Map-based Cloning And Functional Analysis Of Chalkiness Gene OsPK2 In Rice (Oryza Sativa L.)

Starch is the most abundant?60%-90%?element which supports energy in rice seeds.Starch content and structure have great significance for the formation of rice yield and quality.The biosynthesis of starch is a precised and complicated process.The overall network for starch biosynthesis mechanisms and specific details are still unclear.Therefore,it is important to explore,identify and analysis new factors that directly involve or indirectly affect the starch biosynthesis.In this study,we identified a chalky mutant namely ospk2 with obvious phenotypes from the Zhonghua11 mutant library used by EMS mutagenesis.Main results are as follows:1.The ospk2 mutant in this study showed a typical white-core phenotype;starch granules in the middle part of the endosperm became smooth and loosely packed with spherical or oval and smooth surface;compared with the wild-type,the ospk2 mutant showed a significant lower grain-filling rate during seed development;the total starch and amylose contents of mature ospk2 seeds were significantly decreased;and the protein and lipid contents were markedly increased;the ratio of the short and intermediate chains of amylopectin?DP6-12?were decreased,the intermediate and long chain?DP13-25?were elevated,and the fatty acid content was increased;the gel consistency was decreased significantly;mutant rice flour has the largest difference in the degree of dissolution swelling in the 4M urea solution.These results demonstrated that the OsPK2 gene has an important effects on rice endosperm starch.2.Transmission electron microscopy of early developing endosperm?3-9d?showed that the formation of compound starch granules and amyloplast development were abnormal in endosperm of ospk2 mutant.Both small amounts of starch granules which can normally expand to form normal developing amyloplasts and large quantities of small,individual,scattered grains of starch which cannot form compound starch granules were found in ospk2 endosperms.Starch grains loosely packed and the gap among the particles were distinct.Above results displayed that OsPK2 gene may play an important role in the normal formation of compound starch granules in endosperm cells during grain development.3.F₂ population was used for fine mapping which developed from cross between ospk2 mutant and NJ-11?indica varity?.The ospk2 locus was located in the short arm of chr7 with a 90.8kb genomic region between markers Ind18 and Ind20.Sequencing of gDNA of 9 ORFs in this region,a single base substitution?C became T?was found in the fourth exon of ORF4?LOC_Os07g08340?,resulting in the serine?S?of the 296^th amino acid changed to Leucine?L?.Functional complementation,overexpression,RNA interference,and knockout results demonstrated that LOC_Os07g08340 was the target gene responsible for the mutant phenotype which named OsPK2?Pyruvate Kinase 2?based on its putative biological function.The OsPK2 gene encodes a putative pyruvate kinase with 578 amino acids which contained two domains,PK and PK_C.4.OsPK2 was a constitutively expressed gene which expressed in roots,stems,leaves,panicles and endosperm at different developmental stages,and the expression level in ospk2 was lower than that in the wild-type.Results of qRT-PCR analysis,GUS tissue staining and western blot all confirmed the same results.5.Subcellular localization showed the OsPK2 was localizated to plastid?chloroplast?,and the first 1-97aa of Os PK2 was necessary for its chloroplast localization,and mutation in OsPK2 did not affect its protein localization.6.OsPK2 exhibited PK activity;the PK activity and pyr content in mutant?His-ospk2,10DAF endosperm and leaves?was significantly lower than the wild-type.7.Both yeast two-hybrid assay and BiFC assay confirmed that OsPK2 can interact with three other putative PKp to form a complex.8.Several genes related to multi-metabolic?such as glycolysis and gluconeogenesis pathways,fatty acid metabolism,and starch synthesis pathways?changed in ospk2.For instance,the expression of sucrose synthesis genes were significantly increased in the ospk2 mutant,while the transcription levels of most of the genes related to starch biosynthesis were significantly reduced.We speculated that mutation in OsPK2 may caused metabolic disordered.These indicated that the OsPK2 gene was important in the whole metabolic pathway.