ABSTRACT
Canola [Brassica napus L.] is an important oilseed crop. A serious problem in cultivation of this crop and yield loss, are due to fungal disease stem rot caused by Sclerotinia sclerotiorum. The pathogenesis-related [PR] proteins have the potential for enhancing resistance against fungal pathogen. Thaumatin-like proteins [TLPs] have been shown to have antifungal activity on various fungal pathogens. In this study, the tip gene isolated from cereal rye [Secale cereal L.] was introduced into canola plants. The amplified DMA fragment [about 500 bp] was analyzed and confirmed by restriction pattern and cloned into pUC19 and designated as pUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains no intron. The tip gene was predicted to encode a protein of 173 amino acids with an estimated molecular mass of 17.7 kDa. The deduced amino acid sequence of TIP showed a significant sequence identity with TLP from S.cereal and other plants. We used a transgenic over-expression approach in order to investigate anti-fungal activity of expressed TLP on Sclerotinia sclerotiorum. TLP was overexpressed under the CaMV35S constitutive promoter in [Brassica napus, R line Hyola 308]. Transformation of cotyledonary petioles was achieved via Agrobacterium tumefaciens LBA4404. The insertion of transgene was verified by the polymerase chain reaction [PCR] and genomic DMA dot blotting. Antifungal activity was detected in transgenic canola lines using detached leaf assay. The size of lesions induced by S. sclerotiorum in the leaves of transgenic canola was significantly retarded when compared to that detected in non-transgenic plants