Development Of New Rice Lines With Low Cadmium Content By Knocking Out OsNramp5 Gene

Cadmium is a heavy metal that is harmful to both plants and animals.Excessive consumption of cadmium can seriously affect human health.Rice is one of the important food crops,with more than 60%of the population relying on rice as the staple food.Meanwhile,rice is also a kind of crop that is easy to enrich cadmium.With the aggravation of heavy metal pollution in cultivated soil,cadmium rice incidents occur every now and then.In addition,zinc and selenium are two essential trace elements beneficial to human beings.The content of zinc and selenium in bio fortified staple foods is an important direction of functional agriculture at present.OsNramp5(natural resistance-associated macrophage protein 5)is a key gene involved in cadmium uptake or transport in rice.Knockout of OsNramp5 can significantly reduce cadmium accumulation in rice,but its effects on other agronomic,yield and quality traits as well as stress resistance are not well understood or studied,which affects the application of OsNramp5 in the cultivation of new rice varieties with low cadmium and high quality and safety.In this study,the OsNramp5 gene of Nanjing 46(NJ46),a japonica rice variety with good taste in Jiangsu province,was knocked out by CRISPR/Cas9 technique,and then the absorption and enrichment ability of cadmium,manganese,selenium and zinc,agronomic phenotype,yield and quality traits,and stress resistance of the knockout lines were evaluated in detail.The results will provide a theoretical reference for the more scientific and rational application of OsNramp5 knockout lines/mutants to produce low cadmium and high quality nutrient-enriched rice.The main results are as follows:1.Through qRT-PCR,we found that the expression intensity of OsNramp5 gene in NJ46 roots was significantly higher than that in shoots.Application of two different concentrations of cadmium could significantly inhibit the expression of OsNramp5 gene in roots,but for shoots,low concentrations of cadmium induced its expression,while high concentrations of cadmium did not induce its expression significantly.2.Using CRISPR/Cas9,we knocked out OsNramp5 gene in NJ46 and obtained five T0 transgenic plants with different variants of OsNramp5 gene.We designed and developed specific molecular markers for three of these mutants.Combined with marker-assisted selection,some homozygous knockout lines without Hygromycin selective marker gene and CAS9 gene were obtained in three T2 populations.We found that the cadmium content in the whole plant(including grains),and the manganese content in shoots and grains of knockout lines were significantly lower than those of wild-type in both high and low cadmium content soils.In field conditions,we found that foliar spraying of manganese fertilizer could increase the content of manganese in knockout lines to a certain extent,suggesting that the symptoms of manganese deficiency could be solved by foliar spraying of manganese fertilizer.3.By measuring the contents of copper,iron,zinc and selenium in rice grains,we found that compared with the control,the contents of copper and selenium in rice grains of the knockout lines were significantly lower than that of the control,while there was no significant difference in the contents of zinc and iron between the knockout lines and wide-type,suggesting that the knockout of OsNramp5 could affect the absorption of copper and selenium in rice.Furthermore,foliar spraying of zinc and selenium fertilizers could significantly increase the contents of zinc and selenium,respectively,and spraying of both two selenium fertilizers could reduce the content of cadmium in grains.These results indicated that OsNramp5 knockout lines could be used to produce functional rice with low cadmium and rich zinc and selenium.4.Under field conditions,we determined the main agronomic traits,yield and quality related traits of the knockout lines and wild-type under the three different levels of tillering fertilizer levels,and found that the plant height of the knockout lines were slightly lower than the control,the number of grains per panicle significantly reduced in the knockout lines compared with wide-type,resulting in the yield per plant of knockout lines being significantly lower than that of the control in the normal and high concentration of tillering fertilizer treatments,but the yield of knockout lines was not significantly reduced under low tillering fertilizer treatment.In terms of quality traits,we found that the appearance quality of the knockout lines was generally better than that that of the control,and the contents of protein and amylose were significantly higher than that in the control,which led to a significant decrease in the taste value of the knockout lines.5.Under the conditions of high temperature,low temperature and inoculation with R.solani,we compared the stress tolerance differences between the knockout lines and the control and found that the resistances of the knockout lines to sheath blight were significantly lower than that of the control.The change trend of seed setting rate between the knockout lines and the control was the same under extreme high temperature and low temperature conditions,suggesting that the use of OsNramp5 knockout lines in production should pay attention to the prevention and control of sheath blight.