Zn(?),Co(?).And NRAMP2 may have antagonistic effects on NRAMP1.NRAMP3 may play a role in the metal detoxification mechanism of C.reinhardtii,one of which is to reduce the accumulation of Cd(?),Zn(?),and Co(?)in the cell of C.reinhardtii.And it may be located on the vacuole membrane.The metal ions are stored in the vacuole and may also be located on the cell membrane,transporting excess metal out of C.reinhardtii.The expression of ZRT1,ZRT4 and ZIP6 may not be related to NRAMP1.The role of ZRT1 may in transporting Zn(?)is beneficial to the accumulation of Zn(?)in the cells of C.reinhardtii to provide the trace elements needed for its growth.ZRT4 may play a role when Zn(?)enters and leaves C.reinhardtii cells.ZIP6 may not play a key role in the transport of Zn(?)and may be a cofactor of one transporter.