| Polycomb group (PcG) proteins are a class of conserved transcriptional repressor, which formed in plants and animals evolution process. PcG proteins function by forming large protein complexes. In Arabidopsis, the Polycomb Repressive Complex 2 (PRC2) have been identified, which contain four core proteins:E(z), Su(z)12, ESC and P55. These proteins form FIS complex, play a role in the process of central cell without fertilization initiation endosperm development.Eulaliopsis binata is a apomixis plant, the central cell without fertilization autonomous division formed in endosperm. In this study, we cloned E(z) homologues from the Eulaliopsis binata and compared the nucleotide and amino acid sequence differences; analysis of E(z) gene expression patterns; investigated DNA methylation level and analysis of function by ectopic expression of transgenic Arabidopsis and rice. The main results are as follows:(1) Cloning and sequence analysis of EbEZ genes:two full-length cDNA clones were obtained after RACE-PCR and named EbEZ1 and EbEZ2, respectively. Through southern blotting experiments confirmed that there are two copies of the E(z) gene in Eulaliopsis binata genomic. The full-length cDNA of EbEZ1 gene 3300 bp, DNA length 9600 bp, contains 16 exons and 15 introns; the EbEZ2 cDNA full-length 3130 bp, DNA length 9500 bp, and also contains 16 exons and 15 introns. Using NCBI Protein Structure online web analysis EbEZ protein domain, the result showed that there were five typical conserved domain exist in EbEZ1 and EbEZ2 proteins, respectively EZD1, EZD2, SANT, CXC and SET domain. Phylogenetic analysis illustrated that, EbEZ1 was highest identical to maize ZmMEZ1; and EbEZ2 was highest identical to maize ZmMEZ3.(2) Isolation and cis-acting element analysis of EbEZ promoter:we cloned EbEZ1 and EbEZ2 gene promoter by Genome Walking technology. In their promoter sequences, some response element in sensitive to environmental changes present. In addition, also contains some of the endosperm, pollen or seeds with a tissue-specific expression of the components sequences.(3) Expression analysis of EbEZ gene in Eulaliopsis binata:Real-time PCR analysis of EbEZ gene expression patterns found that EbEZ1 and EbEZ2 have different expression level in roots, stems, leaves, flowers and seeds, but in the inflorescence and seeds higher than in vegetative organs. RT-PCR results showed that, EbEZ1 expression level in ovaries before flowering and development of early seed was significantly lower than mature phase of the seed.(4) Methylation analysis of EbEZ gene promoter:use of online analysis program EMBOSS CpG island tools analysis showed that, in EbEZ1 promoter presence two CpG islands region but there is only one CpG island in EbEZ2. Analysis EbEZ1 promoter methylation status by Bisulfite Sequencing, results showed that the methylation of the ovary before flowering is the highest and in the early seed development but also to maintain a high methylation state. But the degree of methylation significantly reduced in the mature seeds, and consistent with the EbEZ1 expression in these developmental stages.(5) The function analysis of EbEZ1 gene:we transformed EbEZ1 into Arabidopsis and rice, in transgenic Arabidopsis the middle leaves and growth points was inhibited. The number of flowers and floret glumes and pollen fertility has changed in transgenic rice, compared with wild-type. Phenotype observation of transgenic plants discovered that EbEZ1 genes may be involved in regulation of plant vegetative and reproductive development. |
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