Brassica campestris L. ssp. chinensis M

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452 plants. Control of pollen acceptance by adhesion is particularly important in species with a dry stigma, such as members of the <strong>Brassica</strong>ceae. 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 ARTICLE IN PRESS 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. Q. Zhang et al. SLR-BP, except the difference in length of 5 0 in the upstream region and 3 0 in the downstream region (unpublished data).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 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 <strong>Brassica</strong> 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
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Brassica campestris L. ssp. chinensis M

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