| Leaf blade is a main organ of photosynthesis and responsible for over ninety percent of rice yield. Increasing rice leaf area index and light use efficiency is of great significance to improve rice production and its quality. Improved rice leaf morphology is one of the important goals of the plant type breeding. The study about development mechanism of blade is also important to plant type breeding. Current national researchers have made many achievements in the study of the regulation of the rice leaf. But the regulating mutual relations between the cloned leaf regulatory genes have not yet sorted out, and the regulation mechanism of rice leaf development remains to be further studied. In this study, we find a mutant dw1 with dwarf plants and wider blades from the rice restorer line Jinhui10 mutant library by EMS. This paper performed systematic studies in morphological characteristics detection, identification of photosynthetic pigments and characteristics, cytological observation, genetic analysis, gene fine mapping and candidate gene identification etc. The main results were as follows:1. Under cultivation conditions of the field, Compared to wild type Jinhui10, in the whole growth period, dw1 showed dwarf, dark green leaves, shorter pour 1 leaf, 2 leaf, 3 leaf and internodes, wider blade, more severe wrinkles on the blade section, wider grains, increased large vascular bundles and small vascular bundles, and increased spacing between adjacent small vascular bundles. 2. The plant height, panicles, panicle length, panicle setting rate of mutant dw1 were significantly reduced compared to wild type. Grain length has no significant difference. Mutant dw1 grain width is significantly widened. Therefore, grain weight of dw1 also very significantly increased compared with the wild-type. 3. The chlorophyll a, chlorophyll b and total chlorophyll content were not significantly different between the wild-type and dw1. But the carotenoid content of dw1 significantly and very significantly reduced. The stomatal conductance and transpiration rate of dw1 significantly lower than the wild-type electrode, while the net photosynthetic rate and water use efficiency increased significantly. 4. Compared to wild type Jinhui10, large vascular bundles of dw1 whole becomes large, thin-walled cells and vascular bundle sheath cells increased, mesophyll cell layers increased. Speculating mesophyll cell layers cadogan cause dw1 has darker leaf color while the total chlorophyll content was no difference. The number of mesophyll cells between adjacent two small vascular bundles of dw1 is 11, two more than the wild type. 5. Genetic analysis showed that mutant trait was controlled by a recessive nuclear gene. The regulate gene was located on chromosome 6 between indel markers Ind06-9 and Ind06-10, with 16 kb physical distance, containing three annotated genes. Gene sequencing and alignment of annotated genes shows LOC_Os06g03710 exon has A-T base substitution at 1675 th base. Initially proved LOC_Os06g03710 as a candidate gene in DW1. 6. By identification and gene mapping of allelic mutant dw1-1, the results show a consistent target range between dw1-1 and dw1. Sequencing of LOC_Os06g03710 comparison of the wild type Xinong1 B and dw1-1, it found that exon of dw1-1 inserted a guanine(G) between the 822 th bp and 823 th bp, and a C-T base substitutions occurred at the 888 th bp, leading frameshift mutation from 275 th codon and stop at 297 th codon, resulting in an advanced translation termination. This result further confirmed mutant phenotypes of dw1 and dw1-1 caused by a mutation from gene LOC_Os06g03710. 7. The DW1 gene in wild-type has low expression at seedling stage, tillering stage and heading stage, with the highest expression at the seedling stage, secondly expression at heading stage and lowest expression at tillering stage. The DW1 gene at tillering stage has low expression level in the heart leaf, the higher expression in the whole leaf, and the highest expression in part of leaf sheath, but the expression levels are very low on the whole. 8. The mutant dw1 is insensitive to exogenous IAA, while the blade of wild-type was widened and significantly reduced expression of DW1 gene when treated by exogenous IAA, indicating DW1 gene expression is inhibited by IAA, and DW1 gene plays a role in the process of auxin-mediated leaf development. For expression analysis of leaf development related genes and genes of some narrow-leaved shows, other narrow leaf gene and related genes were up-regulated relative wild type except the narrow leaves genes NAL1 and PIN with down-regulated. Where in NAL7 gene up-regulation is extremely significant, and the CYCD3 gene which coding cyclin was significantily upregulated in mutant dw1. |
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