Enhancement of Trichoderma endochitinase secretion in tobacco cell cultures using an α-amylase signal peptide

An α-amylase signal peptide from rice was synthesized and fused with endochitinase ( ech 42) gene cloned from Trichoderma virens. The chimeric gene was designated as PSP α-amyech 42, and this was transferred to a plant transformation vector, referred to as pMASGK. Leaf explants of tobacco cv White Burley were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying ech 42 with its own signal peptide( ech 42SP) and PSP α - amyech 42(pMASGK) separately. Putative transformants were selected on Murashige and Skoog (MS) medium, supplemented with 1 mg/l bezyladenine (BA), 0.5 mg/l naphthalene acetic acid(NAA), and containing 200 mg/l kanamycin and 200 mg/l cefotaxime. Transformation was further confirmed by PCR with specific primers and Southern blot hybridization. Endochitinase secretion was quantified in 1-week-old cell suspension cultures obtained from 3-week-old callus cultures of transformants carrying PSP α - amyech 42, transformants with ech 42 and of control (untransformed) plant. Callus cultures of PSP α - amyech 42 showed higher endochitinase activity (9–12 times) than those carrying ech 42SP (7–8 times) in both medium and cell extracts. Media collected (200 μg of total protein) from PSP α - amyech 42 suspension cultures in Potato Dextrose Agar plates showed growth inhibition of 73 and 53% against Sclerotium rolfsii and Rhizoctonia bataticola , respectively, whereas media collected (200 μg of total protein) from ech 42SP suspension culture showed inhibition of 14 and 24% against Sclerotium rolfsii and Rhizoctonia bataticola , respectively.