Phenotypic Analysis And Gene Cloning Of Rice Floury Endosperm Mutant B454

The main components of dry matter in rice grain are starch,protein,lipid and so on.The content of starch accounts for 80%,and it is also the most important energy storage material.It is composed of amylose and amylopectin.Starch biosynthesis pathway is a complex process involving a series of enzymes.Although some regulatory factors have been found in rice starch biosynthesis pathway,the molecular mechanism of starch biosynthesis is still unknown.More genes related to starch biosynthesis need to be identified to further study starch biosynthesis pathway.In addition,the accumulation level of dry matter in rice grain depends on the accumulation of starch,and the quality of rice food is affected by the properties of starch,so the yield and quality of rice ultimately depends on the synthesis of starch.At present,through the generation of endosperm mutants,the study of these mutants will contribute to a deeper understanding of starch biosynthesis process,and some desirable mutants can be used for the cultivation of new rice varieties.A stable genetically derived silage endosperm mutant b454 was screened from a mutant library of chemically induced indica rice variety W017.By phenotypic analysis of b454 and cloning of the mutated gene,the b454 mutant gene is an unreported gene that affects the biosynthesis of starch in rice endosperm.The main findings are as follows:1.The endosperm appearance of b454 showed a white core and abdominal shrinkage.Scanning electron microscopy showed that the starch granules of wild type W017 were polyhedron,closely arranged and uniform in size;the starch granules of mutant b454 were loosely arranged in the white part of the center,mostly single starch granules,with abnormal shape and gaps between the granules.The 1000-grain weight of b454 was significantly lower than that of wild type,and there was no significant difference in grain width and thickness between wild type and b454.2.The total starch content of mutant b454 was not significantly different from that of wild type,but its amylose content was significantly decreased;the fat content was significantly increased;the starch viscosity and amylopectin length distribution of b454 changed to some extent.3.Semi-thin section observation of endosperm at 9 days after flowering showed that the compound starch granules in the central part of endosperm of b454 were abnormal,mostly scattered as single starch granules;they could not form normal tortoise shell structure,and starch could not be filled normally;these results indicated that b454 mutant gene participated in the formation of compound starch granules in the central part of endosperm of rice.4.Compared with the wild type,the short chain with degree of polymerization（DP）less than 10 in amylopectin increased significantly,the middle chain of DP 10-14 decreased significantly,and the long chain with DP greater than 14 did not change much.At the same time,the decrease of GBSSI protein content in the mutant also explained the reason for the decrease of amylose content.5.A hybrid combination of b454 and indica rice variety Nanjing 11 was developed.The mutant gene was located in the region between markers C1-4 and C1-9 in centromere region on chromosome 1 using the F2 population.Due to the large interval,two mutation sites were finally found using the whole genome sequencing,which may affect four candidate genes,including LOC_Os01g27440,LOC_Os01g29210,LOC Os01g29220 predicted from https://rapdb.dna.frc.go.jp/and Os01g0388400 from https://rice.plantbiology.msu.edu/.At present,no genes related to starch synthesis have been reported in these four candidate genes.Sequencing revealed that the b454 mutant had a single base mutation in LOC_Os01g27440 gene compared with the wild type.Another mutation site had a deletion of 176 base,and an additional 13 base sequences were inserted at the deletion site.The deleted fragments were located in the promoter regions of LOC_Os01g29210 and LOC_Os01g29220 genes,which might affect their expression level.The deleted fragments overlapped with Os01g0388400 partly and might change the protein structure encoded by the gene.Quantitative RT-PCR analysis showed that compared with the other three candidate genes,the expression of LOC_Os01g29220 decreased to the largest extent in the mutant.Overall,b454 gene is a new gene involved in rice endosperm development.The gene affected starch synthesis in rice endosperm,resulting in 1000-grain weight of b454 significantly lower.The b454 mutation can also cause abnormal starch granules in rice endosperm,affect the arrangement of starch granules,and lead to the production of floury endosperm.At the same time,the mutation of b454 affects the expression of starch synthesis-related enzymes.