| Embryo development from one-celled zygote to mature multicellular embryo is an important part of the life cycle in higher plants. During early embryogenesis, the first asymmetric zygote division producing the apical and basal daughter cells with distinct developmental destiny is very critical. Many genes essential or required for embryo development, specific asymmetric cell division and cell differentiation during different developmental processes, such as root hairs, stoma and pollen tube formation, had been successfully identified by genetic screening, and the deatailed fuctional analysis of these genes provides us with valuable information for understanding certain critical mechanisms of embryo development and asymmetric cell division in higher plants, However, the regulation of asymmetric zygote division and subsequent apical and basal cell fate decision still remain to be elucidated. In this study, we manually isolated the tobacco(Nicotiana tabacum) zygotes, two-celled proembryos, and its apical and basal cells in vivo by micromanipulation technique, and obtained symmetrical two-celled proembryos from tobacco zygote culture. Combined with SMART PCR cDNA synthesis, suppression-subtractive hybridization (SSH), macroarray screening, bioimformatic analysis, semi-quantative and quantative RT-PCR, as well as in situ hybridization (ISH), we carried out a systemic and comparative investigation of the differential gene expression in the two different zygotic division processes:the zygote asymmetric division in vivo and symmetric division in culture, and in the apical cell and basal cells of two-celled proembryos, and the main results are described as follows: 1. The differential gene expression between the tobacco two different zygotic division systems, the zygote asymmetric division in vivo and symmetric division in culture were investigated at the global transcriptional level, and further anlysis of the resultant candidate transcripts were carried out. We isolated the zygotes and asymmetric two-celled proembryos from ovaries in vivo by micromanipulation technique, and the symmetric two-celled proembryos from zygote culture supplement withβGlcY, and then used SMART PCR to amplify their cDNAs respectively. Combined with the techniques of suppression-subtractive hybridization (SSH) and macroarray analysis, we constructed the subtracted libraries of zygotes and the two kinds of two-celled proembryoes, and screened them with dot blot hybridization. After the sequencing of the differentially expressed clones, a total of 1610 EST representing 685 non-redundant unigenes were obtained and used for further analysis. Gene ontology (GO) term analysis for cell biological process reveals that the transcripts involved in response to stimulus, biological regulation, localization and anatomical structure formation are differentially expressed between the asymmetric and symmetric zygote divisions. Putative tobacco homolog search against those identified genes involved in asymmetric cell division and embryogenesis in Arabidopsis shows that we successfully identify some transcripts related to regulation of cell division and embryo development. Subsequent protein sequence aligment of some candidate transcripts revealed the conservation of certain regulatory mechanisms in different species of animals and plants. Further verification by quantitative real-time PCR demonstrated that we successfully isolated some up-and down-regulated differential genes during zytoge division, some of which displayed special expression patterns in the zygotes, asymmetric and symmetric two-celled proembryos. These differentially and specifically expressed transcripts may be involved in the regulation of asymmetric zygote division, and initiate the early embryo development and apical-basal polarity formation. Expression analyses with different tissues and organs also reveal some potential roles of these candidate transcripts in fertilization, seed mutation and organ development.2. The differential gene expression between the apical and basal cells of two-celled proembryos was investigated at the global transcriptional level, and the resultant candidate transcripts were further anlyzed. We developed an optimizational procedure for isolating the in vivo apical and basal cells of the two-celled proembryos in tobacco, and then performed a comparative transcriptome analysis of the two types of cells by suppression subtractive hybridization (SSH) and macroarray screening techniques. After sequencing, we identified 797 differentially expressed ESTs corresponding to 299 unigenes. Transcript composition analysis reveals that the apical and basal cell differentially expressed transcripts are involved in different functions, and homolog search successfully identifies putative tobacco transcripts involved in embryogenesis and seed development. By quantitative real-time PCR, we validated the differential expression of 40 detected genes, with 6 transcripts of them specifically expressed in the apical or basal cell. Expression analysis also revealed that some transcripts displayed cell specific activation in one of the daughter cells after zygote division. These differential expressions were further validated by in situ hybridization (ISH) technique. The results show that some differential or specific transcripts in the apical and basal cells of two-celled proembryos were successfully isolated, and the identification of these transcripts reveals that these two daughter cells possess distinct transcriptional profiles after zygote division. Combined with the zygote experession, we suggest that there are two different possible mechanisms controlling the differential gene expression, including preferential gene inheritance and cell specific gene activation. Tissue expression pattern analysis also reveal that some potential roles of these candidate genes in embryogenesis and post-embryo development.Our transcriptional analysis on the zygotes, two-celled proembryos and its apical and basal cells provide us important information about gene expression in these cells, and further functional works on the differentially or specifically expressed genes will promote the elucidation of molecular mechanisms of zygote division, early embryonic apical-basal polarity formation and the cell fate decision. |
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