| Rice, wheat and maize are known as three major food crops in the world. The endosperm of rice is one of the main sources of human food. In fact, the yield and quality are mainly determined by the development status of endosperm. As people's demand for the quality and yield of rice is getting higher and higher today, the researches on the endosperm developmental mechanism become a major task to the plant biologists since it is necessary for improving the yield and the quality of rice, and may lay a theoretical foundation for solving the grain crisis.The process of the early development of rice endosperm is critical to the seed development, although it is very short, compared with the whole process of endosperm development. The rice primary endosperm nucleus come from the fusion among a sperm nucleus and two polar nuclei of the central cell. A lot of free endosperm nuclei are generated after the primary endosperm nucleus constant mitosis, and then distribute along the embryo sac wall in the syncytium. When the endosperm develops to a certain stage, the free endosperm nucleus begins cellularization. When the cellularization process is completed, the endosperm enters into the stage of differentiation and maturation. The developmental process may need about 4 days from double fertilization to the completion of the cellularization. This developmental stage contains several important developmental events, including triple fusion after fertilization, the initiation of the primary endosperm nucleus division, the initiation and completion of endosperm cellularization. Each of these stages is accompanied by a lot of important physiological and biochemical activities, including the activation and turnoff of crucial genes, of which the imprinted genes have been drawn a great attention recently.Rice endosperm is triploid, its genome has two copies from maternal chromosome and one copy from paternal chromosome. The expression of many genes is not equal between the paternal and maternal allele, some genes are only paternal allele expressed but maternal allele has no transcript. On the contrary, some genes are only maternal allele expressed but paternal allele has no expression. This epigenetic phenomenon is called genome imprinting.Differential expression of imprinted genes in endosperm can properly regulate the distribution of hormones and nutrients in the embryo and endosperm, and transfer all kinds of resources to the offspring. The imprinted genes are important for the regulation of the growth and development of rice endosperm. As reported, if some imprinted genes are mutated or abnormally expressed, endosperm development process can be disturbed and grain crops may be destruction.The most closely related genes in rice endosperm development are six PcG gene, including OsFIE1, OsFIE2,OsEMF2a,OsEMF2b, OsCLF, OsiEZ1. The expression products of these six PcG genes constitute the PRC2 polycomb protein complex. The six PcG genes affect cell proliferation and differentiation as well as determine cell fate by means of a series of epigenetic modification of chromatin related target genes by mediating trimethylation formation of 27 lysine on histone H3(H3K27me3), which will regulate a series of morphogenesis of the rice reproductive organs and developmental behaviors of the endosperm.In this study, we have focused on the expression patterns of these six genes in the early stage of rice development, and discussed the possible imprinting effect of OsFIE2, in order to obtain a deeper understanding of the characteristics and functions of the imprinted genes during the early development of the rice endosperm. The main results are as follows:1. To study related genes in the early development of rice endosperm, we must isolate the rice early free nuclear endosperm that should be pure and without maternal contamination. Extraction of the trace RNA from the few endosperm cells should also be performed for the analysis and screening of imprinted gene. Because of the special characteristics of rice endosperm at the early stage, the isolation technology and collection procedure for the early endosperm have been very difficult. After repeated trials, we created a reliable isolation technique that can accurately and effectively collect the early endosperm at 24 and 48 hours after pollination. Theendosperm obtained by this method has a high purity after being tested by RT-PCR and SNP analysis along with other techniques. This method can be widely used for the screening of imprinted genes and the identification of the endosperm specific expressed genes in the crop plants.2. Based on the previous studies and our method for collection of the early endosperm, we re-analyzed the expression patterns of the six PcG genes at the 1?15 days after pollination endosperm. Some of these genes have been identified previously as non imprinted genes since both parent alleles are expressed. Our analysis shows that these genes are imprinted genes of single parental allele expression in the early stage of endosperm development. Our results indicated that the early stage of endosperm development is essential for the identification of imprinted genes.3. Our analysis revealed that the expression pattern of the parental allele was dynamically changed as the endosperm development process, and the dynamical change of the expression pattern in the first three days after pollination was the most obvious, implying that imprinted genes may have important regulatory effect during this early period.4. It is found that the same gene may have different transcripts with different expression patterns of the parent alleles. One gene simultaneously appears the expression pattern of imprinted genes and non imprinted genes, which raises a new question for the recognition of imprinted genes.5. We also revealed that the expression pattern of the imprinted genes can be changed when one of the parents is replaced by different materials for hybridization or exchange the place of the parent materials for reciprocal cross. We confirmed that there are some strong imprinted genes, which provides new clues for the interpretation of filial generation phenotypes.6. It is tested that the expression pattern of the imprinted genes under the conditions of physical or chemical stress during the endosperm development. It is found that stress growing conditions can affect the development of rice, but it does not affect the expression pattern of imprinted genes.7. In order to explore the imprinting effect of PcG gene, we cooperated with the Beijing Plant Institute of the Chinese Academy of Sciences, and created eight OsFIE2 single nucleotide mutants of Zh11, and screened a heterozygous rice line that produces a mutation at the last base of the sixth intron. This mutation destroys the "GT-AG" splicing rules of the intron, resulting in the 215bp base of the sixth entire intron of OsFIE2 can not be cut in the mRNA mature processing. Mutated OsFIE2 encounters termination codon in the translation of the protein, leading to premature termination of translation, and can not produce the normal OsFIE2 protein to combine with other PcG proteins to form a functional PRC2 multi-comb protein complex. We find that the mutant had a serious impact on the normal development of the rice endosperm and ovary. The homozygous mutant seeds were not produced.8. Compared with the wild type Zh11, the self-fertilization of the OsFIE2 heterozygous mutants can produce three types of ovary, which are normaly developed, delayed and aborted type ovules, respectively. It means that some ovaries and endosperm can normally develop, and some ovaries and endosperm are completely aborted, and some ovaries and endosperm are seriously retarded compared to the normal development.9. Two types of ovaries, normal and aborted, could be observed when the reciprocal hybridizations between the OsFIE2 heterozygous mutant and the wild type Zhll (or the wild type 9311, or the OsFIE2 over expression Nip. rice). Several types of ovary, which were developed by self-fertilization and reciprocal hybridizations, were in accordance with Mendel's law of independent separation.10. We observed and compared the internal structure of various types of ovaries by confocal microscopy, and found that the aborted ovaries had been arrested at the 2-nucleus stage of the primary endosperm nucleus, which could not develop further. By analyzing the reciprocal cross between OsFIE2 heterozygous mutant and the Zh11 wild type (or the 9311 wild type), the OsFIE2 allele of the normal wild-type has not expression before 1 dap, but another OsFIE2 allele expresses can not translate normal protein as a result of mutation, which is difficult to combine with other PcG genes protein to form a functional PRC2 complex and play a role. Until the other normal wild-type OsFIE2 allele begin to express, endosperm to restart nuclear division and continue to develop forward.11. The OsFIE2 imprinting effect mainly reflects in blocking continuous division of the primary endosperm nucleus, and indirectly delayed the initiation and termination of the endosperm cellularization, and affect the subsequent development of endosperm. |
PDF Full Text Request