Brassica campestris L. ssp. chinensis M

Journal of Plant Physiology 165 (2008) 445—455 

Characterization and functional analysis 

of a novel PCP gene BcMF5 from Chinese cabbage 

(Brassica campestris L. ssp. chinensis Makino) 

Qiang Zhang a , Jiashu Cao b, , Huizhi Liu c , Li Huang b , 

Xun Xiang b , Xiaolin Yu b 

a 

Institute of Horticulture Science, Henan Academy of Agriculture Science, Zhengzhou 450002, P.R. China 

b 

Laboratory of Cell & Molecular Biology, Institute of Vegetable Science, Zhejiang University, 

Hangzhou 310029, P.R. China 

c 

Institute of Biology Science, Zhejiang University, Hangzhou, 310029, P.R. China 

Received 16 April 2007; received in revised form 18 June 2007; accepted 26 June 2007 

KEYWORDS 

Brassica campestris 

ssp. chinensis; 

Brassica campestris 

Male Fertile 5 

(BcMF5); 

Characterization; 

Functional analysis; 

PCP 

Summary 

Corresponding author. Tel./fax: +86 571 86971188. 

E-mail address: jshcao@zju.edu.cn (J. Cao). 

ARTICLE IN PRESS 

0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. 

doi:10.1016/j.jplph.2007.06.020 

Brassica campestris Male Fertile 5(BcMF5), a novel member of the pollen coat 

protein class A (PCP-A) gene family, was identified from Brassica campestris L. ssp. 

chinensis Makino (Chinese cabbage-pak-choi). Temporal and spatial expression 

analysis showed that BcMF5 is a late-expressed PCP gene related to the process of 

determining pollen fertility. Functional analysis by hairpin RNA (hpRNA)-mediated 

RNA interference also showed that the expression of BcMF5 is inhibited, which 

resulted in the low germination ability of the pollen and also in an abnormality of the 

pollen exemplified by a collapsed germination furrow. This demonstrates that the 

expression of BcMF5 is closely related to the tapetum. Further, the expression profile 

of the BcMF5 promoter in Arabidopsis was also analyzed. This analysis indicated that 

the BcMF5 promoter began expression in the early stage of anther development and 

drove high levels of glucuronidase (GUS) expression in anthers, pollen, and the 

pollen tube in the late stage of pollen development, but did not drive any expression 

in petals, sepals, or pistils. Together with the functional analysis, the hypothesis that 

BcMF5 may have a sporophytic or gametophytic expression pattern is presented. 

& 2007 Elsevier GmbH. All rights reserved. 

Introduction 

www.elsevier.de/jplph 

Chinese cabbage (Brassica campestris L. ssp. 

chinensis Makino) is one of the most important

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 


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

for 3 min, 94 1C for 30 s, 40 1C for 30 s, and 72 1C for 1 min, 

followed by one cycle of 72 1C for 7 min. Afterwards, the 

Actin transcripts were amplified as an external control. 

Construction of the hpRNA-mediated RNA interference 

plasmid vector 

The oligonucleotide primers 5 0 -GCAGGATCCGTCTCGT- 

CATGCTCACAA-3 0 (forward) and 5 0 -GAACCCGGGGTGTC- 

CTTTGCCAGTATC A-3 0 (reverse) were used to amplify the 

459-bp sense fragment containing the intron of the 

BcMF5 gene. The forward primer was added, a Bam HI 

restriction endonuclease site at its beginning, while the 

reverse primer Sma I restriction endonuclease site at its 

beginning. The oligonucleotide primers 5 0 -GTAGGATCCG- 

TA GTGGCGAATGCTCAA-3 0 (forward) and 5 0 -GCGTCTA- 

GAGTGTCCTTTGCCAGTATCA-3 0 (reverse) were used to 

amplify the 159-bp antisense fragment of the BcMF5 

gene. Its forward primer was added, a Bam HI restriction 

endonuclease site at its beginning, while the reverse 

primer Xba I restriction endonuclease site at its beginning. 

