Construction Of Low-cadmium Rice Mutant By Knocking Out The OsNramp5 And OsLCD Genes Using The CRISPR/Cas9 System

At present,the pollution of cadmium(Cd)in paddy feild has been a serious issue.Rice is capable of enriching Cd form paddy soil.As an important staple food,rice has become the main source of Cd intake in human.It is of great significance to construct and breed rice varieties with low-Cd accumulation for ensuring food security.In present study,using the CRISPR/Cas9 system,the genes including OsNramp5 and OsLCD,which control Cd absorption and transport in rice,were knocked out in two conventional rice vaireties including yuexijingsi miao 2(YJSM2,indica)and yuxiangyouzhan(YXYZ,indica)in Guangdong province,and one japonica rice,zhonghua 11(ZH11)to create low-grain-Cd mutant.The main results are as follows:1)After knocking out the OsNramp5 gene,the concentration of Cd in brown rice,root and straw in the Cd-stressed rice plants was significantly decreased as compared to that of the wild type.The concentrations of Cd in brown rice in the YJSM2 and YXYZ mutants with0.5 μM Cd supply were decreased by 57～61% and 56～62%,respectively,as compared to the wild type.Additionally,the concentration of Cd in brown rice in YJSM2 with 2.0 μM Cd supply was decreased by 76～78%.It was also observed that the seed setting rate,root length and dry weight in the Cd-stressed mutants decrease significantly,however,the plant height,the effective tiller number and the dry weight of straw are unchanged in comparison with that of the wild type.In addition,the concentration of Mn in brown rice and straw in the Cdstressed mutants decreased significantly,while the concentration of Fe,Cu and Zn were unchanged as compared to that of the wild type.By increasing the concentration of Mn in the culture solution,it was observed that the adverse effects on the growth of rice seedlings due to less uptake of Mn are improved.2)Among the mutants produced by knocking out of the OsLCD gene,except for YJSM2,the concentration of Cd in brown rice under Cd stress was significantly higher than that of the wild type.Under 0.5 μM stress condition,the concentration of Cd in brown rice in the YXYZ mutants and the ZH11 mutants were increased by 115-149% and 60-64%,respectively,as compared to that of the wild type,while the Cd concentration in brown rice of the YJSM2 mutants were not changed significantly.Under 2.0 μM Cd stress condition,the Cd concentration in brown rice in the YXYZ mutants and the ZH11 mutants were increased by 66-84% and 165-291%,respectively,while the Cd concentration in brown rice of the YJSM mutants were decreased by 25%.It was also found that the seed setting rate,root length and root dry weight were significantly decreased in the OsLCD gene-knockout mutants as compared to that of the wild type,however,the plant height,straw dry weight and effective tiller number were not affected.In addition,the contents of Fe,Mn,Cu and Zn in brown rice in the OsLCD gene-knockout mutants were not changed as compared to that of the wild type.3)In the mutants with double knockout of the OsNramp5 and OsLCD genes,the Cd concentration in brown rice was significantly lower than that of the wild type under Cd stress.The Cd concentration in brown rice in the YJSM double knockout mutants were decreased by 71-75% and 76% under 0.5 and 2.0 μM Cd stress,respectively,as compared to that of the wild type.Moreover,the Cd concentrations in the straw and root in the double knockout mutants were also decreased significantly.In addition,the seed setting rate,root length and root dry weight in the mutants were decreased significantly,however,the plant height,straw dry weight and effective tiller number were not changed significantly.The content of Mn in the brown rice in the double knockout mutants was decreased significantly,while the contents of Fe,Cu and Zn were not significantly changed as compared to that of the wild type.By increasing Mn concentration in the hydroponic solution,the growth of the double knockout mutants was improved under Cd stress condition.The comparison of the Cd concentration in brown rice between the double knockout mutant and the OsNramp5-knockout mutant OsNramp5 showed that the Cd concentration in rice grains may be mainly controlled by the process of Cd absorption in the roots OsNramp5.In summary,the rice mutants with low grain-Cd were successfully constructed using the CRISPR/Cas9 system in present study,and the results also indicated that Cd uptake in the roots may be a key process to control grain Cd accumulation in rice,thereby laying the foundation for low-Cd molecular breeding in rice;at the same time,the mutants with increased Cd accumulation were also constructed,which can be used for the phytoremediation in Cd contaminated soils.