| Rice is one of the four major food crops in China, of which more than 50 percent of people live on rice. Photosynthesis is one of the most important factors affecting the yield of rice. Chlorophyll which fuctions in photosynthesis, is an important pigment in plant chloroplasts. Chlorophyll, not only turns arganism green, but also plays a key role in light energy absortion, transimission and convertion. Rice leaf color mutation is relatively common, and the mutated genes do often directly or indirectly affect chlorophyll synthesis and degradation, thus affecting the photosynthesis of rice leaves, cutting or even causing plant to death. Recent years, numerous studies have shown that, leaf color mutant plants were ideal material for the understanding of structure and function of genetic regulation mechanism of chloroplast develop ment, photosynthesis mechanism, and crop marks and other characters of the study.In this study, a new rice mutant was identified with stable inheritance tentatively named as yellow-green leaf 13(ygl13), which was derived from the progeny of ethyl methane sulfonate(EMS)-treated restorer line Jinhui 10(Oryza sativa). Morphological characteristics, the photosynthetic pigment contents and the agronomic traits were measured systematically. Transmission electron microscopy was conducted to analyze the ultrastructure of the mesophyll cells and chloroplasts of the ygl13 mutant and wide-type plants. The ygl13 was crossed with indica sterile line Xinong1 A whose plant and leaves were normally green, F1 and F2 were used for genetic analysis and gene mapping. The putative genes in the fine mapped region were analyzed, and the candidate genes were sequenced. The main resoults are as follows : 1. Morphological analysisMutant ygl13(yellow-green leaf 13) displayed yellow- green leaf color compared with the wild type. This phenotype was kept until the maturity stage. And the photosynthetic pigment contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid were decreased significantly at the seedling and the booting stages, the decreaseing level was 41.9%, 43.8%, 42.3% and 33.5%, respectively at the seedling, and 33.3% ~ 41.6%, 42.5% ~ 46.8%, 35.4% ~ 43.5% and 30.8%-41.6%, respectively at the booting stages, in comparision with the wide type. 2. Ultra- microstructure observationThe results from transmission electron microscope demonstrated that there were no significant difference in cell structure of the chloroplast between the ygl13 and the the wide type. But the structure o f the chloroplast developed abnormally with poor thylakoids, less grana stacks and scattered distribution while the the chloroplast of wide type exhibited normal. 3. Investigation of agronomic traitsThe resoult of the mleaf length and width of the flag leaf, the second leaf and the third leaf between the wild-type and mutant ygl13, demonstrated that the leaf length of the flag leaf, the second leaf was highly significantly increased by 34.75%, 22.62%,respectively in ygl13. Compared with the wide-type, plant height of ygl13 decreased by 12.33%, as well as the seed setting decreased by 26.06%. Panicle length, effective panicles per plant, filled grain number per panicle and 1000-grain weight showed no significant difference. 4. Genetic AnalysisGreen Indica Xinong 1A was srossed with yellow-green mutant ygl13 as the male parent, F1 plants were all normal green. Separation occured in the F2 population. There were 602 yellow-green plants and 1942 normal green plants. Genetic analysis demonstrated that the mutant trait was controlled by a single recessive gene as the number of green seedlings verses that of yellow- green seedlings approached 3:1(χ2=2.35<χ20.05=3.84). 5. Gene targeting moleculeScreening test of linkaged marker depended on BSA. Genetic mapping o f the target gene was conducted using 602 recessive individuals from the F2 segregation population. The target gene was linkaged with RM6429 and RM310. Finally, YGL13 was mapped on the short arm of chromosome 8 between In Del marker ID43 and ID69, with 44 and 6 recombinants, and with an interval of 318 kb. 6. Analysis and cloning of candidate genesSearching NCBI, gramene, Rice Genome Browser and the National Rice Data Center, the 52 annotations genes in this region were analyzed. And there was an acyl Co A binding protein, two Os FBDUF43-F-box and DUF domain containing protein, two pentatricopeptide, a MYB family transcription factor, a zinc finger C3HC4 type domain containing protein, a RN A polymerase sigma factor, two ribosomal protein L13, a rubredoxin family protein, a mitochondrial N ADH-ubiquinone, a abhydrolase domain-containing protein, a CSLF6-cellulose synthase- like family F, a secretory carrier-associated membrane, a cytidylyltransferase domain, a DNAJ domain containing protein, a transmembrane 9 superfamily member, a lissencephaly type-like homology motif, a nuf2 family protein, a mps one binder kinase activator- like 1A, a DNA polymerase delta subunit, a stress- induced protein, three hypothetical protein, twenty expressed protein and six transposon or retrotransposon protein. The mutant and its wild type revealed a single base change(G1005A) of the Os SIG1 gene(LOC_Os08g06630) in the encoded product resulted in a premature stop codon and protein truncation with 334 residues not the primary protein with 520 residues in the mutant ygl13. The gene YGL13 was allelic to Os SIG1 which was documented as a plastid sigma factor previously in rice. 7. The expression analysis of the related genesThe relative expression level of genes involved in chlorophyll metabolism and photosynthesis showed a trend of mixed disorder in the ygl13 mutant compared with WT. The expression level of the gene YGL13 decreased in ygl13 mutant than that in the wild-type parent. |
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