Expression Patterns of PR proteins with Different Extract Methods during Germination of Rape Seed (Brassica napus L.)

This study was conducted to investigate the expression patterns of pathogenesis-related proteins (chitinase, β-1,3-glucanase and peroxidase) using activity staining of native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE during germination of rape seed (Brassica napus L. cv. Saturnin). The crude enzymes were extracted by distilled water (DW, pH 6.0) and 100 mM K-PO4 buffer (pH 7.0). The expression patterns of chitinase isozymes changed clearly on 10% native-PAGE gel with DW and K-PO4 buffer extract and on 12% SDS-PAGE gel with K-PO4 buffer extract, except for 12% SDS-PAGE conducted using DW during germination. The active bands of the chitinase isozymes were observed as four major bands (ch1, ch2, 86, and 78 kDa) and three minor bands (71, 60, and 54 kDa) on 10% native-PAGE gel conducted using DW and K-PO4 buffer extract. The two active bands on the 12% (w/v) SDS-PAGE gel presented as 34 and 29 kDa with DW extract, whereas one active band of 34 kDa was observed when the K-PO4 buffer extract was used. Active bands of β-1,3-glucanase isozymes changed slightly on 10% native-PAGE gel with DW and K-PO4 buffer extract during germination. The active band of β-1,3-glucanase isozymes were shown to have a high molecular weight (G1 and G2) on native-PAGE gel with DW extract at 0, 1, 2, and 3 days after germination, but not at 4 and 5 days. One active band of β-1,3-glucanase presented as G1 in the K-PO4 buffer extract. Active staining of peroxidase was stronger earlier in the DW extract than K-PO4 buffer extract at 2 days. The active bands showed as P1 and P2 in both DW and K-PO4 buffer extract at 5 days after germination.