Helliwell Chris A, Wesley S Varsha, Wielopolska Anna J, Waterhouse Peter M
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. Corresponding author; email:
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.
Funct Plant Biol. 2002 Oct;29(10):1217-1225. doi: 10.1071/FP02033.
A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.
植物生物学后基因组时代的一个主要挑战是确定植物基因组中所有基因的功能。解决这个问题的一个直接方法是减少或敲除一个基因的表达，期望看到一个暗示其功能的表型。插入诱变是这类研究的一个有用工具，但受到基因冗余、致死性敲除、无标签突变体以及无法将插入元件靶向特定基因的限制。许多研究小组已经证明，使用编码发夹RNA（hpRNA）的反向重复转基因构建体在植物中进行基因沉默的有效性，并且与插入诱变相比有几个优点。在本文中，我们描述了两种改进的pHellsgate载体，它们有助于快速生成编码hpRNA的构建体。pHellsgate 4允许从单个聚合酶链反应产物一步生产hpRNA构建体，而pHellsgate 8需要通过中间载体的两步过程。我们表明这些载体在沉默拟南芥中的三个内源基因，即开花位点C、八氢番茄红素去饱和酶和乙烯不敏感2方面是有效的。我们还表明，来自两个基因的序列构建体可以沉默这两个基因。
