Map-based Cloning And Functional Analysis Of A Carbon And Nitrogen Metabolism Gene OsCNM1 In Rice

Rice is one of the main crops in China,and its yield and quality have attracted more and more attention.In this study,a mutant cnm1 with stable genetically defective phenotype with carbon and nitrogen utilization was selected from the mutant pool induced by EMS in Zhongjiazao 17（YK17）.Intensive research on this mutant revealed that the carbon and nitrogen metabolism of mutant cnm1 was affected,which not only resulted in a decrease in yield but also caused a decline in rice quality.The main results are as follows:1.The plant height,number of tillers,number of grains per panicle,and yield per plant of the mutant cnm1 were significantly decreased.The roots were stunted of mutant cnm1 and the grain filling rate was significantly reduced.At the maturity stage of the mutant cnm1,there was an obvious greeding-green phenomenon,and the contents of chlorophyll a,chlorophyll b and carotenoids in the flags of the mutant were significantly increased.The grain shape of mature seeds is altered,with a very significant reduction in grain length,grain width and thousand-grain weight,and chalkiness was observed.The contents of amylose and total protein in the endosperm of the mutant cnm1 decreased,while the contents of soluble sugar increased significantly.The gelatinization temperature of starch in endosperm of mutant cnm1 was significantly increased,and the viscosity and expansion characteristics were significantly changed.In the process of development,the compound starch granules of the mutant cnm1 were abnormal,showing a cracking state and loose arrangement,so there was a gap between the compound starch granules.2.Under different nitrogen level conditions,the plant height,dry weight and root-shoot ratio of cnm1mutant hydroponics seedlings were lower and nitrogen content were higher than that wild-type in the same treatment.Under different nitrogen level conditions,the number of tillers,seed setting rate,grain number per panicle,yield per plant,dry weight of different tissues,total dry weight and nitrogen accumulation of cnm1 mutant potted plants were lower and nitrogen content in different tissues were higher than that wild-type in the same treatment.3.The F₂ population obtained from the cross of the cnm1 mutant and Nip was used to perform map-based cloning,and finally locate the mutant gene at a physical distance of about 62.3 kb on the long arm of chromosome 10.There are 9 ORFs in this interval.After sequencing analysis,it was found that only the 7th exon of ORF4（LOC＿Os10g25130）had a single base substitution（G-A）,resulting in the 135th glycine(Gly¹³⁵)changed to arginine(Arg¹³⁵).It was confirmed that OsCNM1 was the target gene by CRISPR/Cas9 knockdown experiment.A search of the gene revealed that OsCNM1 is allelic to OsAlaAT1,an alanine aminotransferase gene based on cloning.4.The expression pattern of OsCNM1 is constitutive,with the highest expression in the endosperm of 6 DAF.OsCNM1 protein,which consisted of 483 amino acids,is localized in the cytoplasm.The amino acid sequence of the Os CNM1 protein contains a transamination domain at positions 83-470,and the homologous protein is alanine aminotransferase.Phylogenetic analysis showed that OsCNM1 protein has high similarity with alanine aminotransferase in gramineous plant such as corn,wheat,millet,millet,and low similar with alanine aminotransferase in non-gramineous plants such as dendrobium,pineapple,lettuce.5.An alteration of a highly conserved amino acid site in OsCNM1 resulted in an extremely significant decrease in alanine aminotransferase enzyme activity,and thus affected the synthesis of amino acids,which reduced the amino acid content of the cmn1 mutant mature seeds.6.In the cnm1 mutant,the expression of many carbon metabolism-related genes is disturbed,and the expression level of most of the carbon metabolism-related genes is reduced,so that pyruvate cannot be fully used for carbohydrates synthesis,so it accumulates in leaves and endosperm.7.The enzyme activity of alanine aminotransferase was extremely significantly decreased in the cnm1 mutant and the ability to catalyze the conversion ofα-ketoglutarate and alanine to pyruvate and glutamate was reduced,allowingα-ketoglutarate to accumulate in tissues.Whenα-ketoglutarate accumulated,it significantly increased the enzyme activity of glutamate synthase（GOGAT）,enabling the plant to take up more assimilated nitrogen and thus increase the expression of genes related to nitrogen metabolism.