Impact of Insulators On Single Copy Transgene Expression Variability in Sugarcane.

Sugarcane, a tropical C4 grass in the genus Saccharum(Poaceae), accounts for nearly 70% of sugar produced worldwide. Sugarcane is also an important feedstock for biofuel production. Brazil is currently the largest global producer of sugarcane-derived ethanol for fuel. In the U.S., Florida is the largest producer of cane sugar followed by Louisiana, Texas, and Hawaii. Sugarcane is a highly polyploidy and frequently aneuploidy interspecific hybrid. Its traditional breeding is difficult due to its complex genome, poor fertility, and the long breeding/selection cycle. Genetic transformation technology for the generation of transgenic sugarcane will complement traditional breeding in the development of advanced cultivars. Transgenic sugarcane plants with improved agronomic and value-added traits have already been reported. Commercialization of transgenic sugarcane is expected in the near future, which will benefit the U.S. and global sugar industries, in addition to the new cellulosic biofuel industries. 

Delivering the desired gene expression level is critical to basic plant biological research and crop improvement. Prediction of transgene performance is difficult due to random insertion into the genome and associated position effects, as well as variegated, ectopic, and silenced gene expression. In addition, enhancer–promoter interference may disturb the specificity and strength of promoters in applications where gene stacking is necessary for crop improvement.

Insulators have been identified which a) establish genomic barriers and thereby protect genes from the neighboring heterochromatin regions; b) block the activity of far-away located enhancers.  These two properties make effective insulators desirable in transgenic vector construction to ensure the stable and high expression of transgenes or precise comparison of different promoters by shielding them from neighboring enhancers with the help of insulators.

Our objective is to explore the influence of insulators on transgene expression in sugarcane.

    The series of experiments include: 1) Construct vectors which a) contain insulators to shield the transgene (nptII) expression cassette from the neighboring chromatin region and b) control vector which carries the nptII expression cassette without insulators. 2) Generate and identify single-copy transgenic lines from vectors with and without insulators following biolistic gene transfer. 3) Evaluate insulators’ performance through comparison of nptII expression levels of single copy lines of insulator or insulator lacking constructs.

So far independent transgenic lines with or without insulators flanking the nptII expression cassette were generated and NPTII expression of these lines was confirmed by NPTII-ELISA. Data on transgene copy number and relative expression levels following quantitative NPTII-ELISA will be presented.