The PCR product was then cloned into a pGEM-T 

Easy Vector (Promega, USA). Ti binary vector pBI121 

containing the A9 promoter of Arabidopsis was used for 

plasmid construction. The sense fragment containing the 

intron was restricted by Bam HI and Sma I restriction 

endonucleases, and the antisense fragment by Bam HI, 

Xba I, and A9-pBI121 vectors which simultaneously 

underwent the same process by Xba I and Sma I. The 

sense and antisense fragments of the BcMF5 gene were 

then reclaimed from agarose and were cloned into the 

restricted A9-pBI121 vector constructing pBIA9-RMF5 

(Figure 1). Finally, the plant-expressing vector was 

transferred into Agrobacterium tumefaciens strains 

LBA4404 through the freezing–thaw method. 

Shoot regeneration and plant transformation 

The plant regeneration system of flowering Chinese 

cabbage was based on modified methods (Cao et al., 

2000). In all the experiments conducted in this study, all 

the constituents were added to the MS medium, and the 

pH was adjusted to 5.8 before autoclaving at 1 kg cm 2 

and 121 1C for 15 min, except silver nitrate solution and 

antibiotics, which were both filter-sterilized. Cultures 

were maintained in a tissue culture room under a lighting 

regime of 16 h/8 h (L/D, 40 mEm 2 s 1 )at2571 1C. 


Functional analysis of a novel PCP gene BcMF5 447 

RB 

Antisense 

A. tumefaciens strains were grown in a shaking 

incubator for 36–48 h in 100 mL of a liquid YEP medium 

containing 50 mg L 1 kanamycin and 25 mg L 1 rifampicin. 

The grown bacteria were collected by centrifugation 

and then suspended in a transformation buffer consisting 

of MS salts and vitamins. The optical density (OD) of the 

bacterial suspension was adjusted to approximately 

A600 ¼ 0.6 prior to incubation. After the explants of 

petiole with cotyledon were pre-cultured on the shoot 

induction medium for 3 d, they were incubated in the 

bacterial suspension for 15–20 min. Excessive fluid was 

removed by placing explants on filter paper. The explants 

were subsequently co-cultivated on a shooting medium 

containing 10 mg L 1 kanamycin for 3 d, and were then 

transferred to a shoot induction selective medium 

supplemented with 10 mg L 1 

of kanamycin and 

300 mg L 1 of ampicillin. Finally, the resulting plants 

were transferred to soil in pots. 

PCR analysis 

Genomic DNA was extracted from approximately 

0.5–1 g of fresh young leaves of putative transformed 

and non-transgenic plants. PCR was later performed in a 

GeneAmp PCR system (Applied Biosystem). The primers 

used for the amplification of a 371-bp-fragment of the 

uidA gene were 5 0 -ACGTCCTGTAGAAACCCAACC-3 0 and 

5 0 - TCCCGGCAATAACATACGGCGT-3 0 (Ying et al., 1999). 

The reaction mixture was composed of 1 Taq polymerase 

buffer, 2.0 mM MgCl2, 0.06 mM dNTPs, 0.5 mM of 

each primer, 1 unit Taq polymerase (Sangon), and 

approximately 50 ng of total plant DNA as template in a 

total volume of 25 mL. The reaction parameters were 

95 1C for 3 min, followed by 30 cycles of 95 1C for 30 s, 

58 1C for 30 s, and 72 1C for 60 s. A final extension 

step was carried out at 72 1C for 7 min. The PCR products 

were then subjected to electrophoresis using a 1.0% 

agarose gel. 

DNA gel blot analysis 

The genomic DNA of the transgenic line was digested 

with EcoRI for DNA hybridization to confirm the integration 

of the antisense gene of BcMF5 into the recipient 

genome. The digested genomic DNAs were fractionated 

on 0.8% agarose gel and then transferred onto a 

nylon membrane (Amersham), before being hybridized to 

intron Sense 

NOS-pro NPTII NOS-ter A9-Pro 

GUS NOS-ter 

Hind III 

Sph I 

Pst I 

Xba I 

Figure 1. Structure of the plant expression vector pBIA9-RMF5. The T-DNA region of pBI101 containing the NPTII (Kan R ) 

gene is illustrated, followed by the nopaline synthase polyadenylation site (NOS-ter). The 159-bp antisense fragment 

and the 459-bp sense fragment with the intron of the BcMF5 were directionally inserted between the A9 promoter and 

the b-glucuronidase gene (GUS). 

