| As an important process during angiosperm pattern formation, embryogenesis is always an active field of research in development biology. Zygotes follow predictable cell division patterns and finally form mature embryos. Many highly regulated gene expression and signal transduction events are involved in this important process. Mechanism of plant gene regulation could also be revealed from research in embryogenesis.Asymmetric zygotic division in higher plants results in the formation of an apical cell and a basal cell. These two embryonic cells possess distinct morphologies and cell developmental fates. It has been proposed that unevenly distributed cell fate determinants and/or distinct cell transcript profiles may be the underlying reason for their distinct fates. However, neither of these hypotheses has convincing support due to technical limitations. Transcription profile analysis of apical and basal cell would help reveal the mechanism of cell fate determination in apical and basal cell.Plant embryo is deeply embedded in ovule tissues, and it is very difficult to isolate the embryo from surrounding maternal material and endosperm. But we isolated pure apical and basal cells from tobacco two-celled proembryo, construct cDNA library from apical/basal cell respectively, and then obtain EST sequences from these two libraries. We also check transcription level of some genes in apical cell/basal cell/zygote, try to find out the mechanism of apical/basal cell fate determination. Main results are:1. Isolated apical/basal cells from two-celled proembryo with laser microdissection. Two transcription inhibitors (Actinomycin D and Cordycepin) were added to isolation solution to prevent stress triggered gene expression. The isolated viable apical/basal cells were confirmed by FDA staining. cDNA libraries were constructed by SMART cDNA Library Construction Kit, from 72 apical cells and 88 basal cells. The titers of the unamplified apical and basal cell libraries were 2.5x106 and 2×106 pfu ml"1, respectively. More than 99% of the clones had an insert, indicating that the libraries were of high quality. The insert size of the cDNA clones in the apical cell cDNA library ranged from 0.3 to 1.7 kb (centered around 0.6 kb), compared to 0.3 to 1.7 kb in the basal cell cDNA library (centered around 0.5 kb). This is the first cDNA library constructed from apical/basal cell of two-celled proembryo; it provides great material for transcription profile analysis of apical and basal cell.2. Clones from apical/basal cell cDNA libraries were randomly picked and sequenced from 5’ends with 5’sequencing primer.2,772 ESTs from the apical cell cDNA library were assembled into 2,072 non-redundant clusters; 2,776 ESTs were assembled into 1,950 non-redundant clusters for the basal cell cDNA library. Several clusters containing ESTs related to ribosomal proteins, chaperones, ubiquitin, calmodulin, and histones appeared in both the apical and basal cells as abundant clusters, suggesting that apical and basal cells share the majority of their most abundant transcripts. Functional categories showed a high degree of similarity between the two cell types, with most of the annotated clusters and ESTs were related to protein synthesis, protein fate, transcription, metabolism, cell cycle and DNA processing in both cell types. Thus, we found that difference between apical and basal cell transcription profiles is not significant, and some embryogenesis related genes are differentially expressed between the two cell types. These differentially expressed genes may contribute to the cell fate determination of apical/basal cell. 3. We select candidates from apical/basal cell ESTs and examine their expression level in apical cell/basal cell. RT-PCR identified two genes that only expressed in basal cell. Then we use more accurate qPCR to examine the expression level in apical cell/basal cell. We found some genes that are expressed at significantly different level between apical and basal cell, and also found two ESTs that were expressed specially in apical cell. Thus we confirmed that transcripts of selected embryogenesis-related genes were cell-type preferentially distributed. And these differentially expressed genes would be great materials for revealing the mechanism of apical/basal cell fate determination.4. Many EST are shared between apical/basal cell and zygote. Then we use qPCR to examine expression level of the ESTs in zygote. Most of these differentially expressed genes are also present in zygote, but the two genes specially expressed in apical cell are not detected in zygote. These result confirmed that some zygotic transcripts are portioned into daughter cells, and cell type specific de novo transcription occurs in apical cell after zygote division. These differentially distributed transcripts and cell specific de novo transcription may contribute to the different expressed genes between apical and basal cells.5. We select an EST named contig500 which expressed both in apical and basal cell, and obtain its full-length cDNA sequence by RACE. contig500 is similar to several arabidopsis proteins, which are all from MAN gene family. AtMAN7 is the most similar one in protein sequences. Then we cloned promoters of all MAN gene family members, and constructed GUS vectors for examining activities of these promoters. We also construct overexpression and amiRNA vectors, and obtain corresponding transformed arabidopsis plant, to reveal function of AtMAN7 during embryogenesis. |
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