Relationship Between Expression Levels Of Zinc/iron Transporter And Cd Uptake And Translocation By Amaranth Exposed To Ca-deficiency,Fe-deficiency And Zn-deficiency Under Saline Environment

In recent years,As the development of our country,land resources have become extremely scarce,so people have to develop agricultural land in salinized soil.However,previous studies show that salt ions can activate heavy metals in soil and the low utilization of nutrient elements in saline soil can increased accumlation of heavy metals in plants,poses a great threat to food security.Two cultivars of edible amaranth,were selected to conduct a hydroponics culture.The concentration of Cd,Ca,Fe,Zn were measured in calcium（Ca）,iron（Fe）,and zinc（Zn）deficiency under saline environment,respectively,in order to investigate their accumulation in edible amaranth.The expression levels of 19 metal transporters in root were determined.These were used to expound the relationship between the uptakes and accumulation of Cd and the expression levels of the 19 metal transporters.The relationship was verified through genetic engineering technology.Our research provided theoretical foundation for studying consumption risks of crops grown in Cd-polluter soils with Ca/Fe/Zn deficiency and salinity stress.The major information about the study and conclusions was list as bellow:（1）There is no difference between two amaranth cultivar of uptake and accumulation in Ca-deficiency,Fe-deficiency and Zn-deficiency under saline environment,indicated that the two amaranth cultivars had the same mechanism of Cd uptake and accumulation.Compared with control groups,Ca-deficiency treatment significantly strengthened Cd uptake by roots,but inhibited root-to shoot Cd translocation.Fe-deficiency treatment significantly inhibited the uptake of Cd by roots and the translocation of Cd to the shoot.Zn-deficiency treatment significantly enhanced the uptake and transportation of Cd.Cd,Ca,Fe,Zn uptake by per unit root mass obtained by calculation.A very significant positive correlation between cadmium and zinc uptake was observed in the CK,Ca-deficiency and Fe-deficiency treatment,besides cadmium was significantly higher in plant treat with Zn-deficiency than the control group,which indicate that Cd²⁺was uptake by Zinc transporter in amaranth.（2）Based on the transcriptional sequence annotation,qRT-PCR technique was used to determine the relative transcriptional level of 12 genes related to the regulation of Cd uptake in roots and to explore the relationship between it and the Cd uptake.The results show that,A significant positive correlation between the expression of zip2,zip4 and Cd_net（P<0.05）,and a very significant positive correlation between the expression of ZIP8,ZIP11 and Cd_net（P<0.01）.Multiple linear regression analysis on the relative transcriptional level of zip2,zip4,zip8 and zip11 gene and Cd uptakebyperunitrootmass,andwegetabestequation Y=39.217+0.837X₁+14.214X₂+34.641X₃（R²=0.873）.The equation can better fit the difference of Cd absorptive capacity of plants under different treatments,which indicate that amaranth can affect the uptake of Cd by plants by regulating the expression of zip4,zip8 and zip11 gene.（3）qRT-PCR technique was used to determine the relative transcriptional level of7 genesrelated to the Cd sequestration in the vacuoles under different treatments to explore the relationship between it and the ability of cadmium intercepting in root(Cd_stay).The results show that,A significant positive correlation between the expression of HMA3 and Cd_stay（P<0.05）,and a very significant positive correlation between the expression of mtp10,abcc3,opt3 and Cd_stay（P<0.01）.High expression of hma3,mtp10,abcc3 and opt3 enhance the ability of cadmium intercepting in root under Ca、Fe deficiency condition.However,low expression of hma3,mtp10,abcc3and opt3 do not reduce the the ability of cadmium intercepting in root under Zn deficiency condition,this may be due to the low expression of the gene that regular Cd loading xylem under Zn deficiency.These results indicate that the expression of hma3,mtp10,abcc3 and opt3 in amaranth plays an important role in root interception of plants,but not the only factor.（4）A complete CDS sequence of zip2,zip4,zip8 and zip11 were cloned from amaranth,encoded 347,407,351 and 354 amino acids respectively.The CDS sequence of these four genes were complete agreement which show that they have the same function in two amaranth.Homologous protein alignment and transmembrane structure show that ZIP2 protein in amaranth may have the same function as AtZIP2in transferring Zn²⁺,ZIP4 protein may have the same function as NcZNT1 in transferring Zn²⁺and Cd²⁺,ZIP8 protein may have the same function as OsZIP8 in transferring Zn²⁺.ZIP11 protein may have the same function as OsZIP2 have no ability to transfer Zn²⁺.As the functions of OsZIP8,AtZIP2,and OsZIP2 proteins have not been reported on Cd transport,the ability of amaranth ZIP8,ZIP2,and ZIP11 to transport Cd is also needed to be further studied.（5）Expression of zip8 and zip11 genes in yeast enhance the sensitivity of Cd and increase the content of cadmium in yeast,which suggestd that ZIP8 and ZIP11 protein are plasma membrane not tonoplast transporters transferring Cd²⁺in yeast.Only zip8genes restored the zinc absorption facility of the yeast,by which the mutant’s Zn accumulation increase significantly,indicating that ZIP8 protein can transport Zn in yeast.None of thems can restored the Fe absorption facility of the yeast under low Fe treatment and influencing its Fe accumulation,suggested that ZIP2,ZIP8 and ZIP11protein have no ability to transferring Fe²⁺.