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. 2006 Mar;18(3):676–687. doi: 10.1105/tpc.105.038240

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Copyright © 2006, American Society of Plant Biologists

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Figure 7. — Processing of B-atp6/orf79 mRNA and Suppression of the ORF79 Production by the Rf Genes.

(A) Transcript profile of B-atp6/orf79 in transgenic plants with Rf1a assayed by RNA gel blot analysis. The relatively low signal intensity of the 1.4- and 1.0-kb bands was due to the relatively smaller portion of the DNA fragment III probe (see Figure 1A) hybridizing to atp6. The 1.4-kb band (N-atp6) also served as a loading control. ML, maintainer line.

(B) CR-RT-PCR analysis of the 5′ and 3′ termini of the primary N-atp6 transcript from a maintainer line (lanes 1 and 2), the processed B-atp6 (lanes 3 and 4), and orf79 (lanes 5 and 6) mRNA fragments from an Rf1a transgenic plant using the primer pairs P5/P6, P7/P6, and P9/P10, respectively (see Figure 1A). The RNA samples were treated with (lanes 1, 3, and 5) and without (lanes 2, 4, and 6) TAP before the RNA ligation reaction. M, molecular weight marker.

(C) Transcript profile of B-atp6/orf79 in transgenic and fertility-restored hybrid plants with Rf1b.

(D) Transcript profile of B-atp6/orf79 in fertility-restored hybrid plants with Rf1a alone or both of Rf1a and Rf1b.

(E) Immunoblot analysis probed with an antibody to ORF79. Proteins were prepared from anthers containing microspores of a CMS-BT line (lanes 3 and 4), fertility-restored transgenic plants with Rf1a (lane 1) or Rf1b (lane 2), fertility-restored hybrid plants carrying Rf1a and Rf1b (lanes 5 and 6), and a maintainer line (lane 7). The specific band (lanes 3 and 4) for ORF79 is indicated with an arrow, and the top one marked with an asterisk is a cross-reacting unknown protein.

(F) Immunoblot analysis of ORF79 using proteins prepared from microspores of fertility-restored transgenic plants with Rf1a (lane 1), mitochondria of seedling leaves of the CMS-BT line (lane 2), microspores of the CMS-BT line (lanes 3 and 4), and anther wall tissue not including the microspores (lanes 5 and 6). The cross-reacting unknown protein, as well as ORF79, was not detected in the mitochondria of the seedling leaves of the CMS-BT line (lane 2).