Fine Mapping And Functional Analysis Of A Floury Endosperm Gene In Rice

In this study,we used an alkylating agent N-methyl N-nitrosourea(MNU)to treat japonica cultivar Hwacheong to obtain an endosperm mutant flo(t).First,the phenotypic and physicochemical properties were analyzed.The structure characteristics and function properties of starch between mutants and wild type were compared by extracting the starch from mature seeds,and the target gene was fine mapped,gene cloned and verified by map-based cloning.The main experimental results were as follows:1.The mutant flo(t)endosperm showed white opacity,the seed length was basically the same,the grain width decreased slightly,and the grain weight decreased obviously.Scanning electron microscopy showed that the distance between flo(t)starch granules became larger and the arrangement was loose,while wild-type arrangement was tightly enriched.However,there was little difference in the shape of starch granules.The results of physiological and biochemical determination of mutant seeds showed that the total starch content and amylose content of the mutant seeds decreased compared with the wild type,especially the total starch content,decreased by 17%.Protein content,lipid content compared with the wild type had improved.2.Study on starch properties of mutant showed that the molecular structure of the mutant changed,and the content of long B chain in the amylopectin was significantly decreased.The crystallinity was decreased,the wild type crystallinity was 29.96,and the crystallinity of the mutant decreased to 23.63.The thermodynamic analysis showed that the gelatinization initiation temperature of the mutant was decreased.The chain length distribution of the mutant changed,the degree of polymerization in the short chain branching ratio of 8-14 increased,then short chain of DP3-7,medium short chain of DP16-20 and medium long chain of DP25-37 decreased.3.The F1 seeds were obtained by hybridization between the mutant and Zhonghua11,F2 population was obtained from the F1 population for genetic analysis,the normal endosperm phenotype and the floury endosperm phenotype were statistically analyzed.The result showed segregation ratio was 3:1.It indicated that flo(t)is a mutant caused by single gene recessive mutation.The F1 seeds were obtained by hybridization between the mutant and Indica rice variety Dular,F2 population was obtained from the F1 population as the gene mapping population.The SSR,Indel and dCAPS markers were used to locate the gene within the interval of 94.5kb on chromosome 6.4.There were a total of 16 open reading frames in 94.5kb interval by inqured Rice Genome Annotation Project.By sequencing,it was found that ORF14,that was,LOC_Os06g13810 occurred the mutation in the intron splicing region,changed from G to A,resulting in the insertion of 7 bases in cDNA,which resulted the encoding early termination.5.We constructed the transgenic complement vector of the full-length CDS and transformed flo(t)mutant.The results showed that the T0 seed recovered the transparent phenotype and the scanning electron microscopy analysis showed that the starch grains changed from the disordered state into closely arranged state,which can be determined that the gene lead to the floury phenotype,meanwhile traits can be restored by complementary.The subcellular localization indicated that the protein encoded by the gene is located in the cytoplasm.