Map-based Cloning Of The Gry79 Mutant Gene Controlling Green-revertible Yellow Leaf In Rice

Rice (Oryza sativa L.) is not only an important cereal but also a model plant of monocotyledon in gene function research. The leaf color mutants is a common mutantion of rice, it played an important part in the basic study of rice, such as photosynthesis, biosynthesis of chlorophyll and genetic differentiation and development of chloroplast and so on, and it was also served as a marker character in breeding study. In order to understanding the inheritance of green-revertible yellow mutant 79 (gry79) and analysising the function of the gene, the leaf color character, some important agricultural characteristics, and ultrastructure of chloroplasts in the gry79 mutant and the positional cloning of mutant genes were studied.The main reults were as following.The gry79 mutant was isolated from ethyl methanesulfonate mutagenesis. The mutant developed yellow-green leaves before the three-leaf stage, but gradually turned green from the four-leaf stage onward and appeared normal green like the wild type plants after the six-leaf stage. Although the gry79 mutant grew slowly at early seedling stage, its days to heading increased only 1.4 d. At the maturity stage, except number of productive panicles per plant reduced by 15.2%, its plant height, number of spikelets per panicle, seed setting rate and 1000-grain weight were not affected remarkably. The contents of Chla, Chl b, total Chls and carotenoids decreased by 76.4%,76.9%,76.5%and 58.1%compared with the wildtype at the third -leaf stage, respectively. Transmission electron microscopy analysis revealed that, the chloroplasts displayed well-developed membrane systems composed of grana connected by stroma lamellae In the third-leaf of wild type. However, the grana stacks in the gry79 mutant appeared less dense and lacked well-structured thylakoid membranes. In addition, the mutant chloroplasts contain fewer starch granules compared to wild-type chloroplasts. These results indicated that chloroplast development of the gry79 mutant was suppressed in the first few leaves.Genetic analysis suggested that the yellow leaf phenotype of gry79 was controlled by a single recessive nuclear gene. Utilizing the SSR primers and developed insertion/deletion (InDel) markers, the gry 79 locus was narrowed down to a 71-kb interval between the InDel markers W4 and W5. Within the W4 and W5 region, eight hypothetical genes had been annotated in Rice Genome Annotation Project. We sequenced the eight genes in the gry79 mutant and its wild-type parent, respectively. DNA sequencing results revealed that a genetic mutation occurred only in the Os02g33610 gene, whereas no mutation existed in the other seven genes in the gry79 mutant. The Os02g33610 gene is comprised of 18 exons and 17 introns, whose total length of genomic and cDNA sequences are 7492 bp and 2616 bp, respectively. In the gry79 mutant, a single nucleotide C-to-T substitution occurred in exon 11 of the gene (at position 4209 of its coding region). Next, its cDNA sequence was amplified by reverse transcription (RT)-PCR, and the C-to-T substitution at codon 1160 (corresponding to position 4209 in the coding region) was confirmed in the mutant, which resulted in an amino acid change from Ser-387 to Phe in the encoded protein. Thus, Os02g33610 gene was considered as the candidate gene of gry79, and designated tentatively as GRY79 gene.Searching in the rice genome database revealed that GRY79 is a single-copy gene encoding a protein with a molecular mass of approximately 96 kDa. The GRY79 protein contains an apparent chloroplast-targeting sequence of 36 amino acid residues at its N terminus. Meanwhile, the protein possesses a putative metallo-β-lactamase domain of 202 amino acid residues at its N terminus and a putative trihelix DNA binding domain of 66 amino acid residues at its C terminus, respectively. Multiple amino acid sequence alignment showed that GRY79 has high similarity to sorghum (Sorghum bicolor) and foxtail millet (Setaria italic) hypothetical proteins. In addition, the phylogenetic relationship between GRY79 and its related proteins from other species was analyzed. The result suggested that the rice GRY79 protein is more closely related to the homologues proteins of monocotyledonous plants sorghum and foxtail millet than to those of other species.Genetic complementation experiments were carried out. As result, we successfully obtained 22 independent positive transgenic lines which all showed normal green leaf phenotype at early seedling stage as the wild type did and the Chl and carotenoid contents of the transgenic lines were restored to levels of the wild type, suggesting that the gry79 phenotype was rescued by transformation with the wild-type Os02g33610 gene. Therefore, we concluded that we succeeded to clone GRY79 gene in rice, and that the gry79 mutant resulted from a single base pair mutation in Os02g33610 gene.Quantitative RT-PCR results showed GRY79 expression levels in gry79 at the two and three-leaf stages were significantly lower than those in the wild type; subsequently, the expression in gry79 gradually increased, and eventually closed to the level in the wild type at the nine-leaf stage. The data suggested that the expression level of GRY79 gene significantly decreased in the first few leaves of gry79, which is consistent with the seedling-stage-specific yellow leaf phenotype of the mutant.