Identification And Gene Mapping Of A Yellow-color Rice Mutant Ys94

The chloroplasts of green plants can fix CO2 and transform them into organic substances by photosynthesis. In chloroplasts, Chlorophyll and Lutein are the most abundant pigments and play very important functions in plant chloroplast development and photosynthesis. Mature chloroplasts are developed from the pro-plastids under the co-regulation of both nuclear and chloroplast genes, among them the nuclear genes play a major role. Leaf-color mutants are the ideal material to explore the mechanisms of chlorophyll biosynthesis and chloroplast development. Chlorophyll content and chloroplast development are directly related to the efficiency of plant photosynthesis. So, understanding the mechanism of chloroplast development and pigment synthesis is of great significance to improve the yield of crops. The material used in this study is a yellow-leaf mutant obtained by radiation. We isolated the target gene by map-based cloning and conducted the functional studies on it. Main results as follows: 1. The phenotype analysis of yellow-leaf mutant ys94A yellow-leaf mutant ys94 was isolated by chemical mutagenesis. Compared with wild type cultivars, ys94 mutants display yellow leaf traits in the germination, and both its Chlorophyll a and chlorophyll b decreased significantly. The result of electron microscopic observation revealed that numbers of chloroplast in the ys94 were reduced significantly while there were many single chloroplast dysplasia and degenerated thylakoid lamella. Confocal microscopic analysis of the leaves of wild type and ys94 mutant showed that the number of lighting cells decreased significantly, showing the development of chloroplast was abnormal in mutant ys94. These results are consistent with the yellow-leaf phenotype and reduced chlorophyll content in ys94 mutant. 2. Mapping of the target gene in mutant ys94The phenotype of the ys94 mutant is regulated by a recessive nuclear gene. The target gene was mapped to a 140 kb interval on the short arm of rice chromosome 8 via map-based cloning method. We found that there are 11 predicted genes in the 140 kb interval and there is a nucleotide substitution(T to A) at 490 bp locus apart from the translation initiation site in CDS of the LOC_Os08g12840 gene, which resulted in an amino acid substitution(W163R) in the predicted protein. The gene(LOC_Os08g12840) may affect the synthesis of fatty acids and has not been reported by now in rice, our results of this study laid a foundation for future cloning and functional analysis of the gene. 3. Subcellular localization of the protein encoded by the target geneBy transient expression of target genes in rice protoplasts, we found that both in wild-type and mutant protoplasts, the target gene is located in the chloroplast, suggests that the target gene is functionally related to chloroplast development. 4. Expression analysis of target gene and genes associated with Chloroplast development and photosynthesisBy quantitative RT-PCR(qRT-PCR), we found that the target gene expressed in various tissues and organs with a highest level in young leaves. At normal temperature treatment, qRT-PCR analysis of the leaf of the mutant at its third leaf stage showed that the expressions of genes associated with pigment metabolism and photosynthesis in ys94 were downregulated. The reduce of chlorophyll content in mutant ys94 may be due to the expression disorder of related genes in chlorophyll synthetic pathways and photosynthesis system. The reduction or lack of protein in photosynthetic system probably result in the defects of chloroplast developmental and the failure of photosynthesis system assembly.