Functional Analysis Of Two Cadmium Accumulation Genes In Grains Of Rice(Oryza Sativa)

Heavy metal cadmium(Cd)pollution is a common problem in rice growing areas in southern China.Therefore,the rice produced from Cd contaminated paddy soils has risk with Cd content beyond the standard(>0.2 mg/kg).Long term Cd-polluted rice diet has potential impact on human health.Unfortunately,it will be a long-term and arduous task to remove Cd and other heavy metals from the environment by various remediation methods.Generating cultivars with low Cd accumulation trait in grains is one of the effective approaches to solve the problem of Cd safety in rice.In recent years,some progress has been made in the identification of Cd uptake and transport genes in plants.However,the molecular and physiological mechanisms of Cd uptake,transport and accumulation in rice,especially in rice grains,are still unclear.Therefore,further identification of Cd accumulation related genes and study of their physiological functions can deepen the understanding of the genetic mechanism of Cd transport and accumulation in rice,and provide guidance for breeding low Cd rice germplasm resources through genetic regulation.The root and nodes of rice play important roles in ion uptake and Cd accumulation.In this study,the genes OsCCX2 and OsMDR6 were screened as candidate genes by transcriptome analysis of root and nodes,and cadmium tolerance analysis in yeast mutants.At the same time,the correlation between Cd accumulation-related genes vaiation and Cd accumulation in grains of thermo-sensitive sterile line(TGMS)rice was analyzed.The main results were as follows:1.OsCCX2 is involved in the regulation of Cd accumulation in rice.Firstly,a knockout mutant(japonica rice Nipponbare and indica rice 377)was constructed by CRISPR/cas9 gene editing method.Cd accumulation analysis showed that Cd content in the mutant grains was significantly lower than that in the wild type under different Cd soil conditions,indicating that the gene was involved in Cd accumulation in rice grains.Secondly,the accumulation characteristics of Cd in different parts of rice at different developmental stages were analyzed.The root-to-shoot transport rate of Cd in the mutant was significantly lower than that in the wild type,suggesting that OsCCX2 gene was involved in the Cd transport from root to shoot.The Cd accumulation characteristics of different internodes showed that the proportion of Cd in the uppermost internode of the mutant was lower than that of the wild type,and gradually higher than that of the wild type with the decrease of the internode.The proportions of Cd accumulation of the fourth internode and the below internodes were significantly higher than those of the wild type,indicating that OsCCX2 gene participated in the distribution of Cd in tissues of different node levels above ground.Under Cd stress,the OsCCX2-overexpressed ycfl yeast-displayed higher tolerance to cadmium toxicity than the control.At the same time,the content of cadmium in the yeast cells overexpressing OsCCX2 decreased significantly compared with the empty vector-transformed yeast,suggesting that OsCCX2 might participate in the outward transport of Cd.Based on the analysis of plant subcellular components,Cd content in xylem sap and the distribution and localization of OsCCX2,it is suggested that OsCCX2 may play a key role in regulating the outward transport of Cd from parenchyma cells to xylem.2.OsCCX2 participated in the regulation of calcium homeostasis in plants.OsCCX2 is a hypothetical Na/Ca exchanger.The content of Ca in flag leaf of ccx2 mutant was lower than that of wild type,and the content of Na was higher than that of wild type,while the accumulation of Ca and Na in the flag leaf of OsCCX2 overexpression line displayed opposite tendency,compared with the mutant,suggesting OsCCX2 mght participate in the anti-transport of Ca and Na.By using NMT technology,we found overexpression of OsCCX2 gene increased the calcium outflow signal in roots,which may lead to the change of resting calcium level in cells.When treated with Na,the instantaneous Ca influx signal was detected,the peak value of Ca signal in overexpression plants was found significantly lower than that in control.The calcium signal is also affected in the ccx2 mutant.The Ca root-to-shoot transport rate and Ca distribution ratio in the shoot displayed similar patterns to Cd,suggesting that OsCCX2 participated in Ca transport from root to shoot and Ca distribution in the tissues of different node levels,suggesting that Ca transport system was used to transport Cd upward in the node.The pollen fertility and the seed setting rate in overexpressed OsCCX2 rice decreased significantly.The OsCCX2 ectopic-expressed Arabidopsis plants displayed dwarf stature and could not bear fruit,which might be caused by the disorder of intracellular Ca homoestasis.3.OsMDR6 mediates Cd accumulation in rice grains.When OsMDR6 function was disrupted,the accumulation of Cd in the mdr6 mutant grains decreased significantly.Except for Cd,the contents of Fe and Cu in mutant grains also decreased significantly,suggesting that OsMDR6 might be involved in the transport or accumulation of Fe or Cu.OsMDR6 was highly expressed in roots,and its expression level was induced by Cd.The mutant yeast with expression of OsMDR6 enhanced the tolerance to Cd,however,the Cd content in the yeast cells did not change significantly compared with the control.Considering that the Cd content in vegetative organs such as stems and leaves of the osmdr6 mutant was not significantly different from that of the control as well,it was speculated that OsMDR6 could not transport Cd directly,and might only affect Cd accumulation in rice grains and tolerance of yeast cells to Cd indirectly through other pathways.4.Preliminary study on physiological function of OsMDR6.The grain size and 1000-grain weight of mdr6 mutant were significantly smaller than the wild type control,and the length of main root was slightly longer than that of wild type.Compared with the wild type,the roots of mutants were still longer and less inhibited under high concentration of IAA treatment.However,the mutant and wild type were strongly inhibited by high concentration of NAA treatment,where the root length showed no difference,suggesting osmdr6 is more likely to be an auxin inactivated mutant.The expression of the auxin response genes,such as OsIAA3,OsIAA23 etc,were up-regulated significantly in osmdr6,suggesting that OsMDR6 is involved in auxin transport or signaling.5.Association between sequence variants in cadmium-related genes and brown cadmium accumulation traits in thermo-sensitive genic male sterile rice.Firstly,the grain Cd content of 174 thermo-sensitive genic male sterile lines was measured.The variation of cadmium content ranged from 0.063 to 1.466 mg/kg DW,averaging 0.23 mg/kg DW.Among them,47%of the cadmium content of the material were beyond the standard(>0.2 mg/kg DW),of which some 8%of the materials were severely exceeded(>0.4 mg/kg DW).Secondly,the polymorphisms of the seven Cd genes were detected by using the Nipponbare genomic sequence as the reference sequence.It was found that nine mutation loci located in OsNRAMP5,OsNRAMP1,OsHMA2 and OsHMA3 genes were significantly or extremely significantly related to the Cd accumulation traits in cadmium-contaminated soil.Among the significantly correlated loci,two loci(OsNRAMP1 2573bp;OsNRAMP5 1306bp)had negative effects on Cd accumulation traits in grains,and could be developed into low Cd screening molecular markers in grains.