全 文 ：Received 8 Aug. 2003 Accepted 7 Jan. 2004
Supported by the National Natural Science Foundation of China (30370743), the National Outstanding Youth Science Fund (30225006) and
Pre-investigation Project for State Key Basic Research and Development Plan of China (2002CCA00100).
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Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 578-581
Single-Embryo RT-PCR: Application for Gene Expression Dynamics
and Some Technical Aspects
ZOU Jian-Wei, SUN Meng-Xiang*, YANG Hong-Yuan
(Key Laboratory of Ministry of Education for Plant Developmental Biology, College of Life Sciences,
Wuhan University, Wuhan 430072, China)
Abstract: An extended investigation was carried out for further demonstrating single-embryo RT-PCR
technique, which was previously established in our laboratory as a useful tool in the study of gene expression
of Arabidopsis thaliana (L.) Heynh. Based on the previous work some important technical aspects in the
application of the technique were mainly discussed in the present report. The dynamic expression of three
selected genes, shoot-meristemless (STM), monppteros (MP) and Arabidopsis shaggy-like kinase etha
(ASKh), was care fully compared during the embryogenesis. It was revealed that the technique was
especially useful for tracing the expression dynamics of specific genes during embryogenesis. However,
the quantity of starting material and the duration of DNaseⅠ treatment are critical points for successful
application of the technique. The best condition for eliminating the possible influence of genomic DNA on
data analysis was also tested and discussed.
Key words: single-embryo RT-PCR; gene expression patterns; embryogenesis; Arabidopsis thaliana
There are two body axes of polarity established in higher
plants during early embryogenesis : apical-basal axis and
radial axis (Gerd et al., 1997). The development of higher
plants can be characterized by cell division, expansion and
differen tiat ion along the two axes (Gerd , 1995; 2001). In
recen t years, many genes have been iden tified and
characterized, which are involved in the polarity originat-
ing during embryogenesis (Gerd et al., 1997). To study the
expression dynamics of these genes in single embryos it is
important to correlate gene expression to specific develop-
mental stage of embryogenesis and to peculiar cytological
and morphological feature of the embryo, which offers a
useful clue for further gene function analysis (Karrer et al.,
1995; Adjaye et al., 1999). Recently, we have developed a
method, s ingle-embryo RT-PCR, in Arabidopsis tha liana
for such a purpose (Zou et al., 2002). Present work is the
extending study to investigate gene expression dynamics
in early embryogenesis by the technique. Some important
technical aspects were carefully investigated and discussed
in this report for successful application of the method. Three
important genes , monppteros (MP), shoot-meristemless
(STM) and Arabidopsis shaggy-like kinase etha (ASKh)
involved in the establishment of body axes in higher plants
were used in the experiments. MP is required for correct cell
axialization in the early embryo (Hardtke and Berleth, 1998).
STM plays a role in restricting cells with a shoot meristem
fate from participating in organ formation (Long et al., 1996).
ASKh proteins are involved in signal transduction path-
ways that estab lish cell fate and/or pattern formation in
plants (Dornelas et al., 1999). All these genes are known to
be express ed in specific tis sue of embryos and they were
therefore selected as markers to indicate the gene expres-
sion dynamics.
1 Materials and Methods
1.1 Plant materials
Plants of Arabidopsis thaliana (L.) Heynh. were grown
in a culture chamber (M3750, Forma Scientific, Inc.) under
standard conditions. Develop ing siliques at 2, 4 and 6 d
after pollination (DAP) were selected and cut for the isola-
tion of embryos. A ll siliques were surface-sterilized with
2% sodium hypochloride for 10 min at 22 ℃, rinsed with
sterile water.
Under an inverted microscope, the ovules were isolated
with a scalpel. Then the embryos of different stages were
isolated by a g lass micropipette and transferred into lysis
buffer.
1.2 Lysis of embryos and single-embryo RT-PCR
The procedure used for the lysis of embryos was de-
scribed by Zou et al. (2002). All the lysis containing grinded
embryo cells was us ed as template to perfo rm s ingle-
embryo RT-PCR. The procedure of single-embryo RT-PCR


ZOU Jian-Wei et al.: Single-Embryo RT-PCR: Application for Gene Expression Dynamics and Some Technical Aspects 581
2.3 The influence of genomic DNA and the degradation of
mRNAs
The best condition for eliminating the possible in flu-
ence of genomic DNA on data analysis was also tested. If
the templates had not been treated with RNase-free DNase
Ⅰ, both the desired bands and the bands corresponding
to genomic DNA were visible after the first amplification.
Different durations of DNase Ⅰ treatment were compared
and one hour was turned out to be the shortest time for
satis fied result. When the templates were t reated with
DNase Ⅰ for one hour, the desired bands could be visible
after the second amplification, and the bands correspond-
ing to genomic DNA were no longer visible in both amplifi-
cations (Fig.6). In this case, a second amplification is nec-
essary since after one hour treatment with DNase Ⅰ the
signal strength of desired bands was very weak. This may
be due to that long treatment of DNase Ⅰ cou ld lead to
Mg2+-dependent hydrolysis of the RNA and /or the rem-
nant activity of the DNase Ⅰ after heating the enzyme at 65
℃ might still result in the degradation of the cDNA (Burke,
1997). Therefore, it is important to control the time of DNase
Ⅰ treatment as short as pos sible. Otherwis e, mRNAs of
target genes can be gradually degraded during the RNase-
free DNase Ⅰ treatment.
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(Managing editor: WANG Wei)