Gene Cloning And Functional Analysis Of WRS1 Involved In Rice Endosperm Development

Endosperm is the mainly edible part of rice,and its development status directly determines the yield and quality of rice.Starch is the main component of endosperm,its synthesis and accumulation directly affect rice yield,its amylose content and amylopectin fine structure affects the taste quality of rice.Therefore,it has great value to studying endosperm mutants with starch synthesis defection and exploring the relevant mechanisms affecting starch synthesis in rice.Aminoacyl-tRNA synthetases（aaRS）play an important role in the establishment of genetic code rules.aaRS complete the interpretation of the mRNA chain by assisting tRNA to bind the corresponding amino acids.Such ancient housekeeping proteins have developed diversified functions along with evolution.Biological processes such as gametogenesis and embryo development,early development of plastids,and perception of immune signals and disease defense,which aaRS involved in have been reported in plants.The research related to tryptophanyl-tRNA synthetase（WRS）in aaRS family has not been reported yet.In this study,a stable inherited rice floury endosperm mutant wrs1 was screened by ethyl methane sulfonate（EMS）mutagenesis of N22（Oryza sativa subsp.indica）.The phenotype of wrs1 was analyzed in detail,and the molecular mechanism of floury phenotype was preliminarily discussed.Results of our study are as follows:1.Compared to the wild type,the mutant wrs1 exhibits significant developmental lag and cannot develop into mature plants.The floury grains isolated from the heterozygous mutant（WRS1wrs1）showed shrunken abdomen,and the grain width and thousand-grain weight decreased.The total starch content of the grains decreased significantly,rice starch viscosity and breakdown values were lower than those of the wild type.Observation using semi-thin sections revealed that the compound starch granules of the endosperm of mutant wrs1 became smaller and the starch granules were loosely arranged.2.Select individuals similar with wrs1 mutant from the F2 population of wrs1/Dianjingyoul,using molecular markers to locate WRS1 in a 183 kb region on chromosome 12.Sequencing revealed a single base mutation in exon 6 of the gene Os12g0540900,resulting in the substitution of methionine at a conservative position.Complementation experiments verified that the mutant phenotype of wrs1 was caused by a mutation in gene Os12g0540900,which encodes a tryptophanyl-tRNA synthase.Combining phylogenetic tree analysis and homologous sequence alignment results,this protein is a conserved housekeeping protein widely existed in organisms.3.Combining the results of quantitative real-time PCR and GUS staining experiments,WRS1 was constitutively expressed in rice and higher in developing endosperm.The protein translation process in plants that aaRS involved in can be divided into three systemscytoplasm,mitochondria and chloroplasts--according to the cellular compartment.Through transient expression of rice protoplasts and fluorescence observation of root tip cells of WRS1-GFP fusion protein transgenic complementary families,WRS1 was confirmed to be a cytoplasmic localized protein.4.The mature seeds of wrs1 mutant have floury endosperm with shrunken abdomen which is an apparent starch synthesis defect phenotype.Quantitative RT-PCR was performed on key genes in the starch synthesis pathway.The expression levels of most genes were down-regulated,while the results of western blotting showed an inconsistent trend.The results showed that the WRS1 mutation indirectly caused the key enzymes in the starch synthesis pathway be affected to varying degrees.5.The formation of starch granules also depends on the overall efficiency of plastid and cellular functions.It was found that the protein content of wrs1 grains decreased significantly,and the free amino acid significantly increased.Therefore,the mutation of this gene affects expression of starch synthesis-related genes by influencing the amino acid homeostasis and protein synthesis,which finally causing the abnormal synthesis and accumulation of starch in rice endosperm.