Brassica campestris L. ssp. chinensis M

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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 ARTICLE IN PRESS 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 Q. Zhang et al. 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- ARTICLE IN PRESS Functional analysis of a novel PCP gene BcMF5 449 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
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Brassica campestris L. ssp. chinensis M

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