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

Document Type: Research Paper

Authors

1 National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran

2 Department of Biology, Kharazmi University, P.O. Box 31979-37551, Tehran, I.R. Iran.

3 National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.

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 tlp gene
isolated 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 as
pUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains no
intron. The tlp 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 TLP showed a significant sequence identity with TLP
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
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 DNA 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.

Keywords