TRANSFORMATION OF LTP GENE INTO Brassica napus TO ENHANCE ITS RESISTANCE TO Sclerotinia sclerotiorum

摘要

Rapeseed (Brassica napus L.) is one of the most important economic crops worldwide, and Sclerotinia sclerotiorum is the most dangerous disease that affects its yield greatly. Lipid transfer protein (LTP) has broad-spectrum anti-bacterial and fungal activities. In this study, B. napus was transformed using Agrobacterium tumefaciens harboring the plasmid-containing LTP gene to study its possible capability of increasing plant's resistance. First, we optimized the petiole genetic transformation system by adjusting the days of explants, bacterial concentrations, ratio of hormones, and cultivating condition. Second, we obtained 8 positive plants by PCR analysis of T_0 generation. The PCR results of T_1 generation were positive, indicating that the LTP gene had been integrated into B. napus. Third, T_1 transgenic plants inoculated by detached leaves with myce-lia of S. sclerotiorum showed better disease resistance than non-transformants. Oxalic acid belongs to secondary metabolites of S. sclerotiorum, and several studies have demonstrated that the resistance of rapeseed to oxalic acid is significantly consistent with its resistance to S. sclerotiorum. The result from the seed germination assay showed that when T_1 seeds were exposed to oxalic acid stress, their germination rate was evidently higher than that of non-transformant seeds. In addition, we measured some physiological changes in T_1 plants and control plants under oxalic acid stress. The results showed that T_1 transgenic plants had lower malondialde-hyde (MDA) content, higher super oxide dismutase (SOD), and peroxidase (POD) activities than non-transformants, whereas disease resistance was related to low MDA content and high SOD and POD activities.