Sma I 

Bam H I 

LB

448 

a 32 P-labeling probes according to the supplier’s instructions 

(Beijing Yahui Biomedical Engineering Ltd. Co.). The 

DNA probes were prepared by labeling the NPTII gene 

using a random primed labeling method as described in 

the literature (Sambrook et al., 1989). 

RNA gel blot analysis 

Total RNA was extracted from floral buds of the 

transgenic line and its non-transformed plants using an 

RNA Isolation Kit (GIBCO BRL R , Life Technologies). The 

RNA of 20 mg was fractionated on 1.2% (w/v) agarose gel 

containing formaldehyde and then transferred onto a 

nylon membrane (Amersham) before being probed with 

a 32 P-labeling following the same procedure of DNA 

hybridization. However, the probe DNA resulted from 

the 252-bp open reading frame of the BcMF5 gene. 

Pollen germination test of the transgenic plant and 

scanning electron microscope analysis 

Pollen germination was tested by the culture medium 

containing 15% sucrose, 0.01% HBO3, and 1 mg L 1 GA3 . 

At least 10 flowers from each plant were tested. After 

incubating for 4 h, germinating and non-germinating 

pollen grains were counted, and pollen from the 

transgenic line and the non-transformed plants was 

scanned by an electron microscope, respectively. 

Isolation of the BcMF5 promoter 

Using the same process as that described in Liu and 

Whittier (1995), TAIL-PCR was utilized in order to isolate 

the BcMF5 promoter. Three reverse-specific primers A1, 

A2, and A3 were designed based on the cDNA sequence 

of BcMF5. The primer sequence was as follows: A1: 

5 0 -TTTGGCACATTGGGCTTTA-3 0 , A2: 5 0 -GGCACATGGGCTT- 

TACATT-3 0 , and A3: 5 0 -GACAATTAAGCGATGATCGATA-3 0 . 

The three arbitrary primers used were AD1: 5 0 -NG- 

TCGASWGANAWGAA-3 0 , AD2: 5 0 -TGWGNAGSANCASA- 

GA-3 0 , and AD3: 5 0 -AGWGNAGWANCAWAGG-3 0 . Primers 

were synthesized by Sangon (Shanghai, CN). The PCR 

products were reclaimed using the V-GENE DNA Gel 

Extraction Kit (Hangzhou, CN) and cloned into a pGEM-T 

easy vector (Promega, USA). They were then transformed 

into DH5a-competent cells, before white clones were 

selected and their plasmids were extracted. After 

identification by PCR amplification and digestion by 

EcoR1, the recombinant clone was sequenced by 

invitrogen (Shanghai, CN). 

Expression of the BcMF5 promoter in Arabidopsis 

The 609-bp promoter fragments upstream ATG of 

BcMF5 were inserted before the GUS of pBI121 (Clonetech), 

thereby replacing the original CaMV35S promoter 

to construct the expression vectors of the BcMF5 

promoter. The expression vector was transferred to 

Agrobacterium LBA4404 via the freezing–thaw method. 

It was then transformed to Arabidopsis by the floral 


dipping method. Transgenic plants were selected in an MS 

medium containing 50 mg L 1 kanamycin, and the expression 

pattern of the BcMF5 promoter in transgenic 

Arabidopsis plants was checked by the GUS histochemistry 

assay experiment, according to the procedure of 

Jefferson et al. (1987), and as modified by Kosugi et al. 

(1990). The anther or pollen was placed in a 100 mL GUS 

assay solution and incubated at 37 1C for 4–5 h. The GUS 

assay solution contained 1.9 mM X-Gluc (Sangon, Shanghai, 

CN), 20% methanol, 0.5 mM potassium ferricyanide, 

0.5 mM potassium ferrocyanide, and 0.3% Triton X-100 in 

a 0.1 M sodium phosphate buffer (pH 7.0). 

