Identification And Gene Cloning Of Two Leaf Color Related Mutants In Rice

Leaf is an important organ for photosynthesis in plant, variation of leaf will directly affect the photosynthetic rate and the accumulation of organic matter, thus affecting growth and development of plant. Characterisation of the rice leaf color related mutants provides not only important materials for studying the synthesis, regulation and degradation of chlorophyll as well as the development of chloroplast and the chloroplast-nucleus signaling way, but also the specific germplasm resources for rice breeding. In this study, two leaf color related mutants, lzs and swp were selected from the rice transgenic offspring of ZH-11 and the experiments for the phenotypic identification, gene cloning and biological function analyses of the mutants were conducted. The main results were summarized as follows:Ⅰ:The lethal zebra seedling mutant lzs in rice1. From the two-leaf stage, the new leaves of the mutant lzs were turned from yellow to white, and the contents of chlorophyll a, chlorophyll b and carotenoid were decreased. At the three-leaf stage, the maximal photochemical efficiency of PSII of the mutant leaves were significantly lower than the wild type, the relative electron transport rate and the actual photochemical efficiency were nearly 0, the mutant almost no photochemical activity so that gradually die. Compared to the seedling stage of wild type, the soluble protein of mutant was significantly lower, about 20% of the wild type, while the activities of antioxidant enzymes of POD, SOD, CAT and the content of MDA were significantly increased,1.2 times,4.4 times,2 times and 6.8 times of the ones of the wild type, respectively.2. Genetic analysis indicated that the mutant lzs was controlled by a recessive gene, which was located on the chromosome 3 between the markers RM15857 and RM15887 with a genetic distance of 1.6 cM. The candidate region was then delimited to 49 Kb between the markers RH3-17 and RH3-21 which were on two overlapping BACs by using 16 new developed molecular markers. There were 7 open reading frames in the candidate region, among which there was a gene encoded the serine hydroxymethyl transferase 1, the sequence analysis showed that there was a mutation in the fourteenth exon (T-C), resulted in a new restriction site AflⅡand the amino acid alteration from serine to leucine.3. The candidate 6.8Kb DNA fragment of LZS including the promoter region, 3'non-translated region, integrity length of ORF and 5'non-translated region was selected as a complementary fragment to construct the complementary vector. The agrobacterium-mediated transformation was performed on the heterozygous mutants, and 10 transgenic plants with homozygous mutant background were identified based on the selection of the pre-designed dCAPS markers. The phenotypes of 10 transgenic plants were fully complemented to the wild type. This showed that the gene caused spotted leaves'mutation phenotype was the OsSHMl.4. The sequence analysis revealed that the ORF with the length of 1545 bp, encoded 514 amino acids, had a very high homology with the Arabidopsis AtSHM1. From RT-PCR, it was detected that OsLZS gene were expressed in roots, stems, leaves and flowers, specifically high expression was observed in leaves.Ⅱ:The heading stage with white stripes and seedling stage with white leaves mutant swp in rice.1. Under natural condition, the mutant swp showed all white at the one-leaf stage, and the new leaves were gradually turned to green from tip along midrib at the two-leaf stage. Before the heading stage, leaves were become completely green, after that, the inside and outside palea were always white, but the rachis and small branches were green. The plant height and tiller number of the mutant swp showed significantly reduced, approximately 88.8% and 81% of the wild type plant, respectively. Seed rates were also significantly lower, only 32.9% of the wild-type plants. The grain weight was about 85% of the wild type. 2. At the second-leaf stage, the chlorophyll a, chlorophyll b and carotenoid content of the mutant swp were significantly lower than the wild type, but increased with the gradual growth and development of the plant. At the flowering stage, the chlorophyll content and carotenoid content of leaves showed no significant differences compared to the wild type, respectively, but the significantly lower contents were observed in the hull than that of the wild type. Although the chlorophyll content and carotenoid content of leaves of mutant and wild type had no significant difference at the flowering stage, the photosynthetic rate, stomatal conductance and transpiration rate of mutant were significantly enhanced compared to the wild type.3. Genetic analysis indicated that the mutant swp was controlled by a recessive gene, which was firstly located on the chromosome 3 between the markers RM5711 and RM6574, and the genetic distance was 14.4 cM. The candidate region was then delimited to a region of 87 Kb between the markers STS 7-63 and STS 7-65 which are on two overlapping BACs (P0428D12) by using the developed 48 pairs molecular markers. At the end, the candidate region was finally delimited to 31 Kb region by using 5 new developed CAPS markers, in which only the gene encoded the valyl-tRNA synthetase, was detected.