School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK.
J Microbiol Methods. 2010 Aug;82(2):156-62. doi: 10.1016/j.mimet.2010.05.007. Epub 2010 May 24.
This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5' integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either beta-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.
本文描述了通过开发新的通用载体对现有担子菌分子工具包进行优化。这些载体通过允许启动子、编码区和终止子元件的轻松交换，使基因表达和沉默盒的构建变得简单和快速。这些构建体包含多个克隆位点（MCS），允许使用多种限制酶位点插入任何基因，并可选择 5' 内含子，以实现高效基因表达。我们通过在毛栓菌中进行标记基因表达来测试这些载体。这项工作还扩展了可用于毛栓菌的标记基因范围，首次报告了在毛栓菌中表达 DsRed 和单体红色荧光蛋白（mRFP），并进一步表明在一些标记基因的表达盒中需要内含子。然而，对含有β-葡聚糖酶（GUS）或荧光素酶（LUC）基因的转化体进行分析，无论是否有内含子，都没有检测到标记基因表达。因此，内含子的包含并不能保证表达，可能涉及其他遗传因素。
