Isolation of chitinase gene induced during infection of vicia faba by botrytis fabae

ABSTRACT

The moderately resistant [Giza 716] and the susceptible [Giza 429] faba bean cultivars were used to identify some pathogenesis related proteins [PRs] associated with infection by chocolate spot disease. One isolate of Botrytis fabae purified from a plant sample taken from Nubaria location [Behera governorate, Egypt] was used in the artificial infection experiment. Qualitative and quantitative analyses were carried out on all protein banding patterns of the healthy and the infected faba bean leaves harvested at 8, 24 and 48 hr after inoculation. Data revealed that a 26 kDa protein band was more intensive 8, 24 and 48 hr after inoculation in cultivar Giza 716,. In addition, a 29 kDa protein band appeared after 24 and 48 hr. Furthermore, in cultivar Giza 429, 54 kDa protein bands appeared after 8, 24 and 48 hr post inoculation and 28 and 20 kDa appeared after 24 hr post inoculation.Reverse-Transcription [RT-PCR] showed that chitinase gene is expressed at very early stages in infected faba bean leaves. DNA fragment at molecular weight 900 bp appeared at 8, 24 and 48 hr after inoculation and disappeared in the healthy plants. The amplified products were cloned into pGEM-T Easy vector. Four clones named [PNAM1, PNAM2, PNAM3 and PNAM4] were selected for validation. The recombinant plasmids PNAM1, PNAM2 were verified for the presence of the Chitinase gene coding sequences by using both specific and universal primers in PCR. BNAM1-Chit-EG gene sequence showed 58.15% similarity when aligned with other Chitinase genes published in the gene bank