Genetic Analysis Of The Less-tillering Mutant 6635 And Gene Mapping Of The Yellow-leaf Mutant 505ys In Rice

1. Genetic analysis and gene mapping of the less-tillering mutant 6635 in ricePlant type is closely related to its production. As the unique branching manner, tillering is one of the important constituent elements of plant type. The number of effective panicles is determined by the number of tillering which also affects the yield per unit area. Therefore, the study of tillering is very important for the selecting and breeding the ideal plant and increasing the yield. Rice tillering is the model system for studying the branching of monocots. The research of tillering related genes will not only contribute to elucidating the molecular mechanism of rice tillering, but also improving the rice yield.In this study, by using chemical mutagen EMS to treat rice varieties. Zhonghua 11, we obtained a mutant with stable heritabilities,6635. In order to further understand the causes of the mutant phenotype, we did the following research:By observing the phenotypic characteristics, we found that the number of tillering in 6635 was significantly less than that in wild-type, while there was no significant difference in plant height between 6635 and the wild-type. Genetic analysis suggested that the less-tillering trait of the 6635 mutant was caused by a recessive mutation in a nuclear gene. Mapping was performed with F2 population derived from the cross between 6635 and G46B by using SSR and In/Del markers. The mutant gene was mapped to a region of 5198kb between two In/Del markers:H4 and H5, on the short arm of chromosome 4 of rice, with the genetic distance of 0.53cM and 1.21cM, respectively.2. Map-based cloning and functional analysis of the yellow-leaf mutant 505ys in riceLeaf color mutation is one of the common mutations, which is easy to identify. The mutation of leaf color leads to the anomalies of metabolism of plant photosynthetic pigment in the plants, thus affecting the photosynthesis. The rice leaf color mutant can be used as an important experimental material to study the chlorophyll synthesis and degradation pathways and chloroplast morphology and development and color markers in hybrid rice breeding. Isoprenoids constitute a functionally and structurally diverse family of organic compounds, terpene compounds, and they composite the most abundant secondary metabolite in plants. As one of the biosynthesis pathways of terpene compounds, the MEP pathway plays a very important role in plant growth and development. The study of the key enzymes in this pathway in rice is helpful to elaborate the molecular mechanism.In this study, we obtained a mutant with stable heritabilities,505ys, from japonica cultivarNipponbare through EMS mutagenesis. Based on the previous studies on gene mapping of the mutant, we did the following research:(l)The morphological and agronomic traits of mutant 505ys:the yellow-leaf phenomenon of the mutant existed in the whole growth period. The data of investigating the main agronomic traits showed that the plant height, the average number of panicles per ear of plant and the number of spikelets per panicle all declined compared to the wild-type. The seed setting rate and the thousand grains weight decreased significantly, down 6.5% and 9.8%, respectively.(2)Measuring the photosynthetic pigments in mutant 505ys:We measured the amount of chlorophyll a, chlorophyll b and carotenoids in the seedling stage and heading stage, the results showed that the pigment contents of the mutant were significantly lower than that of the wild type at both stages and the total pigment contents were 1.78 and 1.36 mg/g, decreased by 29.92% and 41.63% compared to the wild-type, respectively.(3)Analysis by Transmission electron microscopy (TEM):By observing the leaf of the same part of mutant 505ys and wild-type Nipponbare at the period of 4 weeks by TEM, we found that the the mesophyll cell morphology of the mutant 505ys had great differences compared with the wild type. The chloroplast had no obvious grana lamella stacks. Thylakoid membrane structure was disorder and appeared many vesicles.(4)Analysis and map-based cloning of the predicted gene:We had located the 505ys gene in the interval 182kb on the long arm of chromosome 2. Screening the 24 candidate genes in the interval, we found that a gene encoding a protein involved in the MEP pathway. DNA sequencing results indicated a single nucleotide C-to-T substitution at position 629 of its DNA (correspond with the 508 of its cDNA) in 505ys mutant which caused an amino acid change from L to F. For this reason, we identified that the mutant of the gene caused the yellow-green phenotypes of 505ys mutant, which was named 505YS, provisionally.(5)Genetic complementation experiments:We constructed the transgenic expression vector, and then transferred the resulting plasmid, pC2300-actin-505YS, into the 505ys mutant mediated by Agrobacterium. The phenotype and pigment contents of the transgenic plants (TP) turned to be normal. At the same time, we transferred the empty vector pCAMBIA2300-actin into 505ys as contrast. The transgenic plants (CK) showed the same phenotype and pigment contents with the mutant. The results suggested that the yellow-green phenotypes of 505ys mutant was caused by the mutant of the gene 505 YS.(6)Expression analysis of the gene 505YS:We analyzed the expression of gene 505 YS in different organs at seedling stage and booting stage by RT-PCR. The result showed that the expression of gene 505YS was all detected in roots, leaves, stems, leaf sheaths and young panicles and the expression in leaves was slightly higher than that in other organs.(7)Subcellular localization:We constructed the expressing vector of 505YS-eGFP fusion protein, and the resulting construct and the empty 35S-eGFP vector (as a control) were transformed into rice protoplasts mediated by PEG, respectively. By using a laser-scanning confocal microscopy to examine, the result showed the fusion protein was clearly co-localized with chlorophyll autofluorescence.