Okamoto Takashi, Scholten Stefan, Lörz Horst, Kranz Erhard
Biozentrum Klein Flottbek und Botanischer Garten, Entwicklungsbiologie und Biotechnologie, Universität Hamburg, Ohnhorststr 18, D-22609 Hamburg, Germany.
Plant Cell Physiol. 2005 Feb;46(2):332-8. doi: 10.1093/pcp/pci032. Epub 2005 Feb 2.
In higher plants, a zygote generally divides asymmetrically into a two-celled embryo. As in planta, maize zygotes produced by in vitro fertilization of an egg cell with a sperm cell also develop into an asymmetrical two-celled embryo that consists of a small plasma-rich apical cell and a large vacuolized basal cell. Subsequently, via zygotic embryogenesis, a proembryo and a transition phase embryo are formed from the two-celled embryo. In the present study, we focused on identifying genes that were up- or down-regulated only in the apical or basal cell of two-celled maize embryos after fertilization. First, a procedure for isolating the apical and basal cells from two-celled embryos was established, and subsequently cDNAs were synthesized from apical cells, basal cells, egg cells, two-celled embryos and multicellular embryos. These cDNAs were used as templates for polymerase chain reaction (PCR) with randomly amplified polymorphic DNA (RAPD) primers. Genes with specific expression patterns were identified, and these expression patterns were categorized into six groups: (1) up-regulated only in the apical cell after gamete fusion; (2) up-regulated only in the basal cell after gamete fusion; (3) up-regulated in both the apical and basal cells after gamete fusion; (4) down-regulated only in the apical cell after gamete fusion; (5) down-regulated only in the basal cell after gamete fusion; and (6) constitutively expressed in the egg cell and embryos. In addition, it was revealed that the genes up-regulated in the apical or basal cell (genes in groups 1 and 2) were already expressed in the early zygote, providing the possibility that the transcripts from these genes are localized to the putative apical or basal region of the zygote, or that the transcripts are rapidly degraded in one of the daughter cells after zygotic cell division.
在高等植物中，合子通常不对称分裂形成二细胞胚。与在植物体内一样，通过卵细胞与精细胞体外受精产生的玉米合子也发育成不对称的二细胞胚，该胚由一个富含细胞质的小顶细胞和一个大的液泡化基细胞组成。随后，通过合子胚胎发生，从二细胞胚形成原胚和过渡阶段胚。在本研究中，我们专注于鉴定受精后仅在二细胞玉米胚的顶细胞或基细胞中上调或下调的基因。首先，建立了从二细胞胚中分离顶细胞和基细胞的方法，随后从顶细胞、基细胞、卵细胞、二细胞胚和多细胞胚中合成cDNA。这些cDNA用作随机扩增多态性DNA（RAPD）引物进行聚合酶链反应（PCR）的模板。鉴定出具有特定表达模式的基因，并将这些表达模式分为六组：（1）配子融合后仅在顶细胞中上调；（2）配子融合后仅在基细胞中上调；（3）配子融合后在顶细胞和基细胞中均上调；（4）配子融合后仅在顶细胞中下调；（5）配子融合后仅在基细胞中下调；（6）在卵细胞和胚中组成型表达。此外，还发现顶细胞或基细胞中上调的基因（第1组和第2组中的基因）在早期合子中已经表达，这表明这些基因的转录本可能定位于合子的假定顶区或基区，或者转录本在合子细胞分裂后的一个子细胞中迅速降解。
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