Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct

• Published in: Plant cell reports 2006-09, Vol.25 (9), p.942-952
• Main Authors: Melander, M, Kamnert, I, Happstadius, I, Liljeroth, E, Bryngelsson, T
• Format: Article
• Language: English
• Subjects: Alternaria brassicae; Alternaria brassicicola; barley; beta-glucanase; Biological and medical sciences; Biotechnology; Blotting, Western; Brassica napus; Brassica rapa - genetics; chitinase; Chitinases - genetics; Chitinases - metabolism; disease resistance; Fundamental and applied biological sciences. Psychology; fungal diseases of plants; Fungi - drug effects; Gene Expression; gene expression regulation; Genetic engineering; Genetic technics; genetic transformation; Glucan Endo-1,3-beta-D-Glucosidase - genetics; Glucan Endo-1,3-beta-D-Glucosidase - metabolism; Haploidy; Hordeum - genetics; Hordeum vulgare; Leptosphaeria maculans; Methods. Procedures. Technologies; pathogenesis-related proteins; Plant Diseases - genetics; Plant Diseases - microbiology; plant pathogenic fungi; plant proteins; Plants, Genetically Modified; polymerase chain reaction; Sclerotinia sclerotiorum; Southern blotting; Thanatephorus cucumeris; transgenes; Transgenes - genetics; Transgenic animals and transgenic plants; Transgenic plants; Trichoderma; Verticillium longisporum; Western blotting
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-006-0153-2

A double-gene construct with one chitinase and one β-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and β-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The β-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified β-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.