Brassica campestris L. ssp. chinensis M

interference has been adopted to repress the
expression of BcMF5 by transgenic approaches.
The results showed that the expression of BcMF5
is depressed. Furthermore, not only did the
germination ability of pollen in transgenic plants
decrease, the resulting pollen was also found to be
abnormal with a collapsed germination furrow.
Furthermore, PCPs have been shown to originate
from the tapetum, a specialized layer of cells that
line the anther locule, but act in a gametic manner
(Heslop-Harrison, 1967, 1968; Dickinson and Lewis,
1973a, b; Heslop-Harrison et al., 1973). The tapetum
serves a nutritive function during microsporogenesis
and provides most of the precursors
necessary for the synthesis of the tough sporopollenin,
the outer wall of the pollen grain, the exine
(Doughty et al., 2000). Because the tapetum is
sporophytic, such male-sterile mutants generally
are recessive and all of the pollen grains within an
anther are affected (McCormick, 2004). The A9
promoter used in this paper is a tapetum-specific
promoter identified from Arabidopsis thaliana (Paul
et al., 1992). Thus, it is rational that all pollen of
transgenic plants should be affected. In this paper,
the percentage of abnormal pollen was recorded at
88.37% and close to 100%. This is likely because the
expression of BcMF5 is not wholly inhibited by RNA
interference, which is consistent with 38.74% of
pollen germination. This also illustrated that the
ARTICLE IN PRESS
Functional analysis of a novel PCP gene BcMF5 453
Figure 7. Morphological comparison of the pollen between transgenic and non-transgenic flowering Chinese cabbage
by scanning electron microscope. (A) pollen of non-transgenic plant at a magnitude of 2000 times; (B) pollen of the
transgenic line at a magnitude of 500 times; and (C) pollen of the transgenic line at a magnitude of 2000 times.
Table 2. Abnormal pollen percentage of pBIA9-RMF5, using a scanning electron microscope
Types of plantlets Total no. of pollen
(pellet)
No. of abonormal pollen
(pellet)
Percentage of abonormal
pollen
WT 282 24 7.972.4
pBIA9-RMF5 402 354 88.3775.32
The data in the table are means7SD from four measurements.
expression of BcMF5 has a close relation to the
tapetum.
A promoter has a significant effect on the
location, timing, and strength of gene expression,
and the expression analysis of the promoter
contributes to the understanding of gene function.
The BcMF5 promoter began to express in the early
stage of anther development (Figure 8B) and drove
high levels of GUS expression in anthers, pollen,
and pollen tube in the late stage of pollen
development (Figure 8C–G). Although BcMF5 shares
a high homology to SLR1-BP, the expression pattern
of BcMF5 was different from that of SLR1-BP. An
RNA gel blot analysis showed that high levels of
SLR1-BP mRNA were detected only in anthers at the
late developmental stages. Moreover, in situ
hybridization also showed that the antisense probe
of SLR1-BP hybridized only to the cytosol of the
microspores and not to any sporophytic tissue of
the anther (Takayama et al., 2000a). This showed a
strictly gametophytic expression manner. In contrast,
the expression profile of BcMF5 and its
promoter (Figures 3 and 8) seemed to show a
sporophytic or gametophytic expression pattern,
which is similar to SP11. The SP11 gene is the sole
male determinant in the self-incompatibility of the
Brassica species (Schopfer et al., 1999; Shiba et al.,
2001). It is also specifically expressed in the tapetal
cell of the anther at the early developmental