High-throughput generation of an activation-tagged mutant library for functional genomic analyses in tobacco

作者：Liu Feng; Gong Daping; Zhang Qian; Wang Dawei; Cui Mengmeng; Zhang Zhiguo; Liu Guanshan; Wu Jinxia^*; Wang Yuanying

来源：Planta, 2015, 241(3): 629-640.

DOI：10.1007/s00425-014-2186-z

• Activation tagging
• Flanking sequence
• Functional genomics
• T-DNA
• Tobacco

Tobacco (Nicotiana tabacum L.) is an ideal model system for molecular biological and genetic studies. In this study, activation tagging was used to generate approximately 100,000 transgenic tobacco plants. Southern blot analysis indicated that there were 1.6 T-DNA inserts per line on average in our transformed population. The phenotypes observed include abnormalities in leaf and flower morphology, plant height, flowering time, branching, and fertility. Among 6,000 plants in the T-0 generation, 57 displayed obvious phenotypes. Among 4,105 lines in the T-1 generation, 311 displayed abnormal phenotypes. Fusion primer and nested integrated PCR was used to identify 963 independent genomic loci of T-DNA insertion sites in 1,257 T-1 lines. The distribution of T-DNA insertions was non-uniform and correlated well with the predicted gene density along each chromosome. The insertions were biased toward genic regions and noncoding regions within 5 kb of a gene. Fifteen plants that showed the same phenotype as their parent with a dominant pattern in the T-2 generation were chosen randomly to detect the expression levels of genes adjacent to the T-DNA integration sites by semi-quantitative RT-PCR. Fifteen candidate genes were identified. Activation was observed in 7 out of the 15 adjacent genes, including one that was located 13.1 kb away from the enhancer sequence. The activation-tagged population described in this paper will be a highly valuable resource for tobacco functional genomics research using both forward and reverse genetic approaches.

• 出版日期2015-3
• 单位中国农业科学院