Gene Cloning And Mutation Mechanism Disection Of Rice Floury Endosperm Mutants Ws And D92

Starch is the major component of rice endosperm,and an important factor that makes up the eating quality of rice.The grain size and weight are determined by the accumulation of starch in rice seeds,and the eating and cooking quality are directly affected by the ratio of amylose and amylopectin.Thus it is of great significance in studying the biosynthesis of starch in rice seeds.The biosynthesis of starch is a complicated and sophisticated process carried by various enzymes and influential factors.Although scientists have summarized the basic pathway of starch synthesis based on many studies,lots of participants remain unclear.By observing mutants with deficiency in starch synthesis,we can work out a more precise pathway and thus clarify the biosynthesis of starch in a more comprehensive way.In this study,we obtained two stably inherited floury endosperm mutants ws and d92 from MNU mutants of japonica variety cv.Dianjingyoul(DJY).By phenotypic,physiological and biochemical analysis and gene cloning,the mutation mechanism of mutants were discussed,and the characters of AGPase and UGPase were also referred.The main results are as follows:1.The ws mutant displayed a floury and opaque endosperm.By analyzing the physiological and biochemical characters,ws mutant showed reductions in total starch content and amylose content,and changes of amylopectin structures.SEM(scanning electron microscopy)analysis showed that the starch granules of ws were smaller and loosely packed,which might cause the opaque endosperm,while the wild type showed polytope and closely packed granules.The semi-thin sections also showed a similar result.The results indicated that in ws mutants,starch structures and synthesis were influenced.2.We crossed the ws mutant with an indica variety Nanjingll to generate an F2 mapping population,and the WS locus was mapped to a 95 kb region flanked by markers WQ8-28 and WZ8-17 in chromosome 8.Sequence analysis revealed that the coding region of the small subunit of adenosine diphosphate glucose pyrophosphorylase(AGPS2)had a single nucleotide substitution,resulting in a change in the amino acid sequence.RT-PCR analysis showed no significant difference in the expression levels of genes encoding the AGPase subunits in developing endosperm of mutant,while immunoblot analysis revealed a reduced protein level of the AGPS2b.Meanwhile,the enzyme activity of AGPase in the mutant was decreased to half of the wild-type.These results showed that the mutation of OsAGPS2 in ws mutant caused the decreased activity of AGPase,thus affecting the starch biosynthesis in rice endosperm.3.The d92 mutant displayed a white core endosperm,with its physiological and biochemical characters changed,as well.d92 mutant showed reductions in total starch content and amylose content,and changes of amylopectin structures compared with wild type.SEM analysis showed that the starch granules of d92 were smaller and loosely packed.The semi-thin sections also showed a similar result.The results indicated that in d92 mutants,starch structures and synthesis were influenced.4.We crossed the d92 mutant with an indica variety IR36 to generate an F2 mapping population,and the mutation locus was mapped to a 220 kb region flanked by markers PA-3 and C-8 in chromosome 9.Sequence analysis revealed that the coding region of uridine diphosphate glucose pyrophosphorylase(UGPasel)had a single nucleotide substitution in the 19th intron.With primers designed accordingly,we found that the d92 mutant displayed aberrant splicing,which could not be translated to normal UGPasel.By transgene complementation experiments,the mutant phenotype could be recovered.In this way we confirmed that UGPasel was the mutation gene of d92 which caused its phenotype.5.Subcellular localization of UGPasel was in the cytoplasm,which suggested that it might not directly participate in the starch synthesis in the amyloplast.RT-PCR analysis showed an increase of AGPS2b level,and decreases of GBSSI?BEI?BEIIb?ISA1 levels in d92 developing endosperm.Immunoblot analysis revealed an increased protein content of the AGPS2b in d92 mature seeds,while no significant differences were found in the enzyme activities of UGPase,AGPase and Sus in d92 developing endosperm.Native-PAGE activity staining analysis was performed to examine the starch related enzymes in developing endosperm,and we preliminarily observed a decrease of PHO1 activity.Immunoblot analysis revealed that the PHO1 protein content was also decreased in the d92 developing endosperm.The specific mutation mechanism of d92 mutant remained to be further studied.