Map-Based Cloning And Functional Analysis Of A Key Gene FLO12 Involved In Regulating Starch Synthesis In Rice Endosperm

Starch is the main storage substance of cereal crops,accounting for about 90%of the dry weight of the grain.Therefore,the content of starch in grains is of great significance for the formation of crop yield.In addition,the content and properties of starch are particularly important for the quality of grains in cereal crops.The synthesis of starch in plants is a complex and sophisticated biological process that involves a series of enzymatic reactions,requiring the participation of a large number of enzymes and related regulatory factors.At present,the direct synthesis pathway of starch has been well studied,but little is known about the regulatory mechanism of this pathway.Therefore,cloning and identifying key genes regulating starch synthesis in plants is of great theoretical significance for enriching the regulatory network of starch synthesis,meanwhile,it has potential application value for rice yield and quality improvement.Abnormal endosperm mutatiorns generally associated with starch quality,so abnormal endosperm mutants is the ideal material for study starch synthesis and properties.In this study,by screening the rice abnormal endosperm mutants,we found a floury endosperm mutant,named floury endosperm12（flo12）.Seeds of the mutant showed a floury white-core endosperm.Scanning electron microscopy revealed that the starch granules were loosely arranged.We successfully cloned the FLO12 gene by means of map-based cloning.The FLO 12 gene encodes Oryza sativa alanine aminotransferase 1（OsAlaAT1）.We studied the structure,subcellular localization and enzyme activity of this protein,and conducted an overexpression study of FLO12 in Nipponbare.The main results are as follows:1.The rice flo12 mutant is obtained from the japonica rice cv.Dianjingyoul（DJY）by N-methyl-N-nitrosourea（MNU）mutation.The mature seeds of the flo12 mutant showed opaque and had floury white-core endosperm.Scanning electron microscopy showed that the starch granules in the middle part of flo12 endosperm were loosely arranged and irregular.Thousand grain weight of flo12 was about 88%of the wild type.Physiological and biochemical measurements of the seeds showed that the amylose content of the mutant was decreased and the protein content was increased,meanwhile the amylopectin chain length distribution and starch viscosity characteristics were also changed.These results indicated that FLO12 is important for the starch synthesis of kernel.2.Semi-thin section experiments showed that the wild type starch granules were closely arranged in the compound starch grains.However,most of the starch granules in the mutants were scattered,and some abnormal compound starch grains appeared,including compound starch grains with smaller starch granules inside and compound starch grains with abnormal morphological and less iodine staining.These results indicated that FLO12 plays a key role in the formation of normal compound starch grains in rice endosperm.3.The flo12 mutant was hybridized with an indica rice variety N22 to generate an F2 mapping population,and the flo12 locus was mapped to a 101.3 kb genomic region flanked by the markers Z-22and Z-39 on long arm of chromosome10.RGAP（http://rice.plantbiology.msu.edu/）predicted that there were 14 genes in this interval.Genome sequencing revealed that only a single base substitution from C to T occurred in the 11th exon of the Os10g0390500 gene,resulting in the replacement of the encoded amino acid by serine with phenylalanine.The results of transgene complementation,RNAi,and CRISPR/Cas9 knockouts all proved that Os10g0390500 is the target gene controlling the mutant phenotype.We named it FLO12（Floury endosperm12）.The FLO12 gene encodes OsAlaAT1,which consists of 483 amino acids and has an Aminotran_l2 domain,and in vitro activity assays indicate that the protein has transaminase activity.4.The results of real-time quantitative PCR showed that FLO12 was expressed in all tissues,but it was highest expressed in the developing endosperm.Western blot showed that the FLO12 protein level was continuously increased between 6-15 DAF（days after flowering）,and then gradually decreased.GUS staining results were consistent with qRT-PCR results.Subcellular localization results showed that FLO12 protein localized in the cytoplasm.5.The expression of genes related to starch synthesis in endosperm was detected by real-time quantitative PCR,and we found that the expression levels of most starch synthesis genes in the mutants were lower than in the wild type,including OsSSⅠ,OsSSⅡb,OsSSⅢa,OsSSⅢb,OsSSⅣa,OsAGPL1,OsAGPL4,OsISA3,OsBEI,OsPUL,OsPHOH and OsPPDKB.Western blot analysis showed that the protein content of AGPS2b,Pho1,SBEIIb,PPDKB,SBEI and SSIIa were reduced in the mutant seeds.In addition,zymogram analysis revealed that the Pho1 activity in the mutant was reduced.These results indicated that FLO12 can affect starch synthesis by regulating the expression of starch synthesis-related genes.6.OsAlaAT1 was drived by Ubi promoter and was over-expressed in Nipponbare.The results showed that the grain length and 1000-grain weight of the over-expressed plant seeds increased significantly,indicating the potential application value of OsAlaATl in rice breeding.