Detailed analysis of rice chitinase gene expression in transgenic cucumber plants showing different levels of disease resistance to gray mold (Botrytis cinerea)

摘要

The class I chitinase cDNA (RCC2) of rice driven by the CaMV 35S promoter was introduced into cucumber by Agrobacterium-mediated transformation. The transgenic cucumbers showed varying levels of disease resistance to gray mold. We selected three transgenic lines representing different resistance levels, namely the most resistant line (CR32), an intermediate-resistance line (CR3), and a susceptible line (CR20), and studied the correlation between RCC2 expression and disease resistance. ELISA analysis showed that the rice chitinase levels of CR32 and CR3 were higher than that of CR20. RCC2 expression was then examined in more detail using an in situ indirect fluorescent antibody technique and 3-D fluorescence microscopy (CELL Scan system). A homogeneous distribution of cells expressing RCC2 to a high degree was observed in the epidermal and mesophyll cells of CR32 leaves, whereas the intensity of RCC2 expression in CR3 leaves was lower. However, CR3 often showed high expression of RCC2 in parts of leaf tissue. Rice chitinase was localized within cells but was not detected between cells. Optical microscopy of Botrytis cinerea infection behavior revealed that none of the transgenic lines nor any of the non-transgenic lines inhibited the penetration of B. cinerea. However, fungal growth within leaf tissue was suppressed in the resistant lines but not in the non-transformed or CR20 leaf tissue. Thus, we speculate that high expression and intracellular localization of rice chitinase may be involved in enhancing the resistance of transgenic plants to gray mold.