Heterologous Expression of the Secale cereal Thaumatin-Like Protein in Transgenic Canola Plants Enhances Resistance to Stem Rot Disease

Canola (Brassica napus L.) is an important oilseed crop. A serious problem in cultivation of this crop andyield 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 tlp geneisolated from cereal rye (Secale cereal L.) was introduced into canola plants. The amplified DNA fragment(about 500 bp) was analyzed and confirmed by restriction pattern and cloned into pUC19 and designated aspUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains nointron. The tlp gene was predicted to encode a protein of 173 amino acids with an estimated molecular massof 17.7 kDa. The deduced amino acid sequence of TLP showed a significant sequence identity with TLPfrom S.cereal and other plants. We used a transgenic over-expression approach in order to investigate antifungal activity of expressed TLP on Sclerotinia sclerotiorum. TLP was overexpressed under the CaMV35Sconstitutive promoter in (Brassica napus, R line Hyola 308). Transformation of cotyledonary petioles wasachieved via Agrobacterium tumefaciens LBA4404. The insertion of transgene was verified by the polymerasechain reaction (PCR) and genomic DNA dot blotting. Antifungal activity was detected in transgeniccanola lines using detached leaf assay. The size of lesions induced by S. sclerotiorum in the leaves oftransgenic canola was significantly retarded when compared to that detected in non-transgenic plants.