| Cadmium(Cd)is a kind of heavy metal with strong biological toxicity and carcinogenicity to human beings.It has been listed as a first-class carcinogen by the International Agency for Research on Cancer.However,with the development of modern industrialization,environmental pollution is becoming more and more serious,and the large area pollution of heavy metal Cd in farmland has posed a major threat to the food security of our country.Rice is not only the most important food crop in the world,but also the main dietary source of human Cd intake.Therefore,reducing rice Cd accumulation is an important direction of rice scientific research and industrial development.OsNRAMP5 is the main transporter that mediates the uptake of Cd and manganese(Mn),and its mutant has a strong effect on reducing Cd,but the contentration of Mn is also greatly reduced,so that its stress tolerance may be weakened.Planted in the environment with limited Mn,the Mn contentrations in the mutant can not maintain its normal growth and development,which leads to the decrease of yield and the deterioration of rice quality.Identifying OsNRAMP5 alleles with activities of low Cd uptake and moderate Mn uptake,and analyzing their genetic characteristics and functional variation loci are helpful to understand the genetic basis of Cd and Mn accumulation traits in rice,and provide target loci and theoretical basis for molecular breeding of rice with low Cd accumulation.The previous study of our group found two potential excellent haplotypes of OsNRAMP5,HAP19 and HAP21.Compared with Nipponbare,haplotype HAP 19 had only one amino acid difference,replacing Ser with Phe at amino acid positon 313 of OsNRAMP5.There were two amino acid changes in the OsNRAMP5 of the haplotype HAP21,one was amino acid substitution from(Gly,G)to(Val,V)at position 196,the other was the deletion of glycine(Gly,G)at position 220.In this study,the characteristics of Cd and Mn accumulation of rice cultivars harboring HAP19 and HAP21 were analyzed.Based on the F2 genetic population constructed by corssing HAP19 or HAP21 rice cultivars with high Cd indica rice cultivars,the genetic effects of OsNRAMP5 alleles were analyzed,and the OsNRAMP5 functional variation loci of HAP21 were identified.The main results are as follows:(1)Inthe paddy field with 0.5mg/kg,0.7mg/kg and 0.9mg/kg Cd in the soils,the Cd contentration of brown rice of 7 rice cultivars with haplotype HAP 19 was steadily lower than the national Cd limit standard of 0.2mg/kg,whereas the Mn contentration of brown rice was also lower than that of other cultivars.In the hydroponic culture experiment,the rice cultivars with HAP19 showed the characteristics of low abilities of Cd and Mn uptake and transport,low concentrations of Cd and Mn in shoots and roots.(2)The phenotypic variation of F2 segregated population generated from a cross between rice cultivar of 11009(haplotype HAP19)and indica rice cultivar Huazhan was analyzed.It was found that the Cd and Mn contentrations of shoot and root of F2 segregated population showed the genetic characteristics of quantitative traits.According to the variation of nucleotide 938 in OsNRAMP5 coding region,KASP molecular marker was developed.It was found that this genetic locus was co-segregated with the shoot Cd,Mn contentrations and root Cd contentrations.It was detected that the locus significantly affected brown rice Cd contentrations under two different environmental conditions,in two F2 populations from different genetic backgrounds.This genetic locus could explain up to 43.01%phenotypic variation,but did not affect brown rice Mn contentrations.Through the uptake kinetics experiment,it was found that the Cd uptake activity of HAP19 rice cultivars was closely associated with this genetic locus By analyzing the candidate genes in the region,it is found that there were three candidate genes,OsNRAMP5,OsNRAMP1 and OsHMA3,which affect the accumulation of Cd in rice.By analyzing the Cd and Mn contentrations of F3 individual plants which occurred genetic recombination in this region,it was found that both OsHMA3 and OsNRAMP5-OsNRAMP1 gene clusters contributed to the low Cd accumulation in shoots of HAP 19 rice cultivars,while the low Cd accumulation in roots and shoots of HAP 19 rice cultivars were mainly caused by OsNRAMP5-OsNRAMP1 gene clusters.(3)Through the field experiment,it was found that the Mn contentrations of straw and brown rice of rice cultivars 7885(haplotype HAP21)were higher than that of other rice cultivars.Through the hydroponic culture experiment,the Mn contentrations in shoots of rice cultivars 7885 was relatively high among 26 rice cultivars,and the Mn contentrations in the roots of 7885 was remarkably higher than in other cultivars,indicating that 7885 had the characteristic of high Mn accumulation.(4)The phenotypic variation of haplotype HAP21 in F2 segregation population derived from a cross between rice cultivars 7885 and Huazhan was analyzed.It was found that the accumulation of Cd and Mn contentration in F2 segregation group showed typical genetic characteristics of quantitative traits.According to the variation of nucleotide 587 in the coding region of HAP21,a KASP molecular marker was developed.This genetic locus was co-segregated with Mn contentrationsin shoots,roots and brown rice.The phenotypic contribution rate of this locus to brown rice Mn contentration was 26.73%.Through the uptake kinetics experiment,it was found that the the Mn uptake characteristic of Mn instead of Cd was tightly correlated with the genetic locus where the KASP marker was located in the rice cultivar 7885.(5)The heterologous expression experiment of the point mutant OsNRAMP5 in Δsmf1,a Mn uptake deficient yeast strain were conducted.It was showed that expressing the OsNRAMP5 mutant with an amino acid substitution from G toV at position 196 could complement Δsmf1 growth defects more effectively,compared with expressing wild type OsNRAMP5.Besides,the Mn contentrations were higher in the yeast strain expressing the point mutant OsNRAMP5 than in the yeast strain expressing wild type OsNRAMP5.The growth of yeast strain expressing OsNRAMP5 with amino acid deletion at position(Δ220D)was similar to the positive control expressing wild type OsNRAMP5.Observing the growth of Cd-sensitive strainΔycf1,it was found that the sensitivities to Cd were similar between yeast strains expressing wild-type OsNRAMP5 and yeast strains expressing OsNRAMP5 mutations at different sites.These results reveals that the mutation at position 196 made OsNRAMP5 have higher Mn transport activity,but did not affect its transport of Cd. |
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