Cloning And Functional Analysis Of Critical Genes Involved In Soil Cd Accumulation In Edible Amaranth(Amaranthus Mangostanus L.)and The Effect Of Nitrogen Fertilizer Form On Their Expression

In recent years,Cd-polluted fields had became increasingly prominent environmental pollution,which seriously influenced crop growth and consumption safety of agricultural products.The screening of high/low-Cd accumulating cultivar,analysis of Cd accumulation difference among cultivars,and regulation of agronomic measures had became research hotspots in reducing Cd accumulation in agricultural products grown in the low and moderate Cd-polluted fields.In the present study,two key genes?Am ALMT2 and Am ALMT7?involved in organic acid secretion and two key genes?Am IRT2 and Am Nramp6?involved in Cd absorption were obtained by screening differential marker metabolites and significantly differentially expressed genes based on rhizospheric organic acids and root gene expression profile of low-?Quanhong?and high-Cd?Liuye?accumulating edible amaranth?Amaranthus mangostanus L.?.The full-length sequence of the target genes was obtained using a homology cloning technology and their bio-informatics analysis were performed.Subcellular localization and function analysis of the target genes were implemented using a agrobacterium-mediated transformation of onion epidermal cells,heterogeneous expression in yeast,and genetic transformation of Arabidopsis thaliana,respectively.Study on the effects of nitrogen fertilizer forms on organic acid metabolism and gene expression?Am ALMT2,Am ALMT7,and Am IRT2?in roots of edible amaranth may provide theoretical foundation for regulating of these key gene expression.The main results are as follows:?1?In the soil-pot trail,the roots from high-Cd cultivar exhibited relatively stronger organic acid release ability,higher gene Am ALMT2,Am ALMT7,and Am IRT2 expression?They were 24.65,1.86 and 2.37 folds of low-Cd cultivar,respectively?,and lower gene Am IRT2 expression?It was0.16-fold of low-Cd cultivar?,which indicated that cultivar difference in Cd accumulation may be due to the difference in soil Cd mobilization and absorption.?2?Bio-informatics analysis shown that gene Am ALMT2 and Am ALMT7 may encode organic acid secreted proteins of ALMT family.Gene Am IRT2 and Am Nramp6 had complete functional domains of ZIP family?Pfam:Zip?and Nramp family?Pfam:Nramp?,respectively.Moreover,all the four genes were hydrophobic transmembrane proteins,and the number and location of their transmembrane domains were different from their proximal genes to some extent.Predication of subcellular localization from sequence revealed that four proteins were most likely on the cytoplasmic membrane.?3?The results of subcellular localization in onion epidermal cells shown that GFP?green fluorescent protein?was on the cytoplasmic membrane,which indicated that genes Am IRT2,Am Nramp6,Am ALMT2 and Am ALMT7 were expressed on the cytoplasmic membrane.?4?The results of function verification shown that gene Am IRT2 participated in Cd and Zn absorption,while gene Am Nramp6 participated in Zn absorption,but not Cd absorption.Gene Am ALMT2 mediated the efflux of malic acid,fumaric acid and aspartic acid?They were 4.75,4.92 and 3.69 folds of the wild-type?non-transgenic?,respectively?,but not succinic acid.However,gene Am ALMT7 mediated the efflux of malic acid,fumaric acid,succinic acid and aspartic acid?They were 8.01,1.90,1.62,and 2.87 folds of the wild-type,respectively?.?5?Compared with ammonium nitrogen?N-NH₄⁺?application,edible amaranth under nitrate nitrogen?N-NO₃^-?application had lower Cd accumulation in roots?average 0.45mg/kg?and shoots?average 0.13mg/kg?,higher biomass accumulation,more lateral roots,stronger root cell activity,fewer root xylem vessels,more phloem sieve tubes,and stronger oxidation resistance and leaf photosynthetic capacity.These results implied that edible amaranth was a kind of“nitrate-priority”vegetable.?6?Compared with N-NO₃^-application,N-NH₄⁺application significantly inhibited Ca-Ca M signal transduction pathway in roots,but improved the root plasma membrane H⁺-ATPase activity and the ability of roots to release H⁺.N-NH₄⁺application enhanced GS enzyme activity and glutamine synthetase-glutamate synthase?GS-GOGAT?pathway,thereby synthesizing more glutamate into the tricarboxylic acid cycle?TCA?.Furthermore,TCA cycle and alanine-aspartate-glutamate?AAGM?metabolic pathways were up-regulated by enhanced PEPCase,MDH and ME enzyme activities induced by N-NH₄⁺application.?7?Importantly,N-NH₄⁺application improved genes Am ALMT2 and Am ALMT7expression in roots,and the quantitative results of organic acids secreted by roots showed that N-NH₄⁺application enhanced the organic acids secretion dominated by binary carboxylic acids,such as malic acid,fumaric acid,oxalic acid,aspartic acid and tartaric acid.Additionally,gene Am IRT2expression in roots was significantly increased when N-NH₄⁺with the low and medium nitrogen content was applied.Overall,these results indicated that N-NH₄⁺application may cause a up-regulation of TCA cycle by enhancing the assimilation of ammonium nitrogen to glutamate and phosphoenolpyruvic acid?PEP?to malic acid,thus stimulating a up-regulation of organic acid secretion gene expression and then promoting organic acids release from roots to mobilize rhizospheric insoluble Cd.Therefore,this study revealed the critical genes underlying cultivar difference in Cd accumulation and influencing mechanism of nitrogen fertilizer form on their expression.