Department of Plant Pathology and Microbiology, Texas AgriLife Research, Texas A&M System, Weslaco, TX 78596-8344, USA.
Genome. 2010 Oct;53(10):840-7. doi: 10.1139/g10-064.
The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5' flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking.
更广泛的启动子可用于调控有价值的转基因作物中的表达，这将有益于功能基因组学研究和当前旨在提高生产力的生物技术计划。基于聚合酶链反应（PCR）的基因组步移技术常用于分离启动子或与已知 cDNA 序列相邻的 5'侧翼基因组区域，这些技术适用于那些尚未完全测序的基因组。然而，当直接应用于具有高度复杂和庞大基因组的作物时，这些技术就会出现问题。本文描述了一种衔接子连接介导的基于 PCR 的 BAC 基因组步移方法，用于有效分离多基因家族成员的启动子，如在富含防御和纤维合成的DIRIGENT 基因的甘蔗茎中，这些基因在具有高度多倍体基因组的物种中很丰富。该方法的优势在于使用 BAC 基因组 DNA 作为衔接子连接介导的 PCR 步移模板，高效且特异性地扩增目标启动子。
