Gene Functional Study Of Rice Floury Endosperm Mutant B381 And Phenotypic Analysis And Gene Mapping Of Giant Embryo Mutant N2-52

Starch is an important storage substance for rice and the main constituent of rice endosperm.The synthesis of starch is regulated by a variety of enzymes,which is a complex and delicate process.Abnormal starch synthesis can cause the endosperm to appear partially opaque or completely opaque,affecting its quality.Although multiple genes have been reported to regulate starch synthesis,the biological mechanisms and regulatory networks for starch synthesis are not comprehensively understood.Endosperm mutants are ideal materials for studying the biological mechanisms and regulatory networks of starch synthesis.From the two chemical mutation mutant pools of japonica rice varieties W017 and Ningjing 2,a floury and shrunken endosperm mutant b381,and another giant embryo highly chalky mutant N2-52 were identified,respectively.The function of the mutant gene of b381 was studied,and phenotypic analysis and positional mapping of the mutant gene were performed in N2-52.The main findings of this paper are as follows:1.b381 exhibited a floury and shrunken endosperm with two constrictions.The gene was cloned by Brother Chen Jie earlier.The mutant gene was OsRpoTp,which is a nuclearencoded plastid RNA polymerase gene.Subcellular localization analysis showed that OsRpo Tp was located in chloroplasts.2.Real-time quantitative PCR and Western blot analysis showed that the nuclearencoded plastid RNA polymerase-dependent expression of plastid-encoded RNA polymerases was down-regulated;the amount of protein was also decreased.The expression of b381 light-system functional genes including psbA,psbD and cab,was down-regulated,and the down-regulation of ATPase and NADH inhibited the respiratory pathway.This indicated that OsRpoTp plays an important role in the development of chloroplasts.3.Analysis of plastid editing sites and splicing sites revealed that the editing efficiency of six sites changed,and the splicing of three sites was abnormal.4.The yeast two-hybrid experiment showed that four PPR proteins and two proteins of rice MORF family interacted with RpoTp.5.The amount of AGPS 2b protein in the starch synthesis pathway decreased,and the amount of SS Ⅱa and GBSS I proteins increased.6.Mature seeds of the mutant N2-52 showed giant embryos and high chalkiness.Compared with the wild type,the plant height,chalkiness rate and amylose content of N2-52 were significantly higher;seed setting rate and 1000-grain weight were significantly lower;grain thickness and fat content were significantly lower.Scanning electron microscope observation showed that the starch granules in the chalky part of N2-52 were loosely and irregularly arranged.Semi-thin section observation revealed that N2-52 amyloplasts was loosely distributed and air spaces appeared.RVA analysis showed that the physicochemical properties of N2-52 endosperm starch changed.7.Using the F2 mapping population of N2-52 and the indica rice variety N22,the mutant gene was located in a 42-kb region on the short arm of chromosome 7 with a total of five Open Reading Frames(ORFs)in the interval.Five candidate genes in this region were sequenced and only ORF 3,the Giant Embryo(GE,Os07g0603700),of which the 1529th base in the second exon was changed from C-to A-,resulting in premature translation stop.8.Western blot analysis showed that in the mutant,the amounts of four starch synthesisrelated enzymes decreased,and the amount of one enzyme increased,so the starch synthesis pathway of N2-52 was abnormal.Sucrose synthase activity assay showed that the enzyme activity of N2-52 was significantly higher than that of the wild type.In summary,we obtained a floury endosperm and albino leaf mutant b381 and studied the function of the mutant gene.At the same time,six related proteins were selected for an in-depth functional study.The mutation of the GE gene may cause the synthesis of starch to be blocked which changed the structure of the starch granules.