Map-based Cloning And Functional Analysis Of A Starch Synthesis Gene FLO16 In Rice Endosperm

Over these years,with the efforts of breeding workers,the yield of cultivated rice in China has increased significantly through the utilization of heterosis and dwarf breeding.However,with the improvement of people's living standards,the requirement of rice quality has also been raised.However,at present,rice quality is limited in Chinese rice production.Therefore,improving quality is currently a very important task.The composition of amylose and amylopectin in rice seeds affects the taste quality.Therefore,it is important to study the synthesis and regulation mechanism of starch in rice.At the same time,starch accumulation in rice seeds often determines grain size.Weight,therefore,is directly related to the yield of rice.Further studies of its developmental and genetic mechanisms are important for improving rice quality and increasing rice yield.The opaque-kernel mutant phenotypes indicate changes in storage metabolites,varying starch content and structure,aberrant SGs,and other abnormalities.Malate participates in the transport of redox equivalents among cell compartments(Kromer and Scheibe,1996;Scheibe,2004).However,the role that malate metabolism plays in starch synthesis in rice endosperm remains unknown.In this study,a floury mutant,flo16,with a deletion of 4 base pairs in the Cytosolic Malate Dehydrogenase gene was characterized.Decreased starch and elevated sucrose levels revealed that the transition from sucrose to starch was disrupted during grain filling in the flo16 mutant.Over-expression of FLO16 led to increased grain weight.Our results demonstrated that FLO16 is essential for starch biosynthesis in rice.1.In this study,a novel mutant flo16 was isolated from the N22 mutant library induced by 60Co irradiation and identified.The flol6 mutant had a floury endosperm and the filling rate during endosperm development was significantly lower than wild type.At the same time,the starch content in flol6 was decreased,and its starch viscosity characteristics,gel characteristics and chain length distribution of amylopectin were changed compared with the wild type.It was supposed that the physicochemical properties of starch accumulated in flo16 changed.These results suggest that the FLO16 gene has an important effect on starch biosynthesis in rice endosperm.2.Observations of developing endosperm showed that flol6 mutant endosperm has a large number of scattered starch granules and smaller sized composite starch granules compared to wild type.These results indicate that FLO16 is necessary for the formation of normal compound starch grains in rice endosperm.3.The FL016 gene was mapped to the 88 kb region between markers 188-21 and 188-2 on the long arm of chromosome 10 using the F2 population of the cross between flo16 and DJY A four base pair deletion in the coding region of Os10g0478200,results in a reading frame shift and a premature stop codon.Transgenic complementation experiments showed that Os10g0478200 restored the phenotype of flo16 endosperm.The base deletion of Os10g0478200 is responsible for flo16.Subcellular localization and zymogram analysis showed that Os10g0478200 encodes a cytoplasmic malate dehydrogenase(CMDH)consisting of 332 amino acids.4.As expected,a protein band was not detected in the flo16 mutant,suggesting that flo16 was a knockout mutant of CMDH.The total MDH activity in flo16 endosperm at 9-12 DAF was less than one-half of that in the.wild type.We evaluated the levels of closely related metabolites.There was an increase in malate levels in young leaves and roots relative to wild type,and levels in grains peaked at 12 DAF.The lack of CMDH activity might result in alterations in substrates and products of malate metabolism and a decrease of starch synthesis in flo16.5.FL016 overexpressing plants showed a significant increase in grain weight and length,and these changes were consistent with gene expression levels,suggesting that overexpression of this gene could increase grain weight.Overexpression of FL016 increased rice grain weight,indicating that optimizing FLO16 expression can be used to increase rice yield.These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.