Map-based Cloning And Preliminary Functional Analysis Of A Key Gene FLO12 Involved In Regulation Of Starch Synthesis In Rice Endosperm

Starch is the major reserve substance of rice(Oryza sativa L.)endosperm.The content and physicochemical properties of starch directly affect the quality indexes of rice.Meanwhile,the accumulation of starch in rice endosperm is closely related to the rice yield.Therefore,it is necessary in theory and application to study the pathway of starch biosynthesis of rice which is the main food crop in the world.In this study,we characterized a novel floury endosperm mutant named flo12 derived from wild type Dianjingyou 1 treated with MNU and observed its phenotype.Further,targeting gene FLO12 was isolated through the method of map-based cloning.1.The brown rice of flo12 had an opaque endosperm appearance.The inner of the endosperm in the flo12 grains was floury white.Scanning electron microscopy observation of the cross section showed that the starch granules of wild-type were polyhedral,anomalistic and densely packed,while flo12 endosperm was filled with spherical and loosely packed starch granules with large spaces.Compared with wild-type,flo12 had decreased 1000-grain weight and grain width.2.Semi-thin section observation showed that the amyloplasts in flo12 endosperm at the middle filling stages were bigger than wild-type and flo12 displayed a large amount of smaller scattered starch granules.3.Physiological and biochemical analyses indicated that compared with wild-type,flo12 showed decreased amylose content,total starch content,and a increased lipid content in endosperm.On the other hand,the swelling power of starch in flo12 was changed.4.A map-based cloning approach was used to isolate the FLO12 gene.We first crossed the flo12 mutant with an indica varity IR36 to generate an F2 mapping population and the FLO12 locus was then mapped to a 93 kb genomic region flanked by the marker LJ4-28 and LJ4-33.Gene sequencing showed a single nucleotide substitution in Os04g0553000,resulting in the change of threonine by isoleucine.5.Genetically modified complementary vector was introduced into flo12 mutant and T0 generation plants harbors both mutation phenotype seeds and transparent seeds at the same time.Meanwhile,the proportion of transparent and opaque seeds probably meet 3:1 separation ratio.Therefore,Os04g0553000 is the candidate gene for the mutant floury endosperm phenotype.6.Temporal and spatial expression analysis demonstrated that FL012 has a constitutive expression pattern with the its highest expression level in leaves.During the endosperm developing,the expression level of FLO12 increased before 12 days after flowering,then gradually reduced.7.The FLO12 protein harbors an N-terminal transit peptide.Subcellular localization analysis indicated that FLO12 was targeted to the chloroplasts.8.The results of Western-blotting showed that the starch synthesis-related proteins had no significant differences between the wild-type and flo12.