1) China National Rice Research Institute, Hangzhou, China
2) Zhejiang Agricultural University, Hangzhou, China
Near-isogenic rice lines K79 and K80 are susceptible and resistant to
Pyricularia oryzae (the pathogen of rice blast), respectively. To determine the
biochemical basis of the differences in blast resistance between the two
cultivars, the separation of soluble proteins was conducted by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE).
One gram of 10-day-old seed lings of each line was ground separately in 1.5 ml 0.25% glycylglycine with silica and PVP. Centrifugation was conducted at 10,000 rpm for 10 minutes, and the supernatants were mixed with equal volums of sample buffer before loading the gel. Sample buffer (0.06 M Tris-HCl, pH 6.8) contained 2% SDS, 5% 2-mercaptoethanol and 10% glycerol. Running buffer consisted of 50 mM Tris-Hcl (pH 8.4), 0.1% SDS and 0.38 M glycine. Samples were run in the 9% stacking gel for 2 hours at 80 volts and in the 18% separation gel for 5 hours at 160 volts. The gel was stained in Coomassie blue R-250 solution for 2 hours, then destained overnight.
The SDS-PAGE pattern (Fig. 1) showed that a difference between the number of protein bands exists between K79 and K80, i.e., several bands near cathode in resistant K 80 cannot be observed in susceptible cv K79. The genes coding for these proteins must be constitutive, because no inoculation of the pathogen was conducted. The differential protein pattern may be responsible for the resistance of K80, since the agronomic characters of the two near-isogenic lines were identical except for the difference in blast resistance. Further studies are being conducted to characterize the proteins that are present only in K80.
Fig. 1. The SDS-PAGE pattern of protein bands from the near-isogenic
lines K80 (land A) and K79 (lane B).