Fine Mapping And Cloning Of Rice Yellow Green-revertible Mutant YGR121

Rice is one of the important food crops in China and even the world.Because of its soft and glutinous taste and rich nutrition,it is loved by the people of the world.In the rice production process,breeding high-efficiency leaf color marker varieties has application value.Therefore,in-depth exploration of the photosynthetic regulation mechanism of rice leaf color mutants has important research value.In this study,the natural mutant ygr121 of rice yellowing to green leaf color was used as the experimental material to study the differences between the wild type and wild type in agronomic traits,physiological characteristics,photosynthetic characteristics,photosynthetic pigments and chloroplast ultrastructure.Its leaf color mutant genes were genetically analyzed,finely mapped and cloned.The main findings are as follows:（1）The phenotype of mutant ygr121 before the heading stage of rice,the newly yellowed leaves of the plant gradually turned green from the base to the tip of the leaf;until the heading stage,the leaf color of the plant basically turned green,and the plant was able to grow normally development.The yield composition of mutant ygr121 was not significantly different from that of wild type.In the seedling stage and booting stage,the chlorophyll content of mutant ygr121 was significantly lower than that of wild type.In mutant ygr121,the structure of the lamella granules in the chloroplast was rough and disordered,and the number of lamella membranes was reduced.The number of lamellas in the inner vesicles also decreased,and the starch granules almost disappeared.（2）Compared with the wild type,the antioxidant enzyme activity and malondialdehyde content of the inverted second leaf and inverted fourth leaf of the mutant ygr121 seedling stage were significantly higher than that of the wild type,only the peroxidase（POD）activity was significantly higher than that of the wild type,superoxide disproportionation The enzyme（SOD）,catalase（CAT）activity and malondialdehyde（MDA）content were not significantly different from the wild type.（3）Analysis of the photosynthetic capacity of mutant ygr121 showed that the net photosynthetic rate at booting stage and heading stage were significantly lower than that of wild-type.Later,as the leaf color turned green,the net photosynthetic rate at heading stage was higher than that at booting stage.By measuring the chlorophyll fluorescence parameters of the tip of the inverted first leaf and inverted second leaf of the mutant ygr121 and the wild-type tillering period,the original photochemical quantum efficiency（fv/fm）of the inverted leaf tip of the mutant ygr121 was significantly lower than that of the relatively mature 3 Group of leaves,and the initial fluorescence（F₀）,photochemical quenching coefficient（q P）and actual photochemical quantum efficiency（ФPSII）are significantly higher than those of the other three groups;the non-photochemical quenching of the inverted one leaf and inverted two leaves of the mutant ygr121 The coefficients（NPQ）were significantly lower than the wild type.（4）Mutant ygr121 and wild-type canopy reflectance change curves at the tillering and heading stages are basically the same,and the reflectance curve has a clear change rule,in the"red edge"region 650-750 nm range,the reflectance increases with wavelength rising rapidly,the steeper curve is equivalent to a straight line.The canopy reflection spectrum of rice at heading stage has a distinct peak between 650-750 nm in the"red edge"region.The correlation coefficient of the chlorophyll content of the mutant ygr121 and the wild type is basically consistent with the canopy spectral reflectance,and the correlation coefficients tend to decrease first and then increase and then decrease.In the range of 450-750 nm,the correlation coefficients of the two varieties are quite different,and the correlation coefficients of the two varieties are extremely significant.Within the scope of the wavelength of visible light is less than 650 nm,the mutant ygr121 correlation coefficients above 0.5,the correlation coefficient of wild type under 0.3,the correlation coefficient between the two varieties were very significant differences.（5）The inheritance method of mutant ygr121 is recessive inheritance of single nuclear gene.Through fine mapping analysis,the YGR121 gene was located in the 0.2 c M range between molecular markers WY67 and WY80 on chromosome 1.In-depth research found that LOC＿Os01g17170 is a candidate gene,and the 515th base in the CDS sequence of this gene changed from C to T,thus speculating that the 172nd amino acid of its protein sequence changed from serine to phenylalanine,and predicted its function as Mg protoporphyrin IX monomethyl alicyclic enzyme;expression analysis of the mutant gene YGR121,it was found that its expression level in leaves or ears was significantly lower than that of wild type.（6）The target gene was connected to the p ROX-HA vector,and a YGR121 gene overexpression vector was successfully constructed and introduced into the recipient Nipponbare;through the PYLsg RNA-U6a vector and p YLCRISPR/Cas9-Pubi-H plant binary expression vector,it will carry YGR121 A target of the gene and the sg RNA expression cassette were successfully constructed and verified by sequencing to successfully construct the CRISPR/Cas9 vector of YGR121 gene.