Absorption And Tolerance Of Cadmium In Apple Rootstocks And Cloning And Functional Identification Of Related Gene MhNRAMP1

Fruit trees are perennial plants,and their roots will absorb and accumulate more heavy metals even in soils with low heavy metal content.Cadmium（Cd）was one of the most toxic heavy metals in the environment and the most serious pollution to cultivated land in China.Apple was one of the four major fruits in the world,and its roots come from rootstocks.In this study,we analyzed and compared absorption,transport and tolerance of cadmium six kinds in apple rootstocks（Malus xiaojinensis Cheng et Jiang,M.sieveersii Roem,M.prunifolia Borkh,M.robusta Rehd,M.baccata Borkh and M.hupehensis Rehd）.Futhermore,we studyed on MhNRAMP1 gene isolated and cloned from M.hupehensis,and analyzed on cadmium uptake and tolerance of transgenic MhNRAMP1 yeast and apple callus.The results showed that:1.There was a significant difference in Cd content among six apple rootstocks,and the Cd content in root was much higher than that in shoots.The content of Cd in shoot and root of M.baccata was the lowest,and the absorption rate of Cd²⁺and the transport coefficient（Tf）were the lowest.The expression levels of MdZIPT1,MdIRT1 and MdNRAMP1 genes related to cadmium uptake were also lower than those of other rootstocks.The expression of MdZIPT1,MdIRT1 and MdNRAMP1 in M.sieveersii wild apple was the highest among the apple rootstocks,and the absorption rate and accumulation of Cd²⁺were also the highest.Except M.sieveersii,the expression of gene MdHMA3 associated with the transport of Cd to shoot was down-regulated in the other five rootstocks compared with the control.As a result,it was found that the absorption and accumulation of Cd²⁺by apple rootstocks under Cd stress are consistent with the gene expression.The results showed that the ability of Cd absorption and transport of M.baccata treated with 50μM CdSO₄ was less than that of the other five apple rootstocks.2.After treatment with 50μM CdSO₄ for 14 days,the declince range of root length,fresh weight and root activity of M.sieveersii,and the increase range of root mortality,MDA and H₂O₂ content were the highest after cadmium treatment.The increase range of dry weight and the increase extent of H₂O₂ content in M.baccata were the lowest.The lowest increase of cell death rate in root system of M.robusta.The activities of POD and SOD in roots of M.robusta increased more than others.The decrease of relative chlorophyll content in M.prunifolia was the most,but this change was not significant in M.robusta and M.baccata.Based on the principal component analysis（PCA）,the index of rootstocks was analyzed by membership function.Taken together,the order of cadmium tolerance of six kinds of apple rootstocks from strong to weak was as follows:M.robusta>M.baccata>M.hupehensis>M.prunifolia>M.xiaojinensis>M.sieveersii.3.MhNRAMP1 gene,whose open reading frame（ORF）length was 1638 bp,encoded a protein containing 545 amino acids.The predicted molecular weight of the protein was 59.12kD.It was located in plasma membrane and has 12 transmembrane structures.The gene MhNRAMP1 was identified to be specifically located in the plasma membrane,which was a membrane protein.It could be expressed in roots,stems and leaves,the highest expression was in the root,the next was the leaf,the lowest in the stem.The expression level in root was up-regulated with cadmium stress,and slightly up-regulated in stem,but decreased significantly in leaf site.4.The yeast expressing MhNRAMP1 gene under cadmium treatment showed poor growth rate and more serious toxicity in point plate test and growth curve test.It was verified that the expression of MhNRAMP1 gene could increase the cadmium sensitivity of yeast.It was proved that the expression of MhNRAMP1 gene could increase the cadmium sensitivity to yeast.Further determination of cadmium content in yeast showed that MhNRAMP1 gene could significantly increase cadmium accumulation in yeast compared with unloaded yeast.The results indicated that MhNRAMP1 gene was involved in the absorption of cadmium in yeast,promoted the accumulation of cadmium and increased the sensitivity of yeast to cadmium.5.Under cadmium treatment,the fresh weight of callus with over-expressed gene MhNRAMP1 decreased and the electrical conductivity increased.The cadmium content in apple callus was detected by Cd fluorescence staining and MS-ICP.The results showed that the overexpression of cadmium content in apple callus was higher.The cadmium uptake rates of wild-type and transgenic apple callus were measured by non-destructive microassay.The cadmium uptake rate of over-expressed apple callus was higher than that of wild-typ.Overexpression of MhNRAMP1 gene increased cadmium-sensitive and cadmium content,reduced cadmium tolerance in apple callus.