Development of transgenic Egyptian cotton varieties using bacterial fructosyl transferase gene coding for fructan accumulation

摘要

Fructans are polyfructose molecules that function as non-structural storage carbohydrates in several plant species. Owing to their solubility, fructans may help cotton survive periods of osmotic stress induced by drought or cold. We developed transgenic Egyptian cotton plants (Gossypium barbadense), from long staple variety, Giza 85, and extra long staple variety, Giza 88 by introducing the bacterial sacB gene of Bacillus subtilis coding for fructosyl transferase enzyme fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene and the selectable marker gene bar encoding phosphoinothricin acetyl transferase enzyme which detoxifies Basta herbicide. The transformation was achieved by particle bombardment using shoot meristematic apices. R_0 plants acclimatized in the greenhouses were submitted to preliminary screening for chimeric transformants by painting the plant leaves with basta herbicide (200 mg/L) to confirm Bar gene integration. The bar gene was found to be incorporated into 30 percent of transformed cotton plants. Further analyses on the basta positive- basta putatively transformed cotton plants were performed using Southern blot analysis. cpy-sacB chimeric gene was labeled and used as a probe. Polymerase Chain Reaction (PCR) revealed the presence of the intact cpy-sacB chimeric gene in 8 and 7 cotton plants from both Giza 85 and 88, respectively. Genomic Southern blot analysis confirmed the integration of cpy-sacB gene into the plant genome. cpy-sacB was found to be integrated into only two plants out of one thousand plants of each cotton variety. Reverse transcriptase -polymerase chain reaction (RT-PCR) was carried out to permit the detection and quantification of RNA transcripts into the cotton plant genome.A 1.7 Kb cDNA fragment was detected using RT-PCR, which confirms the transcription of the chimeric cpy-sacB gene. Polyfructose molecules were detected in plant cotton tissue by HPLC and the increase of dry weight in fructose level was determined in putatively transgenic plants transformed with the fructosyl transferase gene compared to non-transgenic ones.