Preliminary Study On Location Cloning And Function Of Nitrogen Utilization Related Gene OsDNU1 In Rice

Rice is one of the most important food crops in the world,Improving rice yield is of great significance to safeguarding Chinese food security.Nitrogen is the most important nutrient element for plant growth and development,Appropriate application of nitrogen fertilizer can increase rice yield.excessive application of nitrogen fertilizer not only increases the production cost of agriculture,but also causes a series of problems such as environmental pollution.Therefore,It is of great significance to study how to improve the nitrogen use efficiency of crops to ensure Chinese food security and sustainable development of agriculture.Our study screened a nitrogen-utilizing mutant in the EMS mutagenized mutant library of Shuhui 527,the main manifestations are that the plant height is reduced,the 1000-grain weight is reduced,Leaf yellowing during tillering,similar to rice nitrogen deficiency phenotype.cloning a P4-ATPase Gene Os DNU1 by Mutmap Sequencing,The gene function complementation,gene knockout and nitrogen content determination during the whole growth period were used to verify the biological function of the gene.The main results of this study are as follows:1.A candidate gene for controlling the dnu1 trait of the yellowing mutant was cloned by resequencing and co-segregation analysis,and a P4-ATPase was encoded.The mutant dnu1 had a single base mutation G/A in the 607 th base of the first exon,resulting in the replacement of glycine with aspartic acid,named Os DNU1.2.The CDS complement mutant of wild-type Os DNU1 linked to the rice ACTIN promoter indicates,it was found that the leaf yellowing was restored;further using the Crispr/Cas9 knockout gene Os DNU1 in the background of Kasalath,the knockout mutant also showed phenotypes such as leaf yellowing,reduction of1000-grain weight,and reduction of plant height,it is proved that the gene Os DNU1 is the target gene of the mutant dnu1.3.The expression profile of the gene Os DNU1 showed that the gene was constitutively expressed and expressed at the root of seedling stage,leaves and stems at different stages,GUS staining showed that the gene was highly expressed in root cells,leaves and stems.4.Nitrogen treatment showed that the yellowing phenotype of the mutant was more serious under nitrogen-free conditions.while the yellowing phenotype of the mutant was partially restored under high nitrogen conditions,Explain that the phenotype of the mutant is indeed caused by defects in nitrogen utilization.5.The use of hydroponic treatment materials to detect the expression levels of genes related to the nitrogen pathway indicates,the expression of nitrogen pathway gene in dnu1 was significantly higher than that in R527.Under low nitrogen stress,R527 could rapidly respond to nitrogen deficiency signals,the expression levels were significantly up-regulated.In contrast,dnu1 did not show obvious up-regulation,this indicates that the mutant dnu1 has been responsive to nitrogen deficiency in the body due to defects in nitrogen utilization,and is not sensitive to exogenous low nitrogen stress.