Figure 6.

BNM2 BURP domain expression analysis. A, RNA gel-blot analysis of BNM2 gene expression in rapeseed pollen and MDE cultures. Total RNA was isolated from microspores at the start of culture (0); from embryo cultures after 4 d in culture at 32°C (E); from nonembryogenic heat-stressed microspore cultures (NE) obtained by culturing microspores for 1 d at 25°C, followed by 3 d at 32°C; from pollen cultures after 4 d at 25°C (P); and from purified MDEs at the globular (G), heart (H), torpedo (T), 21-d cotyledon (C1), 28-d cotyledon (C2), and 28-d cotyledon stage (C3). B, RNA gel-blot analysis of BNM2 transcripts in developing seeds. RNA samples were isolated from whole seeds collected at specific days after pollination (DAP). These developmental time points correspond approximately to the globular (7 DAP), heart/torpedo (14 DAP), early cotyledon (21 DAP), midcotyledon (28 DAP), and mid-to-late cotyledon (35 and 42 DAP) stages of development. C, RNA gel-blot analysis of BNM2 transcripts in nonseed tissues. RNA samples were isolated from mature leaves (L), roots (R), and stem (S), flowers at anthesis (F), small flower buds (SB, <3 mm), large flower buds (LB, 5–7 mm), and anthers (A) and pistils (P) and from flowers at anthesis. For A, B, and C, 10 μg of total RNA was loaded per sample. Ethidium bromide staining of RNA was used to compare sample loading. D to G, mRNA in situ hybridization analysis of BNM2 expression in MDEs (D and E) and seeds (F and G). The probe used for RNA gel-blot analysis and mRNA in situ hybridization detects at least two duplicated copies of the BNM2 gene in the amphidiploid rapeseed genome.