Function Analysis And Application Research About Gene RH4 In Flavonoid Metabolism In Oryza Sativa

Abnormal color hull in rice is both an excellent genetic material for research on flavonoid biosynthesis and a favorable genetic tool for hybrid rice in mixed direct seedling.Therefore,cloning color hull gene is very important for theory analysis and application.A natural mutant rh4(red hull 4)with stable red hull phenotypes was taken as the study subject.The study analyzed the morphological and physiological characteristics of RH4.RH4 was cloned by map-based cloning with forward genetics,whose biological functions were researched through methods of genetics,biochemistry,bioinformatics in this study.The research identified the action site and control mode of RH4 in flavonoid biosynthesis and evaluated the potential of red hull resources employed in mixed planting breeding in hybrid rice.The main research results were as follows:(1)Flavonoids were significantly accumulated in the rh4 hull which were reddish brown from heading,but the glume base and apex of the lemma were normal color which showed yellow.The content of flavonoids and anthocyanin in rh4 was higher 2.2 and 1.81 times than WT,respectively.However,the chlorophyll content was decreased by 1.7 times.These results implicated that the red hull phenotype in rh4 may be associated with the metabolism of flavonoid and anthocyanin and chlorophyll.(2)LOC?Os12g10880 was found as the target gene of rh4 by map-based cloning.rh4 was controlled by a single recessive nuclear gene.RH4 was accurately located on chromosome 12.Genetic complementation restored the mutant phenotype,which confirmed that the red hull phenotype in rh4 was controlled by LOC?Os12g10880.(3)RH4 were localized in the nucleus and part of the cell membrane and expressed in multiple tissue.RH4 contained a transmembrane domain,an ?-helix and a signal peptide.RH4 was found to be localized in the nucleus and part of the cell membrane by subcellular localization.RH4 was expressed in roots,leaves and hulls by GUS staining.(4)RH4 regulated flavonoid biosynthesis.Transcriptome,proteome,phosphorylated modification and metabolome were focused on the flavonoids biosynthesis.In rh4,phenylalanine ammonia-lyase(PAL2,PAL4,PAL5,PAL7),coumaroyl-Co A ligase(4Cl5),chalcone synthase(CHS),anthocyanins synthase(ANS2),anthocyanin reductase(ANR2),flavonoids transporters(ABC reporter),glutathione transferase(GSTU1,GSTU4),UDP-glycosyl transferase(UDPGT3)were raised in transcriptional and translational levels,but chalcone synthesis isomerase(CHI)was lowered.Sixty-two differential metabolites were involved in the flavonoid biosynthesis,in which the contents of flavonols such as syriline-3-O-glucoside,3,4',7-trihydroxyl flavonoids and cosmosiin were significantly increased,suggesting that these substances may contribute to the upregulation of total flavonoids content and have a certain relationship with the pigment deposition in the glumes.(5)RH4 interacted with Os DSK2 a to regulate flavonoid biosynthesis.Six proteins interacting with RH4 were obtained by yeast two-hybrid(Y2H).Os DSK2 a,which interacts strongly with RH4,is involved in the ubiquitination degradation pathway.Bimolecular fluorescence complementation(Bi FC)and Pull down experiments confirmed the interaction between RH4 and Os DSK2 a,Os DSK2 a and Os CHS in vitro and in vivo.Os DSK2 a had the function of E3 ubiquitin ligase by in vitro ubiquitination test,and Os CHS was ubiquitinated by E1,E2(His-ATubc10),Osdsk2 a,His-Ub and ATP.Therefore,a model of RH4 involved in flavonoids was proposed: RH4 regulates the biosynthesis of flavonoids by mediating the interaction between Os DSK2 a and Os CHS.When RH4 was mutated,RH4 could not bind to Os DSK2 a,thus inhibiting the binding of Os DSK2 a to Os CHS.The accumulation and expression of CHS increased,and the metabolic flow changed,leading to the large amount of flavonols such as large cosmosiin accumulated in the glume,thus making the glume show reddisd-brown.(8)The resources controlled by RH4 have potential application in the seed production of hybrid rice by mixed direct sowing(MDS).In addition to the 1000-grain weight(TGW),the yield and traits of P88S/G-4 and Y58S/G-15 in MDS were significantly higher or lower than those of traditional seed production(SST).The actual yield(AYP)of the two combinations in MDS were also significantly higher than that of SST.The hybrids were successfully colour sorted at a correctness rate of more than 99.90% on a machine,which indicated that the hybrid purity of MDS was in line with relevant regulations.RH4 was suitable for mechanical mixed sowing seed production and had a broad application prospect.