Results 

Sequence composition of BcMF5 

Through the combination of the cDNA-AFLP and 

the RACE methods, the full-length BcMF5 cDNA 

sequence was determined. It contained an ORF of 

249 bp encoding a small, 83-aa protein with a 

putative 26-aa hydrophobic signal peptide in the 

N-terminal and a conservative eight-cysteine amino 

residue (Figure 2). Database searches revealed that 

the amino acid sequence of BcMF5 is the same as 

that of PCPA2 and SLR-BP (Figure 2B). Its genomic 

structure was determined by PCR by using primers 

whose design is based on the extreme 5 0 and 3 0 ends 

of the coding region. The DNA of BcMF5 had a 648bp 

length containing a single 268-bp intron, whose 

size and location were identical to those of PCPA2 

and SLR1-BP (Figure 2A), suggesting that BcMF5 

belongs to the class A PCP gene family and might 

play a role in the pollen–stigma adhesion process. 

Expression of BcMF5 by RT-PCR 

Using the cDNAs of the I–V flower buds, open 

flowers, germinal silique, and flower stems and leaves 

of both the fertile line and sterile line of ‘ZUBajh97- 

01A/B’ as templates, RT-PCR was performed using a 

pair of primers located in the coding region to analyze 

the temporal and spatial expression patterns of 

BcMF5. The results showed that BcMF5 expressed in 

stage III flower buds (diameter: 1.6–2.2 mm), stage IV 

flower buds (diameter: 2.2–2.8 mm), and open flowers 

in the fertile line, and is not expressed in the male 

sterile line at all (Figure 3). This demonstrated that 

BcMF5 belonged to the late-expressed PCP gene which 

decided pollen fertility. 

Plant regeneration and selection of 

transgenic plants 


An earlier study developed an efficient method 

for plant regeneration from petiole with the

Bajh97-01A/B 

Actin 

cotyledon of the Chinese cabbage-pak-choi (Cao 

et al., 2000). In this study, the MS medium, with half 

strength of NH 4 + and supplemented with 4 mg L 1 

6-BA, 1 mg L 1 NAA, and 7.5 mg L 1 silver nitrate 

solution, was found to be optimal in obtaining a high 

frequency of shoot regeneration in flowering Chinese 

cabbage. The optimum conditions for gene transformation 

to flowering Chinese cabbage were estab- 



Figure 2. (A) Nucleotide sequence of BcMF5 and its deduced amino acid sequence. The shadowed region is the intron of 

BcMF5; the underlined amino acid is the putative signal peptide of BcMF5. (B) Alignment of the amino acid sequences of 

BcMF5, PCPA2, and SLR-BP. Asterisks indicate eight conservative cystine residues of PCP. 

m1 w1 m2 w2 m3 w3 m4 w4 m5 w5 mF wF mSi wSi mS wS mL wL 

Figure 3. RT-PCR results of BCMF5 in different organs of ‘Bajh97-01A/B.’ The symbols m1, m2, m3, m4, m5, mSi, mS, 

and mL indicate stage I flower bud (o1.0 mm), stage II flower bud (1–1.6 mm), stage III flower bud (1.6–2.2 mm), stage 

IV flower bud (2.2–2.8 mm), stage V flower bud (2.2–2.8 mm), open flower, germinal silique, stem, and leaf of sterile 

line, respectively, while w1, w2, w3, w4, w5, wF, wSi, wS, and wL indicate stage I flower bud (o1.0 mm), stage II flower 

bud (1–1.6 mm), stage III flower bud (1.6–2.2 mm), stage IV flower bud (2.2–2.8 mm), stage V flower bud (2.2–2.8 mm), 

open flower, germinal silique, stem, and leaf of fertile line, respectively. Action external control. 

lished as follows: 3 d for pre-culture, LBA4404 strain 

for A. tumefaciens, 10–15 min of infection, and 3 d 

for co-culture. Explants gathered after co-culturing 

under optimum conditions were inoculated into the 

regeneration medium containing 10 mg L 1 kanamycin, 

before the Kan R shoots were obtained (Figure 4). 

Afterwards, the explants were sub-cultured on the 

same medium every 3 weeks, before finally obtaining

450 

the BcMF5 transgenic plants of flowering Chinese 

cabbage. 

Integration and transcription confirmation of 

the hpRNA of the BcMF5 gene in transgenic 

plants of pBIA9-RMF5 

To confirm the hpRNA of the BcMF5 gene in the 

regenerated plants of flowering Chinese cabbage, 

19 T0 plants of pBIA9-RMF5 were subjected to PCR 

analysis with the primers specific for the uidA gene. 

