Map-based Cloning And Functional Analysis Of FLO2 Gene Regulating Starch Synthesis In Rice Endosperm

Rice(Oryza sativa L.)is one of the most important food crops in the world.Its endosperm is mainly in the form of starch.Disruption in the starch synthesis pathway often led to adverse effects on the rice quality.Therefore,the rice starch synthesis defect mutants are used to clone related genes.It is of great significance to improve the rice quality and yield.In this study,ethyl methane sulfonate(EMS)was used to treat ZH-11(Zhonghua 11)to obtain a floury endosperm mutant named as flo2.The physicochemical properties and phenotypes of the flo2 mutant were analyzed.An F₂ population for gene mapping was generated by crossing the flo2 mutant with Nanjing-11(Indica).The mutant gene was cloned and functional validation of the FLO2 gene was carried out by CRISPR/Cas9 technology.The main results of this research are as follows:1.Phenotype analysis of flo2 mutant found that there is no obvious difference in the agronomic traits except rice appearance quality between the flo2 mutant and wild type.The grain appearance of the flo2mutant was milky and its endosperm is chalky compared to translucent wild type.Cytological observation through semi-thin sections revealed that starch granules were loosely packed and had abnormal development in flo2 mutant.Consistent with these results,TEM analysis also identified irregular,broken,unfilled amyloplast structures of endosperm at 10 DAF(Days after fertilization)during grain development.Further,SEM analysis identified small,round and loosely packed starch grain formation in mature mutant endosperm.2.Physicochemical analysis of flo2 mutant found that the total starch and amylose contents in flo2mutant decreased significantly.The chain length distribution for amylopectin in starch granules were also disturbed in mutant.The proportion of short chains with degree of polymerization ranging from 9 to 21were significantly decreased,whereas the short and intermediate chains with DP range of 6 to 9 and 21-36 were increased respectively.The long chains with DP ranging from 37-44 were also increased in flo2mutant.3.An F₂ mapping population was developed by a cross between flo2 mutant and Nanjing-11 and fine mapped the candidate gene in a region of 135.5 kb between the molecular markers RM348 and Indel9 on chromosome 4.Gene Sequencing analysis identified a point mutation(A to T)in exon 14 that caused a stop codon.4.Expression analysis of gene by quantitative real-time PCR revealed the expression of FLO2 in most vegetative tissues including root,stem,shoot,leaf,leaf sheath and seeds.Among all these parts,the expression of FLO2 was higher in seeds.During grain development,the predominantly expression was found at 15 DAF,however expression was higher in wild type as compared to flo2 mutant during grain development and at maturity.5.RNA-seq analysis revealed that FLO2 gene was involved in starch and sucrose biosynthesis pathway.A total of 2341 differentially expressed genes(DEGs)were identified for a comparison between wild type and flo2 mutant,out of which 1529 were upregulated,while 812 were downregulated.Our findings suggest that FLO2 is involved in the starch biosynthesis pathway and in grain development of rice.The mutation in FLO2 gene generated abnormal starch granule formation and disrupted amyloplast structure due to impaired starch biosynthesis pathway subsequently caused chalkiness in grain endosperm.