A versatile zero background T-vector system for gene cloning and functional genomics.

摘要: With the recent availability of complete genomic sequences many organisms, high-throughput and cost-efficient systems for gene cloning functional analysis are in great demand. Although site-specific recombination-based systems, such as Gateway technology, extremely useful efficient transfer DNA fragments into multiple destination vectors, two-step process is time consuming expensive. Here, we report a zero background TA system that provides simple high-efficiency direct PCR-amplified with almost no self-ligation. The improved T-vector takes advantage restriction enzyme XcmI to generate T-overhang after digestion negative selection marker ccdB eliminate self-ligation transformation. We demonstrate feasibility flexibility technology by developing set transient stable transformation vectors constitutive expression, silencing, protein tagging, subcellular localization detection, promoter fragment activity plants. Because can be easily adapted specialized expression other general, cost-efficient, platform complements genomics.