Independent Transgenic Tobacco Research Articles

Expression of active hen egg white lysozyme in transgenic tobacco

A full-length complementary DNA clone of hen egg white lysozyme (HEWL) was inserted into pBI121 vector lacking the β-glucuronidase (GUS) gene and used to transform tobacco. The HEWL gene had its own 18-amino acid signal peptide. HEWL activity was detected using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. The enzyme was active against Micrococcus luteus cells and migrated at the same molecular mass as purified mature HEWL. The protein was also found by immunodetection of HEWL after SDS-PAGE. Twenty-seven independent transgenic tobacco plants were analyzed for the expression of HEWL by a modified lysoplate assay for quantifying HEWL activity. Twenty-five plants exhibited HEWL activity up to 30 ng of HEWL per mg of leaf tissue. When using successive intercellular fluid (IF) extracts to study the localization of HEWL, it was found that no more than 10% of HEWL could be recovered in IF extracts. This is contrary to several extracellular proteins, such as the pathogenesis-related proteins. However, purified HEWL was recovered with the same yield as in transgenic tobacco leaves when it was injected into normal leaf tissue. Transgenic tobacco plants did not exhibit a phenotype change due to the expression of HEWL. The HEWL gene could be a useful reporter gene because the activity of the protein can be quantified by a simple assay.

Expression of bacterial chitinase protein in tobacco leaves using two photosynthetic gene promoters

A bacterial chitinase gene from Serratia marcescens (chiA) was fused to (i) a promoter of the ribulose bisphosphate carboxylase small subunit (rbcS) gene and (ii) two different chlorophyll a/b binding protein (cab) gene promoters from petunia. The resulting constructions were introduced into Agrobacterium Ti plasmid-based plant cell transformation vectors and used to generate multiple independent transgenic tobacco plants. ChiA mRNA and protein levels were measured in these plants. On average, the rbcS/chiA fusion gave rise to threefold more chiA mRNA than either cab/chiA fusion. We investigated the influence of sequences around the translational initiation ATG codon on the level of ChiA protein. The rbcS/chiA and cab/chiA fusions in which the sequence in the vicinity of the translational initiation codon is ACC ATGGC gave rise to transformants with higher levels of ChiA protein than those carrying a cab/chiA fusion with the sequence CAT ATGCG in the same region. This difference in translational efficiency is consistent with previous findings on preferred sequences in this region of the mRNA. In those transformants showing the highest level of ChiA expression, ChiA protein accumulated to about 0.25% of total soluble leaf protein. These plants contained significantly higher chitinase enzymatic activity than control plants.