Brassica campestris L. ssp. chinensis M

446
vegetable crops in China and in other countries due
to its high yield and ability to cover huge planting
areas. Since a variety of male-sterile lines (genic
male sterile (GMS) and cytoplasmic male sterile
(CMS)) have been selected, they have become one
of the best materials in exploring the pollen
development process. The ‘ZUBajh97-01A/B,’ a
Chinese cabbage (B. campestris ssp. chinensis cv.
Aijiaohuang) GMS AB line, was selected in 1987,
because it was discovered that its progeny constantly
segregates into one sterile and one fertile
type during reproduction (Cao et al., 2006).
In this system, the fertile plant is normally used
as the male sterile plant’s maintainer line to
reproduce the A line. This procedure has been
available for more than 10 years, and through it,
the character of male sterility can be steadily
maintained (Cao et al., 2006). Using the complementary
deoxyribonucleic acid-amplified fragment
length polymorphism (cDNA-AFLP) technique, the
transcriptional profiles in the flower buds of the
male sterile line with fertile line have been
compared. In addition, some pollen preferentially
expressed genes, such as CYP86MF, BcMF2, BcMF3,
and BcMF4 have also been identified (Ye et al.,
2003; Yu et al., 2004; Wang et al., 2004, 2005; Cao
et al., 2006; Liu et al., 2006). The cDNA-AFLP
technique, combined with the rapid amplification
of cDNA ends (RACE) method, is used to identify
BcMF5 from the fertile line of ‘ZUBajh97-01A/B.’
Its deduced amino acid showed the same sequence
as that of the PCP-A2 and SLR-BP1, two family
members of pollen coat proteins (PCPs). Furthermore,
their length and the position of the intron
are also found to be identical in the two PCPs.
Moreover, both PCP-A2 and SLR-BP1 have been
hypothesized to interact with the S-locus glycoprotein
(SLG) and the S-locus related protein 1 (SLR1)
(Hiscock et al., 1995; Takayama et al., 2000a), and
both SLG and SLR1 have been shown to play a role
in pollen adhesion in Brassica by transgenic plants
(Luu et al., 1997, 1999), thus contributing to the
pollen–stigma adhesion process. Consequently,
BcMF5 is a candidate of the PCP family and may
also be involved in the interaction between pollen
and the stigma. However, its precise biological
function in pollen development of B. campestris
remains unclear.
This study thus sought to explain and analyze the
function of BcMF5 through the loss-of-function
approach, and then to determine its role in the
process of pollen development. To characterize
its temporal and special expression profile in
‘ZUBajh97-01A/B,’ the reverse transcriptase-polymerase
chain reaction (RT-PCR) technique was
applied, and the results indicated that BcMF5 is a
ARTICLE IN PRESS
late-expressed PCP. To understand the role of
the promoter in the process of transcription
control, the BcMF5 promoter was first identified
through thermal asymmetric interlaced polymerase
chain reaction (TAIL-PCR), which aided in distinguishing
the expression profile of the promoter in
Arabidopsis.
Materials and methods
Plant material
The B. campestris ssp. chinensis var. communis Tsen et
Lee cv. Ai’jiaohuang ‘ZUBajh97-01A/B,’ the GMS A/B
line, is a stable system such that the proportion of the
progeny of the fertile plants and the sterile plants is 1:1,
and male sterility is controlled only by a pair of nuclear
recessive genes. In this study, the male sterile line was
reproduced continuously by its maintainer line for more
than 10 years, and the variety cv. flowering Chinese
cabbage was used for functional analysis.
The seeds were sown in the field station in Zhejiang
University. During the flowering stage, the pods, flowers,
and stems with five different sizes of flower buds were
harvested. The size standards of the flower buds were as
follows: stage I (diameter o1.0 mm); stage II (diameter:
1–1.6 mm); stage III (diameter: 1.6–2.2 mm); stage IV
(diameter: 2.2–2.8 mm); and stage V (diameter
42.8 mm). At the same time, the plants’ fertility levels
were also investigated. Their fertility was judged
primarily based on the observed length of the filament,
color of the anther, and the existence of pollen. Such
fertility was confirmed through the results of the
inflorescence observation of the pollens. The mixture of
the RNA of 15 plants at the same stage was separately
used as A/B line templates for RT-PCR analysis.
RNA isolation and reverse transcription of RNA
Total RNA was extracted using the Trizol reagent
according to the manufacturer’s instructions. RNA deposition
was dried for 5–10 min in room temperature,
before it was dissolved by diethyl pyrocarbonate water
and stored at 75 1C for use. The first and second strands
of cDNA were synthesized using the SMART TM PCR cDNA
Library Construction Kit (Clontech, USA).
Analysis of gene expression by RT-PCR
Q. Zhang et al.
One pair of primers located in the coding region
(forward: 5 0 -AATGTAAAGCCCAATG-3 0 ; reverse: 5 0 -ATA-
AATTCTTTTCATTGA-3 0 ) was used to analyze the temporal
and spatial expressions of BcMF5. For the RT-PCR, the
reagents used were cDNA template 0.23 mL, 10 PCR
buffer 1.5 mL, 25 mM Mg 2+ 1.2 mL, the sense primer
(20 mM) 0.18 mL, the reverse primer (20 mM) 0.18 mL,
10 mM dNTPs 0.3 mL, and Taq-Pol (5 U mL 1 ) 0.2 mL, ddH 2O
11.21 mL. Amplification conditions were 28 cycles of 94 1C