Brassica campestris L. ssp. chinensis M

More documents

Recommendations

Info

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
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. ARTICLE IN PRESS 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
Page 1: Journal of Plant Physiology 165 (20
Page 5 and 6: Bajh97-01A/B Actin cotyledon of the
Page 7 and 8: interference had a significant role
Page 9 and 10: interference has been adopted to re
Page 11: interacts with S-locus glycoprotein

Brassica campestris L. ssp. chinensis M

Create successful ePaper yourself

Delete template?

Save as template?