Map-based Cloning And Funtional Identification Of Leaf Color Gene Yellow-green Leaf-4 In Rice

Rice is one of the most important food crops in the world.Efficient photosynthesis is the guarantee of high yield of rice.The only organelle that can be used for photosynthesis is chloroplast.The map-based cloning and functional analysis of leaf color mutant play an important role in the study of chloroplast formation,function,chlorophyll anabolism process and so on.In this study,A new rice yellow-green leaf gene YGL-4 was cloned by the method of map-based cloning.This gene encodes a key enzyme-lumazine synthase(LS)in the riboflavin synthesis pathway.The enzyme catalyzes ARAPD and DHBP into DMRL in riboflavin biosynthesis.Although there are many studies on LS genes in various microorganisms and plants,the vast majority is related to the association with growth or disease resistance,and how LS gene's changes can change the phenotype of leaf color is not clear.In this study,the possible role of the LS gene affecting the phenotype of leaf color in plants was shown.The main results are as follows:1.The yellow-green leaf mutant ygl-4 and the indica rice variety N22 were crossed for the map-based cloning,and the F1 generations were obtained to get the F2 generation.In the F2 population,551 extreme individuals were selected according to the yellow-green leaf like phenotype,and the ygl-4 gene from the mutant was finally located in the 88.7 kb region.The mutations occurred on the LOC_Os04g42000 gene by a single nucleotide subsititute,resulting in a single amino acid change.The mutation site was located in a highly conserved region of the gene.The gene encodes a key enzyme in the riboflavin synthesis pathway,i.e.lumazine synthase(LS).The phenotype of leaf color on the mutant could be complemented by external riboflavin application.Subcellular localization of the YGL-4 gene showed the gene was located in the chloroplast.2.Transmission electron microscope showed the cell of mutant had more double membrane vesicles compared to wild type implying the cell was prematurely aged.The Quantitative RT-PCR analysis showed that expression levels of the genes from ALA to Pchlide pathway in the chlorophyll biosynthesis was up-regulated in the mutant.So,we speculated LS gene changed leaf color in rice might due to its abnormal expression,which resulted in the decreased riboflavin and ALA accumulation in the dark environment.Then,increased ALA would cause overproduction of photo-oxidation under light,which induced the cell age prematurely and leaf yellowish.To confirm this theory,the wild type and mutant were used to observe the autofluorescence reaction of Pchlide after 7 days of dark treatment.The results showed that the mutant had much more abnormal accumulation of Pchlide in the dark condition than the wild type.In addition,the ROS content in the leaf of the mutant was higher than that of the wild type after the plant moving to light after7 days of dark treatment.It can be reasonable that the basic pathway of LS gene controlling the change of leaf color is to reduce the riboflavin synthesis in plant thereby causeing the abnormal accumulation of ALA in the dark environment.