Gene Cloning And Functional Analysis Of Rice Floury Mutant N199

The endosperm of rice occupies the majority of mature seed volume,and is composed mainly of starch,storage proteins,fats and trace elements.Its development and enrichment not only determine the weight of the seeds,but also determine the quality of the rice.Most of the nutrients required for endosperm development are derived from sucrose produced by photosynthesis,and after the completion of basal embryonic morphogenesis,excess sucrose will be rapidly converted into starch for storage in the endosperm.Floury endosperm mutants are good genetic resources for dissecting amyloplast development and starch biosynthesis,as well as their regulatory mechanisms.In this study,n199,a floury endosperm mutant derived from an indica variety N22 by N-methyl-N-nitrosourea（MNU）mutagenesis was used for phenotypic identification,gene cloning and functional studies,and we obtained the following findings.（1）The n199 appeared a white-core endosperm but a normal outer portion.Scanning electron microscopy（SEM）analyses of mature seeds showed round and loosely packed,compound starch granules in n199 endosperm.In addition,the semi-thin sections of 7 DAF developing endosperm also revealed similar results,which had more single-grain starch granules,fewer composite starch granules than wild-type at the same period.There’s no significant difference in the total protein content of n199,and the total lipid content increased,the total starch content and amylose content decreased,and the distribution of amylopectin length changed significantly.These results indicated that the amyloplasts was abnormal in the endosperm of n199.（2）Dry matter accumulation and soluble sugar（sucrose,fructose,and glucose）content were measured in endosperm of N22 and n199 at different development stages,which showed that grain filling was delayed in n199 early development endosperm,with sucrose degradation.（3）q PCR analysis of 30 starch synthesis related genes showed that all the genes were up-regulated except Os PPDKB（FLO4）in n199.And western-blot showed that the levels of GBSSI,SSIIIa,AGPL2 and Os PPDKB（FLO4）were down-regulated,while the levels of AGPS2b and SBEI were increased in n199,and the levels of Pho I,SSIIa and SBEIIb had no significant differences were found between the two species.These results suggested that starch synthesis was abnormal in the endosperm of n199.（4）Using F₂ populations of n199/9311,the mutant gene N199 was mapped in a region of 676 kb between the locus of N199-1 and N199-13,which were both on the long arm of chromosome 3.After resequencing and sequencing for verification,we confirmed a single base substitution at the end of exon 8 of gene Os03g0294200.A base 1139 G in the coding region of gene Os03g0294200 was replaced by A in n199,resulting in a mutation from arginine（R）to histidine（H）at amino acid position 380 on the encoded protein.In the gene description on the NCBI website（http://www.ncbi.nlm.nih.gov/）,Os03g0294200 encodes a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase（F2KP）,therefore we named the gene as Os F2KP2.（5）The 35S::Os F2KP2-GFP complementation was constructed in p CAMBIA1305and transgene complementation experiments were performed.The complementary vector p CAMBIA1305 of 35S::Os F2KP2-GFP was constructed and transferred into n199 for transgenic complementary experiment.The results showed that the full-length CDS（coding sequence）of this gene in N22 could recover the endosperm floury phenotype of n199,demonstrating that this candidate gene Os F2KP2 is the target gene controlling the floury endosperm of n199.（6）Further studies on the expression pattern of Os F2KP2 and subcellular localization of Os F2KP2 revealed that the gene was constitutively expressed and Os F2KP2 protein was localized in the cytoplasm.（7）The q PCR of 32 genes related to glycometabolism showed that,31 genes were down-regulated in n199 except Os PEPC2,which was not significantly different,indicating an overall suppression of endosperm glycometabolism.In summary,rice endosperm floury mutant n199 is a new rice endosperm amyloplast dysplasia mutant,which has defects in starch synthesis and accumulation in the endosperm,and its endosperm floury phenotype is caused by Os F2KP2 gene mutation,which encodes a new key factor involved in endosperm development in rice seeds and may affect endosperm filling by affecting starch synthesis and sugar metabolism.Exploring how Os F2KP2 affects the specific pathway of glycometabolism and starch synthesis is helpful to refine the mechanism of endosperm starch development and lay a theoretical foundation for breeding high-quality varieties.