| Panicle as an organ of rice seeds sitting, its morphogenesis determines grain yield in rice. Therefore, the studying of rice spike is important to the theory and practice in developmental biology and breeding. Five mutants have been identified from Japonica rice "Zhonghua 11" T-DNA insertion mutant library, all of them show the mutation phenotype as semi-dwarf, smaller leaf angle, whorled primary branches, double kerneled, shorter grain length, wider grain width and other mutant phenotypes. The candidate gene, which controls rice spike morphogenesis, has been identified though genetic analysis and gene mapping from these mutants. Genetic complementation experiments have been introduced to analysis the gene function. The role of this gene during young spike development has been analyzed by using electron microscopy observation and in situ hybridization experiments. Characterization and functional analysis of this gene will lead better understanding of the molecular mechanism in morphogenesis of rice panicles, and provide the foundation for genetic improvement of rice yield character.. The main results are as follows:1. We generated F2 mapping populationa by crossing individual mutants and Zhenshan 97. The trait segregated in a classical Mandel model indicated that the mutant traits were controlled by a single recessive gene (Panicle Morphological Mutant 1, PMM1). Employing genetic mapping method, PMM1 was located between RM3866-1 and X4 (InDel) on the long arm of chromosome 4, with physical region about 147kb. The same mutant phenotype of F1 generated by parallel crossing between pmm1-1, pmm1-2, and pmm1-3 verified that the three mutants were allelic mutants. We predicted PMM1 is cytochrome P450 gene which ID is LOC_Os04g39430, and found that pmm1-1 lose a large fragments in PMM1.2..In skotomorphogenesis analysis provided a clear result. After 10 days treatment in dark condition,pmm1-1 plants showed shorter average coleoptiles length, only 63.7% of the average length of wild-type ZH11. There was no growth of the mesocotyl in pmm1-1 plants, meanwhile,53.3% of the ZH11 plants showed mesocotyl growth. Both the mutant phenotypes could be recovered by increasing the BR concentration, the length of coleoptiles of pmm1-1 was elongated and was almost the same as that of wild-type ZH11. When plants were treated with 10-6mol/L BR, all of the wild-type growed mesocotyl, and the pmm1-1 has also been restored to 63.3%. 3. The expression of PMM1 gene has been detected from the roots, stems, leaves and panicles of wild-type plant in booting stage by using semi-quantitative RT-PCR. This gene PMM1 had different degrees of expression in root, stem, leaf, sheath and panicle in wild-type plants, and much high level of expression in panicles. Compare to the wild-type, the expression of this gene in pmml-1 plants was almost undetectable.4. In situ hybridization experiments provided signals in young panicle, especially in the branch and spikelet primordial, and tiny signals in young leaves of wild-type plants. On the other hand, no signal could be detected from pmml-1 plants.5. The SEM test has been applied to different developmental stages of wild-type and pmml-1 plants. There is no visible difference between them in the primordia formation of panicle, primary branches and secondary branch. The difference started from spikelet and flower primordia formation, when the double kerneled phenotype could be observed, which was not the same as multi-ovary grain rice.6. Complementary vector pC2301-PMM1-25N18 and pC2301-PMM1-16O02 for PMM1 gene have been constructed, and transferred into pmml-1 calli by Agrobacterium mediated transformation. The transgenic plant were ready for more experiments. |
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