The Excavation And Functional Characterization Of Key Gene Regulating The Qtl For Cadmium Concentration In Brown Rice (Oryza Sativa L.)

Cadmium（Cd）contamination in brown rice which is resulted from the Cd pollution in field soils has been one urgent issue to solve in the whole society of China.Selecting and breeding the low-Cd rice cultivar,as well as adopting some soil remediation technology or agronomic measures,can decrease the adverse effects of soil Cd contamination and ensure the food safety.The Cd concentration in brown rice is a complex quantitative trait which is controlled by multiple genes and affected by environment and genotype.Excavating dominant genes regulating the Cd transport of low-Cd rice line can provide gainful gene to achieve selecting or developing the rice cultivar with low Cd concentration in brown rice by marker assistant selective breeding technology.Thus,we regard the previously screened low Cd rice line YaHui2816 as the research object in this study.On the basis of revealing the characterization of Cd transport in YaHui2816,the key genes participating in Cd transport and low Cd accumulation in brown rice were excavated and identified by several research approaches including q PCR,homologous cloning,yeast complementation test,subcellular localization,CRISPR/Cas9 gene-editing technology from the predicted candidate genes in the interval of QTL associated with Cd concentration in brown rice.The main results of this research are shown as below:（1）The Cd concentration in stem,leaves and nodes of YaHui2816 at vegetative stage and grain-filling stage were 1.20-to 3.81-fold significantly higher than that of C268A.However,Cd transport from shoot to spike of YaHui2816 was lower than that of C268A.At grain-filling stage,the Cd concentration in ear neck and ear(0.35 and 0.17 mg·kg^-1)of Ya Hui2186 were significantly lower than that of C268A(0.39 and 0.31 mg·kg^-1).YaHui2816 showed strong Cd retention in nodes than C268A,leading to its lower Cd transport from nodes to leaves.Under the regulation of OsPCS1 and OsGST,Cd stress significantly induced the phytochelatin（PC）concentration in nodes of YaHui2816 with PC₂ as the dominant formation,which results in the increase of Cd retention in nodes and decrease of Cd transport from shoot to spike.Additionally,YaHui2816 exhibited weak Cd retention in leaves.YaHui2816 had 1.50-to 10.50-fold significantly higher Cd translocation factor from lower nodes to upper leaves than C268A.Adsorption kinetics and adsorption isotherm experiments of cell wall in leaves indicated that YaHui2816 showed lower Cd adsorption capacity than C268A,leading to its stronger Cd transport ability from leaf to other organs.The cell wall of C268A showed 1.09-to 1.23-and 1.02-to 1.08-fold significantly higher Cd adsorption amount than that of YaHui2816.Chemical adsorption was the main Cd adsorption process of cell wall in leaf of YaHui2816,which can be well-fitted by Freundlich and pseudo-second-order kinetics models.（2）Among the candidate genes in the interval of QTL controlling Cd concentration in brown rice,Cd stress significantly induced the transcriptional level of OS05G0198400（OsZIP7）,OS12G0161100（OsPUB37）and OS12G0162100（OsWNK9）in node of YaHui2816.Their transcriptional level under Cd treatment were 1.36-to 7.60-fold higher than that under CK treatment.The transcriptional level of them in node of YaHui2816 were significantly 1.86-to 136.0-fold higher than that of C268A.Furthermore,the short-term Cd uptake experiment revealed the transcriptional level of them in node of YaHui2816 were increased with the extension of Cd-treated time,indicating that they might be the pivotal candidate gene which participate in the Cd retention in YaHui2816.The homology-based cloning was adopted to obtain the sequence of OsZIP7-YaHui2816,OsWNK9-YaHui2816 and OsPUB37-YaHui2816genes with the c DNA of YaHui2816 as template.The biological information analysis found that OsZIP7-YaHui2816 has 6 transmembrane domains.The sequence aligning and phylogenetic analysis indicated that OsZIP7-YaHui2816 is the homolog of two zinc（Zn）transporter OsZIP4 and OsZIP10.Additionally,OsZIP7-YaHui2816 showed two amino acid substitutions in the coding region（CDS）compared to the OsZIP7 in Japonica or Indica reference genome.No transmembrane domain is predicted in OsWNK9-YaHui2816,which belongs to the Serine/Threonine protein kinase family.The sequence aligning and phylogenetic analysis indicated that the amino acid sequence of OsWNK9-YaHui2816 was same with that of Japonica reference genome but showed seven amino acid deletions compared to the OsWNK9 in Indica reference genome.OsWNK9 showed high similarity with OsWNK6,At WNK5,At WNK6 and At WNK7.Compared to the OsPUB37 in Japonica reference genome,the OsPUB37-YaHui2816 which don’t have transmembrane domain showed three amino acid deletions and one amino acid substitution and was the homolog of OsPUB38,At HCF136 and At PDPK1.