Map-based Cloning And Functional Study Of White-belly Endosperm Mutant Wbe1 And White-stripe Leaf Mutant Wsl3 In Rice

Rice storage protein is the second largest nutrients in rice seed, and it is generally believed that the protein content and quality of rice is negatively correlated, therefore, the study of protein content in rice has important theoretical and practical value. In the first part,wbel (white-belly endosperm 1) mutant was the research material, and storage protein content of each component in the belly endosperm of mutant was changed by SDS-PAGE gel electrophoresis analysis. Transmission electron microscopy (TEM) analysis revealed that there were a large number of protein aggregates in the endoplasmic reticulum of the mutant abdominal endosperm cells, and amyloplast development had also been affected significantly. In addition, quantitative RT-PCR analysis showed that the expression of endoplasmic reticulum stress related genes were upregulated in the mutant endosperm.Therefore, we supposed that the mutation of WBE1 caused the accumulation of storage proteins in the endoplasmic reticulum, and produced an ER stress, resulting in the fromation of white belly. Results from our experiments are summarized as follows:1. wbel mutant had a large and stable chalkness in abdominal endosperm, and its protein content decreased. SDS-PAGE gel electrophoresis analysis revealed that compared with the wild type, wbel mutant accumulated higher levels of proglutelins, accompanied by lower contents of mature glutelin acidic and basic subunits, and the contants of globulin and prolamin was also decreasd in mutant. Western blot analysis revealed that the contents of GluA, GluB, globulin, Pro 13a, and Pro 16k were decreasd. Transmission electron microscopy (TEM) analysis revealed that there were a large number of protein aggregates in the endoplasmic reticulum of the mutant abdominal endosperm cells, and there was less storage protein contained in PSV (Protein Storage Vacuole) of mutant than wild type.2. Semi-thin sections of mutant endosperm amyloplasts in different deleloping days showed that there were abnormal amyloplasts and a large number of single starch granule in the aleurone cells. These results indicate that WBE1 can influence the development of amyloplasts in abdominal endosperm.3. The WBE1 gene was mapped to a 158 kb genomic region on the short arm of chromosome 3 by map-based cloning. Gene prediction showed that this region contains 17 open reading frames. Sequence alignments between wild-type and wbel showed that there were 19bp deletion in the junction of fourth exon and fourth intron of ORF13(LOC_Os03g24460), and resulted in premature translation termination. Complementation tests confirmed that the LOC_Os03g24460 is the target gene. wbel was identified to encode a protein that contains conservative transaminase structure domain and may be involved in the synthesis or metabolic of branched-chain amino. Quantitative RT-PCR results showed that WBE1 was expressed in roots, stems, leaves, leaf sheaths, panicles and different developmental stages of endosperm.4. Subcellular localization in rice protoplasts showed that WBE1 protein was localized in the endoplasmic reticulum. The relative expression level of genes related to endoplasmic reticulum stress by quantitative RT-PCR revealed that there was a severe endoplasmic reticulum stress in the endosperm of the mutant and caused PCD (Programmed Cell Death)in endosperm cells.Chlorophyll is the main photoreceptor pigment, and chloroplast is the main place of photosynthesis. The obstruction of chlorophyll synthesis, metabolism or chloroplast development can cause color variation of rice leaf. Thus leaf color mutants are ideal materials to study the molecular mechanisms of chlorophyll biosynthesis and chloroplast development. In the second part, wsl3 (white-stripe leaf 3) mutant was the research material.we isolated the target WSL3 gene by map-based cloning, and confirmed this result by complementation and RNA-interfernce tests. Results from our experiments are summarized as follows:1.White striped phenotype of wsl3 mutant will appear at the young leaves during seedling stage and flag leaf and leaf sheath at heading stage. The wsl3 gene was mapped to a 50.8 kb genomic region on the short arm of chromosome 1 by map-based cloning. Gene prediction showed that this region contains 9 open reading frames. Sequence alignments between wild-type and wsl3 showed that there were 9bp deletion in the junction of 15th exon and 15th intron of ORF5 (LOC_Os01g01920), and resulted in the deletion of 9 amino acids in mutant WSL3 protein. Then we confirmed this result by complementation and RNA-interfemce tests. WSL3 was identified to encode a protein that contains HD domain,this family protein may have phosphatase activity and participated in nucleic acid metabolism and signal transduction.2. Transmission electron microscopy (TEM) analysis revealed that the mutant contains two kinds of defected chloroplasts, one had a loose chloroplast lamellae structure, and the other did not form a chloroplast lamellae structure. The relative expression level of genes related to chloropphyll biosynthesis by quantitative RT-PCR revealed that the expression levels of the most genes related to chlorophyll synthesis in mutant are downregulated.Therefore, the wsl3 mutation not only affects the chloroplast development, but also the synthesis of chlorophyll, eventually leading to the white stripe leaf.