Both PCR (Figure 5A) and the two transgenic lines 

TB6 and TB13 for DNA gel blot analysis (Figure 5B) 

confirmed that the hpRNA of the BcMF5 gene had 

integrated into the genome of the flowering 

Chinese cabbage transgenic seedling of pBIA9- 

RMF5. Data from the DNA hybridization showed 

that it was a single copy in the assayed transgenic 

lines TB6 and TB13 (Figure 5B). In addition, the RNA 

hybridization results for both TB6 and TB13 

indicated that the expression of the BcMF5 gene 

decreased seriously in the transgenic line of pBIA9- 

RMF5 (Figure 5C), thus confirming that the normal 

expression of BcMF5 in the flowering Chinese 

cabbage was inhibited by hpRNA-mediated RNA 

interference in the transgenic plant. 



Figure 4. The BcMF5 RNAi transgenic plantlets of flowering Chinese cabbage were obtained by the Agrobacterium 

tumefaciens-mediated genetic transformation method. (A) Seedling for 4–5 d; (B) cotyledon-hypocotyl explants during 

co-culture; (C) after Kan R buds were induced. (D) Roots induced from the Kan R seedling; (E) transgenic plantlets of 

flower Chinese cabbage flowering in the test tube; and (F) regenerated plants transferred to the plot. 

Pollen germination test and scanning 

electron microspore analysis of transgenic 

plants 

To further understand the effect of RNA interference 

on pollen development of the transgenic 

plant, the germination ability of the pollen was 

tested (Figure 6). The results showed that the 

pollen germination percentage of the transgenic 

plant of pBIA9-RMF5 was 38.74%. In contrast, pollen 

from the non-transgenic plant could germinate with 

a frequency of approximately 80.24% (Table 1). 

Further, pollen of the transgenic plant of pBIA9- 

RMF5 and the non-transgenic plant was detected by 

use of a scanning electron microscope, and the 

results indicated that there was a significant 

difference between transgenic plants and their 

non-transgenic counterparts (Figure 7). Compared 

with the pollen of the non-transgenic plant, most of 

the pollen of the transgenic plant of pBIA9-RMF5 

was abnormal and the pollen germination furrow 

collapsed. The results showed that the percentage 

of abnormal pollen of pBIA9-RMF5 in the 402 pollen 

grains assayed was 88.37%, while that of the nontransgenic 

plant in the 282 pollen grains assayed 

was 7.9% (Table 2). This illustrated that RNA

interference had a significant role in the pollen 

development of the transgenic plant and resulted 

in lower pollen germination ability as well as pollen 

abnormality. 

Expression profile analysis of the BcMF5 

promoter in Arabidopsis 

We have shown that BcMF5 was specifically 

expressed in the late stage of pollen development. 

To understand the function of the BcMF5 promoter 

in the process of its transcription, a 620-bp 

sequence upstream ATG was obtained from Brassica 

campestris by TAIL-PCR for the first time. A further 

database search did not find its homology sequence, 

showing that it is an unpublished promoter. 

To identify its temporal and spatial expression 

pattern, the 609+3-bp promoter-GUS fusion vector 

was constructed. Subsequently, its expression 

profile in Arabidopsis was studied through GUS 

histochemistry staining (Figure 8). The results 

indicated that the BcMF5 promoter began expres- 



400 

300 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 

1 2 3 4 1 2 3 

Figure 5. Molecular analysis of transgenic flowering Chinese cabbage. (A) shows the PCR results of transgenic flowering 

