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 B...

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Bibliographic Details
Published in:Plant cell, tissue and organ culture tissue and organ culture, 2011-11, Vol.107 (2), p.215-224
Main Authors: Kumari, Archana, Sharma, Gaurav, Bhat, Sumangala, Bhat, Ramesh S., Krishnaraj, P. U., Kuruvinashetti, M. S.
Format: Article
Language:English
Subjects:
Acetic acid
Amylases
Biological and medical sciences
Biomedical and Life Sciences
Biotechnology
Callus
Cefotaxime
Cell culture
Culture media
Dextrose
Endochitinase
Explants
Expression vectors
Fundamental and applied biological sciences. Psychology
Fungi
Genetic transformation
Hybridization
Inhibition
Kanamycin
Life Sciences
Naphthalene
Original Paper
Peptides
Plant Genetics and Genomics
Plant Pathology
Plant Physiology
Plant Sciences
Potatoes
Primers
Proteins
Rhizoctonia bataticola
Sclerotium rolfsii
Secretion
Suspension culture
Tobacco
α-Amylase
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Summary: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.
ISSN:0167-6857
1573-5044
DOI:10.1007/s11240-011-9972-0