| Manganese is an indispensable micronutrient in the growth and development of plants.It is involved in many important metabolic processes in plants,such as photosynthesis,protein and lipid synthesis,regulation of various enzyme activities,and oxidative stress.In alkaline soils,the effectiveness of manganese is reduced.Therefore,manganese deficiency is an important limiting factor affecting crop yield in alkaline soil.NRAMP1 is a highly affinity manganese transporter in Arabidopsis thaliana,which participates in the absorption of manganese under low manganese conditions.In-depth study of the utilization mechanism of manganese in plants has important guiding significance for improving the nutritional status of plants in the absence of manganese.In this paper,an EMS mutagen library constructed from the Arabidopsis manganese transport mutant nramp1 was used as a material to screen a manganese-dependent new leaf yellowing mutant mdg10nramp1(mdg10,Mn-dependent defective growth 10)under low manganese conditions.The mutant was used as a research object for related physiological phenotypic analysis,genetic analysis,and cloning and functional analysis of the target gene.In addition,manganese transporters are also involved in the uptake and transport of cadmium,and soil cadmium pollution can cause severe cadmium accumulation in rice.Knocking out cadmium transporters in rice is an effective way to reduce cadmium accumulation in grains.The cadmium uptake of rice roots is mainly caused by the manganese transporter OsNRAMP5.At present,it is still unclear whether knocking out the OsNRAMP5 gene can reduce the accumulation of cadmium in rice without affecting its growth and yield.Therefore,this study used CRISPR/Cas9 technology to perform targeted knockout of OsNRAMP5 genes in two major cultivars in Jiangsu Province,Nanjing 46 and Huaidao 5,and conducted relevant experimental analysis on knockout materials,hoping to create new ones.Rice low cadmium strain.The specific test results are as follows:(1)Under the condition of manganese deficiency,the new leaf yellowing phenotype appeared in mdg10nramp1,and the chlorophyll content of the new leaves decreased significantly.By adding sufficient manganese,the mutant manganese-deficient phenotype can be fully restored.When mdg10nrampl was treated with iron deficiency,zinc deficiency(2)The OsNRAMP5 gene in two indica rice varieties was knocked out by CRISPR/Cas9 technology,and three independent homozygous deletion mutants without transgenic tags were obtained.Under low manganese culture conditions,the growth potential and chlorophyll content of osnramp5 plants were significantly lower than that of wild type;the growth defect phenotype of this mutant could be fully recovered by adding sufficient manganese.The accumulation of cadmium and manganese on the ground and below the mutant was significantly reduced compared to the wild type.The results of field experiments showed that the content of cadmium in the flag leaf and grain of the mutant was significantly reduced,but some agronomic traits such as plant height,seed setting rate,and number of grains per panicle were affected to varying degrees,which led to a slight decrease in yield.We hypothesize that the growth of mutant plants is affected by a significant reduction in manganese content in the body.Therefore,we suggest that when using osnramp5 to reduce the accumulation of cadmium in rice,it is also necessary to consider the pH and moisture content of paddy soils.In summary:1.Under the condition of manganese deficiency,the manganese content in the new leaves and chloroplasts of widg10nramp1 decreased significantly.We speculate that it may be because the MDG10 mutation affects the RNA shearing,processing and transportation of some manganese transporters.2.The knockout of OsNRAMP5 gene by CRISPR/Cas9 technology can significantly reduce the cadmium concentration in the grain,but at the same time the manganese accumulation is also significantly reduced,which affects the growth and yield of the mutant. |
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