Chinese cabbage. Lane 1: GeneRuler TM 100-bp DNA ladder; lanes 2– 20: PCR products amplified from genomic DNA of 

transgenic flowering Chinese cabbage; lane 21: non-transformed Chinese cabbage plant. (B) DNA gel blot analysis of 

transgenic line TB-6 and TB-13 of flowering Chinese cabbage. Lane 1: the external plasmid control; lane 2: the 

nontransgenic plant; lanes 3–4: the transgenic line of TB-6 and TB-13. The genomic DNA of the transgenic plants and 

their controls was digested by EcoRI and hybridized with 32 P-labeled NPTII-specific probe. (C) RNA gel blot analysis of 

the transgenic line TB-6 and TB-13 of pBIA9-RMF5. Electrophoresis of the floral bud total RNA through gels containing 

formaldehyde (down) and its result of RNA hybridization (upper). Lane 1: floral bud total RNA of the non-transgenic 

flowering Chinese cabbage; lanes 2–3: floral bud total RNA of the transgenic line TB-6 and TB-13 of pBIA9-RMF5. 

sion 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). It did not, 

however, drive any expression in petals, sepals, or 

pistils, which was relatively consistent with the 

expression pattern of BcMF5 in flowering Chinese 

cabbage (Figure 3). 

Discussion 

Pollination in flowering plants is a highly specialized 

process that culminates in the fertilization of 

the ovule by a male gamete (Doughty et al., 2000). 

This reproductive process depends on highly specific 

interactions between pollen and the pistil with 

highly discriminatory interspecific and intraspecific 

recognition, both of which allow the pistil to 

distinguish among genetically diverse ranges of 

pollen grains arriving at the stigma (Takayama 

et al., 2000a). The adhesion of pollen to the stigma 

is the first step in the pollination of flowering

452 

plants. Control of pollen acceptance by adhesion is 

particularly important in species with a dry stigma, 

such as members of the Brassicaceae. In addition, 

PCPs are an extensive family characterized by high 

polymorphism and are gametophysically expressed 

small cysteine-rich proteins. To date, PCP Class A 

(PCP-A) is the only group of pollen coat proteins 

identified that is suggested to play a key role in the 

pollen–stigma interaction and pollen recognition. It 

is also highly polymorphic around the conserved 

cysteine backbone (Doughty et al., 2000). In this 

study, we isolated and characterized a novel 

member of the PCP-A family featuring eight 

conservative cysteine residues from the GMS A/B 

line ‘ZUBajh97-01A/B,’ named as BcMF5. The 

size of its intron and its deduced amino acid 

sequence were found to be identical to PCPA2 and 


Figure 6. Observations on pollen germination of transgenic and non-transgenic flowering Chinese cabbage. (A) and (B) 

are the pollen germinated from the non-transgenic plant, at magnitudes of 160 and 640 times, respectively. (C) and (D) 

are the pollen germinated from the transgenic plant, at magnitudes of 160 and 640 times, respectively. Bars of 

magnitude 160 and 640 times are 100 and 40 mm, respectively. 

Table 1. Pollen germination assay of the transgenic plant of pBIA9-RMF5 

Types of plantlets Total no. of pollen 

(pellet) 

No. of germinated pollen 

(pellet) 

Frequency of pollen 

germination (%) 

WT 812 654 80.2474.27 

pBIA9-RMF5 588 232 38.7475.6 

The data in the table are means7SD from four measurements. 


SLR-BP, except the difference in length of 5 0 in the 

upstream region and 3 0 in the downstream region 

(unpublished data). Temporal and spatial expression 

analysis revealed that BcMF5 expressed 

in stage III flower buds (diameter: 1.6–2.2 mm), 

stage IV flower buds (diameter: 2.2–2.8 mm), 

and stage V flower buds (open flowers) in the 

fertile line of ‘ZUBajh97-01A/B.’ However, it did 

not express in the male sterile line at all, which 

demonstrated that BcMF5 is a late-expressed PCP 

gene related to the process of deciding pollen 

fertility. 

Reverse genetics has become a strong tool in 

the study of gene functions in the post-genomic 

era. Thus, to understand the role of BcMF5 in 

the process of microspore development and 

pollen–stigma interaction, hpRNA-mediated RNA

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 



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

454 

stages, as well as in the microspore at the late 

developmental stages (Takayama et al., 2000b). 

This showed an apparent sporophytic/gametophytic 

expression pattern. In future studies, the 

expression pattern of BcMF5 should be confirmed 

using in situ hybridization. 

Acknowledgments 

This work was supported by the Natural Science 

Foundation of China (No. 30370975) and the 

Key Sci-technology Project of Zhejiang Province 

(No. 2005C12019-02). 

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