| Rice is a staple food crop worldwide, also an important model plant, we know little about its seed development regulation, so the research on genes related to the development of rice seed has important significance in theory and practice. Previous study showed low seed fertility was exhibited in PcG gene OsiEzl and OsCLF RNA interference plants. We observed the development process of embryo and endosperm to OsCLFi by cytological methods, and using In Situ Hybridization to analyze the temporal and spatial expression of OsiEzl and OsCLF which was cytology basis to further analyze function and mechanism of action of OsiEzl and OsCLF. Our study also focused on a seed developmental defect T-DNA mutant, observed the seed development process which providing the cytology basis to the gene cloning and function identification. The main results are as follows:1. Cytological study of rice PcG geneThe finding of OsCLF:We detected the signal in vegetative organs (root, stem, leaf) and reproductive organs (spikelets, anther, pistil and seed). OsCLF expression exhibited obviouly mRNA signal in the development of embryo and endosperm cells at the early stage in close agreement with the quantitative RT-PCR measure, It indicated that OsCLF gene is constitutive expression in the rice tissue, high expression pattern of OsCLF on early seed development implied that OsCLF may participate in regulation of seed development. It turned out that embryo and endosperm cells of OsCLFi plants exhibited some abnormal phenotypes in the development of caryopsis, such as abnormal embryo, cauliflower embryo, the degradation of endosperm cell, not full endosperm cells. The abnormal rate was the highest at1DAP and8DAP, up to33.33%.The results of in situ hybridization of OsiEzl gene:OsiEzl expresses widely in rice vegetative organ such as root, stem and leaf. It has high expression in meristem of root and stem, young leaf cells. It also has hybridization signal in reproductive organs, such as spikelet, anther and seed. Moreover, we detected remarkable signal in embryo and endosperm at4DAP. The hybridization results revealed OsiEzl gene is constitutive expression in the rice tissue and consistent with the quantitative RT-PCR measure.2. Cytological study of rice seed development mutantThe cytological section process of the two mutant strains of seed development showed that the abnormal seed development mainly displayed seeds without embryo formation, abnormal embryo and delayed development of embryo. We observed some uneven distribution of endosperm cell, there were parts of the seed with endosperm cells degraded trend, the abnormal rate of the lowest2%, up to50%.The reproductive development of rice including the formation of female, stamen and fruit are directly related to yield and quality. The correlation between internal development and reproductive organ morphology is unclear. In order to illuminate the relevance of cytological events of pistil and stamen development to the morphology of spikelet lemma in rice, series of observation was made with material of japonica rice Zhonghua11. The analysis results provided a simple ocular reference to ascertain the reproductive developmental stage of pistil, stamen and seed in rice. It would provide cytological and morphological basis for relevant research of reproductive development and molecular mechanism in rice. The results were as follows:1. The correlation analysis of pistil and stamen development process to the morphology of spikeletWe got the length of spikelet lemma corresponding to gametophyte development stage through the observation of the development process of stamen and pistil in rice and the shape and size of lemmas with stamens and pistils change.The length of lemmas was between1.5-2.5mm at the megaspore mother cell stage and the microspore mother cell stage. The length of lemmas was between2.5-3.0mm at the reduction division phase. The length of lemmas was3.5mm when megaspore mother cell and microspore mother cell form. Then it was the process of formation of gametophyte, also was the period of rapid growth of lemmas. When the pollen and embryo sac are mature, the length of lemmas is6.2mm. 2. The correlation analysis of caryopsis morphology and lemmas length to the inner developmental stage of seed development processCaryopsis growth was speed, but lemmas elongation was not obvious1-6DAP. The elongation speed was slow7-8DAP. Caryopsis development and lemmas were extended to the maximum9DAP. Caryopsis were no longer elongation, mainly transverse widening10-21DAP. The color from the beginning almost transparent green beam shallow gradually turned white, then turned opaque light green. To mature, the pills appeared translucent white skin. |
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