| Embryo development is the beginning of the ontogeny and is one of the most impotant parts of life cycle in higher plants. For a long time, because of technical limitation, the study of early embryo development in higher plants has not been thoroughly carried out. Tobacco (Nicotiana tabacum L.) zygote and early embryo isolation as well as in vitro culture are the traditional techniques in our lab. In order to sdudy the molecular mechanism of the key events in early embryo development, such as zygote polarity formation, apical-basal cell fate differentiation and zygote and proembryo gene activation, we constructed the subtracted libraries of zygote and early embryo in the past few years. In addition, we also use some cellular biology methods, such as the zygote in vitro culture, organelle position by fluorescent dye and ultrastructure observation, to analyze the developmental events in early embryo of tobacco. Based on previous results, we find some candidate genes of intrest in the subtracted libraries by analyzing the transcripts expression and function predication. Combind with molecular and genetic and other experimental methods, we study the biological function of one of the candidate genes in tobacco. The main results are described as follows:1. Based on the established tobacco zygote in vitro culture system, we study the role of arabinogalactan proteins (AGPs) in proembryo morphogenesis. Arabinogalactan proteins (AGPs) are a family of extensively glycosylated cell surface proteins that are thought to have important roles in various aspects of plant growth and development including embryogenesis. The previous results in our lab show that AGPs are involved in tobacco egg cell fertilization, zygote division and proembryo development (Qin and Zhao2006). When AGPs were specifically bound by P-glucosyl Yarive (P-GlcY) reagent, the frequency of aberrant division in tobacco cultured zygotes remarkably increased. However, how the AGPs interacted with other factors that are involved in zygotic division and proembryo development still remain unknown. In this study, we investigated the AGPs role on the in vivo and in vitro zygote and proembryo development in tobacco. By using immunofluorescent localization technique with antibody JIM13and β-GlcY staining, the result shows that AGPs mainly distribute in the wall of daughter cells from zygotic division. By using staining with Calcofluor White (CW) to detect cellulose components, the result displays that strong fluorescence is located in the new-formed wall of daughter cells from zygotic division in vivo and the control samples of in vitro culture without β-GlcY-treated; while weak ones in the daughter-cell wall with β-GlcY treatment. By using immunocytochemistry technique with the monoclonal antibodies JIM5and JIM7to recognize respectively low-and high-esterified pectins, the results display that the two pectins locate in the opposite position of zygotes and proembryo in vivo. Furthermore, the reagent FM4-64staining shows that endosomes are distributed in the cell plates of the proembryos, and the kind of localization is affected by the treatment of β-GlcY reagent. Moreover, the proembryo cell-organelle (such as endoplasmic reticulum and mitochondria) changes induced with β-GlcY reagent were observed by using fluorescent dye staining technique. These results imply that AGPs may contribute to the formation of new cell plate, suggesting that AGPs play roles in tobacco zygotic division and proembryo pattern establishment.2. The biological functional analysis of one of the candidate genes (termed RC18) from subtracted libraries were carried out. RC18is one of the genes which were predominantly expressed in the basal cell of2-celled proembryo in tobacco. Expression analyses with different tissues and organs reveal that RC18is predominantly expressed in anthers, ovules of1,5and15days after pollination (DAP) but not in vegetative organs. We have obtained the opening reading frame (ORF) of RC18by using genome walking method. RC18probably encode a glycosyltransferase of cytokinin and we speculate that RC18may play some roles in maintaining the dynamic balance of cytokinin in tobacco. In addition, RNA interference (RNAi) metnods were utilized to reveal the biological function of RC18in tobacco. Phenotypical observation showed that the female gametophyte, male gametophyte and embryo development were affected in RNAi plants and result in sterility. In globular embryo stage, the embryo proper and suspensor morphogenesis were both affected and suspensor abnormalities were more obvioused. In the heart and torpedo embryo stage, the cotyledons development and morphogenesis were affected. The abnormalities appeared in three cotyledons, asymmetric cotyledons and fusion cotyledons and these abnormalities were also obvioused in seedings.Our study in the role of AGPs in proembryo morphogenesis would provide more relevant experimental data to reveal the mechanisms which regulate the zygote asymmetric division and proembryo development. On the other hand, the biological functional analysis of RC18would help to study the gene regulatory mechanism and network. |
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