Phenotypic Analysis And Gene Mapping Of Glutelin 57H Mutant N47 In Rice

Rice is a major cereal crop in the world.Glutelin is one of the most important storage proteins in rice grains,accounting for about more than 60%of the total proteins in endosperm.The glutenin-encoded genes transcript into the endoplasmic reticulum surface polyribosome and then enter the Endoplasmic Reticulum(ER)cavity in the form of co-translational transports,forming 57 kDa polypeptides.The nucleus of Glutenin transcripts into the Golgi apparatus after a series of protein folding and processing.Then,the 57 kDa polypeptides transport into the protein storage vesicle and splits into the protein storage vesicles(PSV)under the guidance of dense vesicle(DV).The mature acidic and alkaline subunits eventually accumulate in the form of proteins.57 kDa polypeptide is an immature state of glutelin.Abnormal protein sorting or processing result in the accumulation of 57 kDa polypeptide which significantly compromises grain yield and quality.Although numerous regulatory genes and factors involved in protein sorting and processing pathways have been identified,the specific molecular mechanisms remain largely obscure.Therefore,the study of rice glutelin mutants and functional characterizations of candidate genes play essential roles for protein transport pathways establishment and rice variety improvement.The n47 mutant with floury endosperm was isolated from the irradiation mutant library of indica var N22.Phenotypic analysis and mapping-cloning of the mutant allele revealed that the candidate gene is a new regulator that affects glutelin transport in rice.The major findings of this work are as follows:1.Compared with the wild type,n47 exhibited floury endosperm and seedling lethality.1000-grain weight of n47 was decreased by 20%with no significant alterations with respect to the seed length,width and thickness.2.Scanning electron microscopy observations showed that starch granules in the endosperm of n47 mutant are irregularly shaped and loosely packed,resulting in the floury endosperm.Abnormal Protein body Ⅰ(PBⅠ),Protein body Ⅱ(PBⅡ)and Paramural body(PMB)structures as well as empty compartment were also observed in n47.3.Total protein and lipid contents in the endosperm of n47 were significantly reduced.However,the starch and amylose contents as well as the gelatinization properties and RVA profiles remain unchanged in n47.4.SDS-PAGE and Immunoblotting analyses revealed that the 57 kDa precursor is significantly accumulated in the endosperm of n47 mutant.Moreover,the protein contents of Binding Protein(BIP),Protein disulfide isomerase(PDI)and Sarlb subunits were increased,while no significant change was found for Sec23c.5.Genetic analysis showed that the phenotypic variation of n47 was caused by a single recessive locus.Subsequent fine mapping based on the F2 segregation population generated by the cross between n47 and Nipponbare diminished the mutant allele to an interval of about 196 kb on chromosome 11,which contains 12 open reading frames.