Activation Tagging

Insertional mutagenesis is one of the most effective approaches to determine the function of plant genes. However, due to genetic redundancy, loss-of-function mutations often fail to reveal the function of a member of gene families. Activation tagging is a powerful gain-of-function approach to reveal the functions of genes, especially those with high sequence similarity recalcitrant to loss-of-function genetic analyses. Activation tagging randomly inserts a T-DNA fragment containing engineered four copies of enhancer element into a plant genome to activate transcription of flanking genes. We recently generated a new binary vector, pBASTA-AT2 , which has been efficiently used to discover genes involved in BR biosynthesis, metabolism, and signal transduction. Compared to pSKI015 , a commonly used activation tagging vector, pBASTA-AT2 , contains a smaller size of T-DNA and a bigger number of unique restriction sites within the T-DNA region, making cloning of the flanking sequence a lot easier. Our analysis indicated that pBASTA-AT2 gives dramatically improved transformation efficiency relative to pSKI015 . In this article, detailed information about this activation tagging vector and the protocol for its application are provided. Three recommended gene cloning approaches based on the use of pBASTA-AT2 , including inverse PCR, thermal asymmetric interlaced PCR, and adaptor ligation-mediated PCR, are described to identify T-DNA insertion sites after selection of activation-tagged mutant plants.