Phenotypic Analysis And Gene Cloning Of Rice Floury Endosperm Mutants B133 And B303

Starch is the main component of dry matter in rice grain,and the most important carbohydrate storage material,as well as the main factor determining the quality of rice.Two stable floury endosperm mutants and b303 were isolated from the mutant library of chemical mutagenesis of japonica rice W017.In this article,phenotypic analysis and fine mapping of two mutant genes of b133 and b303 were carried out,and the function of b133 gene was studied.The main results of this study are as follows:1.The mature seeds of mutant b133 showed floury endosperm.Scanning electron microscopy(SEM)observation of mature seeds showed that compared with wild type W017,starch granules of b133 were loosely arranged,irregular in shape and some of them were spherical.Compared with wild type,grain filling rate of b133 decreased significantly,1000-grain weight decreased by about 25%,grain size decreased significantly;total starch,amylose、galactose lipid content decreased significantly,fat content increased significantly;in addition,the viscosity properties of b133 seed starch changed significantly.Therefore,b133 has a significant effect on the accumulation of storages ubstances in endosperm.The content of soluble sugar in endosperm of b133 was significantly different from that of wild type during grain filling,and the physicochemical properties of starch changed.The activity of soluble starch synthase in mutant seeds decreased,and the expression of starch synthesis related genes decreased.The development of endosperm was observed using semi-thin sections and transmission electron microscopy.It was found that the amyloplast division of b133 was abnormal,but the development of chloroplast was not affected.2.Using F2 mapping population of b133 and indica rice variety Nanjing 11,the mutant gene was located in the interval of about 202 Kb between markers N4-1 and 14-14 on the short arm of chromosome 4.There was a previously reported Floury gene FLO2(Os04g0645100)in the interval.Sequencing analysis revealed that a single base substitution occurred in exon 17 of the gene in b133,leading to premature termination of protein translation,and gene knockout confirmed that Os04g0645100 is the b133 mutant gene.3.Real-time quantitative PCR and Western blot analysis showed that the expression of FtsZ-1 and FtsZ2-1 in b133 decreased significantly,while the expression of ARC6 increased,suggesting that the abnormality of amyloplast division may be related to the decrease of the expression of plastid division-related genes.4.Previous studies have confirmed that FLO2 is located in the nucleus,and the mutation of FLO2 results in the decrease of storage substances in seeds,as well as the significant decrease of gene transcription levels of starch synthesis related genes and other storage substances.Therefore,we speculated that FLO2 gene may act as a transcription factor.Yeast system confirmed that FLO2-N segment has trans activity.5.Yeast one-hybridization confirmed that FLO2 could bind to the promoter of FtsZ2-1,but LUC experiment did not confirm that FLO2 could activate the promoter of FtsZ2-1.Therefore,FLO2 could indirectly regulate the expression of FtsZ2-1 gene.6.GBSSI transcription level and protein products decreased in b133.Because of the presence of self-activating elements in Waxy promoter fragment,FLO2-N fragments could not be verified to bind to Waxy promoter in the yeast system.However,the LUC experiment confirmed that FLO2 could directly regulate the expression of Waxy gene.In conclusion,the mutation of FLO2 gene results in the decrease of storage substances in endosperm,abnormal physical and chemical properties of seeds、abnormal development and division of amyloplasts.FLO2 can directly regulate the expression of Waxy gene as a transcription factor,and indirectly regulate the expression of FtsZ2-1 gene by binding with the promoter of FtsZ2-1.7.Compared with wild type,b303 seeds showed floury phenotype and albino leaf.The 1000-grain weight and total starch of b303 seeds decreased,the amylose content,fat content increased and the viscosity properties of starch also changed significantly.Therefore,b303 has an obvious effect on the accumulation of storage substances in endosperm and the development of chloroplasts.8.Scanning electron microscopy(SEM)showed that starch granules in cross section of mature mutant b303 seeds were loosely arranged and irregular in shape,some of them were spherical.Semi-thin section observation showed that starch granules were elliptic,fewer and sparsely distributed in cells compared with wild type.Transmission electron microscopy(TEM)observation of 9-day seedlings showed that the chloroplast structure of b303 was looser than that of wild type.Seriously damaged,almost no intact thylakoid membrane structure was observed and a large number of small vacuoles appeared in the plastid.9.Using F2 mapping population of b303 and indica rice variety Nanjing 11,the gene was located in the 79 kb region on the short arm of chromosome 5.The candidate gene was identified by sequencing.It is a new gene involved in rice chloroplast development and starch synthesis.In this study,a mutant b303 of floury endosperm and leaf albino was also screened,and the mutant phenotype was analyzed and the map of the gene was cloned to obtain a new gene involved in rice starch synthesis and chloroplast development.Laid the foundation for the next functional study.