Isolation and characterization of genes which are strongly down-regulated after fertilization
--Cordts, S, Kranz, E, Brettschneider, R, Lorz, H, Dresselhaus, T

The molecular characterization of the unfertilized egg cells is a topic of great importance to understand pre- and postfertilization events in higher plants more precisely. In this context it is interesting to answer the question of how much information required for further developmental processes is already deposited in the unfertilized egg cell. Since many plant cells are able to initiate embryogenesis, it is interesting to examine the difference between unfertilized egg cells and somatic cells.

With the intention to isolate genes which are specifically expressed in the egg cell, we have made a differential screening of the RT-PCR cDNA library of isolated egg cells against the RT-PCR cDNA library of in vitro zygotes (18 h after in vitro fertilization) and a conventional cDNA library of 10-day old seedlings. The result of the screening is a number of clones that are strongly down-regulated after fertilization. One group of clones does not show expression in other maize tissues and also lacks homology to known genes. A number of clones from this group belong to the same gene family, but code for different amino acid sequences. This gene family and a further clone of this group were analysed by tissue and single cell-whole mount in situ hybridisation methods (Figure 1). The expression of these clones seems to be embryo sac-specific. A second group of isolated clones that are more strongly expressed in egg cells than in zygotes show no homology to known sequences in databases but are expressed in other maize tissues. Two of them are highly expressed in embryogenic cell cultures. The function of one of these clones (Zmec 88) has been analysed by transformation experiments. This clone is expressed in embryogenic suspension cells, yellow-green leaves and in uni- to bi-nucleate microspore stages. According to this expression pattern the phenotype of transgenic antisense plants in T1 and T2 - generations is partially male sterile and plants have light and transparent spots in their leaves (Figure 2).

To get more information about the functions of the above described clones, we will perform transformation experiments also with the embryo sac-specific genes and study the subcellular localization of the corresponding proteins using antibodies.

Figure 1. Whole mount in situ hybridisation of isolated egg cells with antisense (AS) and sense (S) probes of an embryo sac-specific clone (bar: 50 µm).

Figure 2. Phenotype of a transgenic maize plant, transformed with an antisense construct of the clone Zmec 88 showing leaves with clear spots.

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