| Polycomb group (PcG) proteins are a class of conserved transcriptional repressors which form in the evolutionary process of plants and animals and function as a form of protein complex. PcG protein-FIS complex plays an important role in repressing the central cell initiating endosperm development without fertilization to form autonomous endosperm. Eulaliopsis binata is an apomictic plant with gametophyte apomixis and the endosperm forms without fertilization which is originate from the central cell. To study the relationship between the PcG proteins and apomixis phenomenon, the subcellular localization and spatio-temporal expression of the cloned Eulaliopsis binata E(z) homologues EbEZl and EbEZ2 were analyzed and the function analysis of EbEZ1 and EbEZ2 genes by agrobacteriun-mediated transformation to Arabidopsis and rice to lay a certain experiment basis on the melocular mechanism of Eulaliopsis binata apomixis. The main results are as follows:(1) Subcellular localization analysis of EbEZ gene:transformed constructed CAMV35S::EbEZ1:GFP and CAMV35S::EbEZ2:GFP fusion vectors into onion epidermal cells by particle gun-mediated method. The results revealled EbEZ1 and EbEZ1 were located at cytoplasm and nucleus, suggesting EbEZ1 and EbEZ2 may act as transcription factors and involve in trans-membrane protein activity.(2) Spatial and temporal expression analysis of EbEZ gene:the spatial expression patterns of EbEZ genes were studied by in situ hybridization. The results showed that EbEZ1 hybridization signals were deteced in vegetative organs (root, stem and leaf) and reproductive organs (anther, ovary and seed). However, the expression of EbEZl in vegetative organ was lower than that in reproductive organs. The expression of EbEZ2 was similar with the hybridization of EbEZ1. The hybridization signals of EbEZ2 were also strong in vegetative organs, and strong signals could be detected in free nuclear endosperm, and EbEZ2 expression was highest in embryo and endosperm of mature seed. The expression analysis of EbEZl and EbEZ2 were accordant to the qRT-PCR detection, suggesting that they might participate in regulating vegetative and reproductive development.(3) Ectopic expression analysis of EbEZ1 gene:phylogenetic analysis showed that EbEZ1 was homologous to Arabidopsis CLF. Transformed pCAMV35S::EbEZ1 vector into clf mutant and wide-type Arabidopsis to make ectopic expression of EbEZ1. When EbEZ1 gene was transformed into clf mutant, T2 positive plants showed recovered rosette leaf but cauline leaf remained curly phenotype. Ortherwise, the plants were dwarf and siliques were short, indicating EbEZ1 gene was partial conservation to CLF on the regulation of leaf. While the overexpression EbEZ1 T2 plants revealed curly leaf and elongated prtiole. In addition, the siliques were shorter in transgenic plants than the wild-type one. RT-PCR analysis on related genes in overexpression EbEZ1 T2 plants showed CLF-target genes AG, AP3, FT, FLC and KNAT1 were down-regulated, suggesting EbEZ1 may involved in the regulatory network of Arabidopsis CLF so as to cause abnormal development of leaf, siliques and seeds in the ectopic expression of EbEZ1 plants.When pCAMV35S::EbEZ1 construct was transformed into japonica rice ZHONGHUA 11, positive T2 rice plants revealed reduced tiller number and earing number, and the ripening rate decreased significantly. The cytological study on positive plants seed development revealed abnormal endosperm and embryo in the seeds of positive plants. In addition, the expression detection of OsEZ1 co-expressed genes in T2 positive plants demonstrated PHD1, KNAT1 and MADS8 were up-regulated, indicating EbEZ1 may affect the regulatory network of rice endogenesis gene OsEZ1 so as to result in abnormal seed development in transgenic plants.The phenotype and expression analysis on EbEZ1 transgenic Arabidopsis and rice plants indicated EbEZ1 could play a role in regulating the development of fruit and seed as well as the leaf morphology. |
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