Map-based Cloning Of Leaf Albino Gene BH6 In Rice

Rice is not only one of the most important food crops in the world,but also an important mode of Gramineae crops.Rice has important practical and scientific value.Leaf is not only an important place for rice photosynthesis,but also an important factor which constitutes ideotype.Through positional cloning and functional analysis of genes related to the leaf color both can be used for high-efficiency breeding and the research on physiological and biochemical mechanisms of photosynthesis to increase production.Aminoacyl-tRNA synthetases are key enzymes involved in protein synthesis catalytic process and ensure the accuracy of genetic information in mRNA is reflected in the amino acid sequence of the protein.Moreover,it also has an important role in the regulation of rRNA synthesis and involved in the expression of genes.Currently,the aminoacyl-tRNA synthetase is how to participate in the synthesis of chlorophyll and the mechanism of photosynthesis is not clear.In this study the albino lethal gene BH6 encoding such a class of glycyl-tRNA synthetase and we get the following results through map-based cloning and phenotype analysis:1.The albino lethal bh6 mutant in this study is from Japonica varieties Ningjing 4 chemical mutagenesis.The mutant showed stable albino in the field and light incubator,and die due to unable to turn green in the late stage of growth.This phenotype does not change with changes in temperature.Chlorophyll content and chlorophyll autofluorescence both proved there are obvious differences between wild type and mutant2.We observed the chloroplasts ultrastructure in leaves using electronic transmission microscope,and obvious structural defects was showed in mutant chloroplasts,like abnormal thylakoid membrane structures and more osmiophilic particles,along with the loss of photosynthetic capacity.3.In this study we observed that heterozygous normal seedlings and albino seedlings segregation ratio conform with 3:1 through genetic analysis of BH6,and indicated bh6 was controlled by a recessive nuclear gene.Then we choose heterozygous plants as the female parent and N22 as male parent.We narrowed to a region of 266kb at the marker Bh6-12 to the top of chromosome 6 using 318 albino seedlings of F2 population using map-based cloning.We predicted ORF4 and ORF15 related to chloroplast early development according to the annotation by RiceXPro.Finally we predicted candidate genes Os06g0103600 by sequencing.There is a single base mutation occurred in the sixth exon of this gene.CrispR knockout expression of candidate genes of wild type also showed albino phenotype same as bh6,suggesting that the mutation in Os06g0103600 is the cause of leaf albino.4.We hypothesized that this gene encodes a glycyl-tRNA synthetase through protein sequence analysis and phylogenetic analysis.This enzyme contains four domains whereas point mutation is located in the most conserved a core domain among species and these changes may lead to the loss of enzyme activity.Phylogenetic analysis indicating that this protein widely found in higher plants and it has higher homology.The above results demonstrate this gene is highly conserved in evolution.5.Finally we examined the effect of gene mutation in interaction of chloroplast development regulatory networks.The quantitative analysis of genes related to chloroplast development and chlorophyll synthesis indicates that genes in regulatory networks are affected in different degree.The research of aminoacyl-tRNA synthetase remains unclear in rice.Our results will prelimarily clarify glycyl-tRNA synthetase mechanism in chloroplast development process.We hope to provide some evidence and clues for further work on these enzymes in rice.