（3）The CRISPR/Cas9 gene-editing technology was used to construct the mutant rice lines of three pivotal candidate genes OsZIP7,OsPUB37 and OsWNK9 with them as targeted genes.Homozygous rice mutants were selected to be used for phenotypic analysis of OsZIP7,OsWNK9 and OsPUB37 respectively due to their frame shift and terminal codon in the target region.The hydroponic and field experiments indicated that knockout of OsZIP7,OsPUB37and OsWNK9 respectively significantly reduced the yield performance of rice.Compared to the wild type（WT）,the thousand kernel weight of oszip7,oswnk9 and ospub37 mutants decreased by 3.0～9.6%,6.7～9.1%and 5.2～12.7%,respectively.At seedling stage,only knockout of the OsWNK9 decreased the Cd tolerance of rice.Additionally,knockout of the OsZIP7 and OsWNK9 significantly affected the Cd concentration in brown rice and basal stem（the un-developing nodes）,whereas knockout the OsPUB37 showed no influence in the Cd concentration in brown rice.These observations indicated that only OsZIP7 and OsWNK9 are the key genes regulating Cd retention in nodes and low Cd accumulation in brown rice.（4）OsZIP7 did not participate in the process of Cd uptake in rice.At seedling stage,oszip7mutant lines showed 1.08-to 1.10-fold higher Cd translocation factor from root to basal stem,whereas lower from basal stem to shoot.At maturity,the oszip7 mutant rice showed significantly lower Cd concentration in the internode,leaves and brown rice than WT,which was 58.0～76.9%,58.6～81.9%and 81.9～85.7%of the WT.The calculation of Cd translocation factor found that the oszip7 mutant rice showed higher Cd translocation factor from nodes to leaves（node III-leaf III,noe II-leaf II,node I-leaf I）and from node I to brown rice,indicating that OsZIP7 participates in the Cd transport from nodes to leaves and brown rice.The yeast drop assay experiment found that expressing OsZIP7-YaHui2816 affected the growth of Cd-,Mn-,Zn-sensitive yeast mutants.This revealed that OsZIP7-YaHui2816 had transport activity of Cd²⁺,Mn²⁺and Zn²⁺.Under Cd-treated condition,OsZIP7 participated in the Mn uptake and Mn movement from nodes to leaves and spike.Additionally,OsZIP7 also regulated the Zn concentration in brown rice.The oszip7 mutant showed significantly lower Zn concentration in internode,node II,node I and brown rice than WT,which were 68.7～72.6%,65.2～72.3%,64.8～77.4%and 87.8～91.1%of WT respectively.The OsZIP7-YaHui2816 localized in the endoplasmic reticulum,and its expression in internodes were 2.3-to-4.7 folds and 4.7-to 27.2-folds higher in internodes than other organs.Thus,the OsZIP7-YaHui2816 is responsible for the Cd movement from root to shoot and internode to brown rice,then increasing the Cd concentration in shoot and brown rice.（5）The OsWNK9 did not participate in the process of Cd uptake but participate in the Cd retention in basal stem at seedling stage.The Cd translocation factor from basal stem to shoot of oswnk9 mutants was 1.06-to 1.7-folds higher than that of WT.At maturity,the Cd concentration in node III of mutants was significantly lower than that of WT,which were71.9%～85.2%of WT.No obvious difference was found in the Cd concentration in node II.The Cd concentration in node I and brown rice was 1.25-to 1.27-folds higher than WT.The oswnk9mutants showed higher Cd translocation factor from lower nodes to upper nodes（node III-node II,node II-node I）,node III-leaf III and basal stem-brown rice.These observations indicated that OsWNK9 regulated the Cd retention in basal stem and nodes,then decreasing the Cd transport to brown rice.Expressing OsZIP7-YaHui2816 affected the growth of Cd-,Mn-,Zn-sensitive yeast mutants.This revealed that OsZIP7-YaHui2816 had transport activity of Cd²⁺,Mn²⁺and Zn²⁺.oswnk9 mutants showed significantly lower iron（Fe）concentration in various organs than WT,indicating that OsWNK9 also participated in the Fe accumulation in brown rice.The OsWNK9-YaHui2816 localized in the nucleus and showed higher expression in internodes,leaf sheath and node than that in leaf at grain-filling stage.Thus,the OsWNK9 is responsible for the Cd retention in nodes（especially lower nodes）and internodes,then decreasing the Cd movement to brown rice but increasing the Fe accumulation in brown rice.