Map-based Cloning And Functional Analysis Of White Belly Chalkiness Gene OsWB1 In Rice

Starch is the most abundant constituent in rice endosperm,which represents the primary source of calories in the human diet.Its synthesis and accumulation also influences crop yield and grain quality.Although many key enzymes in starch biosynthesis have been identified and cloned,their sophisticated and complex network regulation system is so far unclear.Therefore,using mutants to explore and clone genes related to starch biosynthesis is still an effective mean,and it is of great significance to elaborate starch synthesis mechanism and further analyze the regulation mechanism.Moreover,it provides a possibility and option for generating of suitable starch content plants.In this study,we identified a white belly mutant oswb1with stable inheritance of chalkiness from the offspring of tissue-culture of a japonica rice variety,Nipponbare,and its regulatory gene Os WB1 was sequentially map-based cloned and its function was analyzed.The main results are as follows:1.The mutant oswb1 displayed an opaque endosperm mainly in the belly and core,and the starch granules became round and loosely arranged in the opaque endosperm.Compared with the wild type,the filling rate of oswb1 mutant was slowed during the whole grain filling stage,and the grain thickness and 1000-grain weight was reduced.At the same time,the total starch,amylopectin and protein contents were significantly decreased,but the amylose,crude fat and sucrose contents were increased,and remarkable changes were observed in gel consistency,RVA profile,fine structure of amylopectin as well as XRD spectrum in the mutant compared to the wild type.The results therefore indicated that the mutant has a significant effect on the content and structure of rice endosperm starch.2.An F₂ separating population developed from the cross between oswb1 mutant and an indica rice variety Nanjing 11 was used for fine mapping.The Os WB1 locus was narrowed down to a 40.2-kb region in the long arm of rice chromosome 10.By sequencing all seven predicted genes within this region,we identified a single-nucleotide mutation from C to T in LOC?Os10g41160,resulting in a premature termination codon at amino acid residue 81 in the mutant.Functional complementarity,overexpression,RNA interference and knockout results confirmed that LOC?Os10g41160 is the target gene responsible for the mutant phenotype of oswb1,which was then named Os WB1.Os WB1encodes a putative expression protein without any known domain and functional annotation,and no homologous genes have been reported so far.In addition,no Os WB1orthologous proteins were found in any other lower plants,microorganisms,animals and humans,indicating Os WB1 is a regulator specific to higher plants.3.Os WB1 was dominantly expressed in the developing rice endosperm and peaked at 15 days after fertilization,but lower or undetectable expression was observed in other tissues.Both GUS staining and RNA in situ hybridization indicated that Os WB1 was highly abundant in the ovular vascular trace end,embryo and aleurone layer,suggesting that Os WB1 may be related to seed development,sugar loading and transport,and endosperm starch development.4.A total of seven important genes involving in rice endosperms starch biosynthesis were identified to interact with Os WB1 by the methods of RNA-seq,q RT-PCR and yeast point-to-point two hybridization experiment.These genes include Os NF-YB1,Os Su Sy3,Os AGPS1,Os AGPS2a,SSI,FLO6 and Os SUT2.Among them,the physical interaction between Os WB1 with Os NF-YB1,Os Su Sy3,Os AGPS1 and Os AGPS2a were further confirmed by Bi FC and Pull-down assays in vivo and in vitro.We speculated that Os WB1may play a pivotal role in regulating rice endosperm starch biosynthesis in the formation of homologous or heterologous complexes.5.Single mutants Os WB1,Os NF-YB1,Os Su Sy3 and Os AGPS1,as well as double mutants of Os WB1 and Os NF-YB1 were generated by CRISPR-Cas9 gene editing technology,and all of them showed chalky phenotypes similar to the oswb1 mutant.Further physicochemical analysis revealed that Os WB1 and Os NF-YB1 share a synergistic effect.6.Subcellular localization showed that Os WB1 is predominantly in the nucleus,and the LUC transcriptional activation of rice protoplast assay indicated that Os WB1 and its truanted proteins have transcriptional activation activity,and the activation region is between 141 and 289 amino acids residues.LUC assay also revealed that Os WB1 and transcription factor Os NF-YB1 can separately or jointly regulate sucrose transporter Os SUT4.EMSA assay further verified that Os WB1 interacts with Os NF-YB1 to form heterodimers and collaboratively regulated Os SUT4.It was speculated that Os WB1 might be a new transcription factor.