Heterologous expression of the Secale cereal thaumatinlike protein in transgenic canola plants enhances resistance to stem rot disease

Abstract

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