Travella Silvia, Klimm Theres E, Keller Beat
Institute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland.
Plant Physiol. 2006 Sep;142(1):6-20. doi: 10.1104/pp.106.084517. Epub 2006 Jul 21.
Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.
插入诱变和基因沉默是确定基因功能的有效工具。与功能获得或功能缺失方法不同,RNA干扰(RNAi)诱导的基因沉默可能使多倍体中的多基因家族和同源基因沉默。这对于六倍体小麦(普通小麦)的功能研究非常重要,其中大多数基因至少有三个同源拷贝,传统的插入诱变无效。我们已将含有编码八氢番茄红素去饱和酶(PDS)或乙烯信号转导子乙烯不敏感2(EIN2)基因片段的双链RNA表达构建体导入面包小麦。转化植株表现出表型变化,这些变化至少在两代中稳定遗传。这些变化与二倍体模式植物中这两个基因的突变表型非常相似。定量实时聚合酶链反应显示,mRNA水平降低与表型严重程度增加之间存在良好的相关性。RNAi沉默对所有三个同源基因具有相同的定量效应。在纯合植株中观察到最严重的表型,这些植株的mRNA减少最强,有趣的是,与杂合植株相比,产生的小RNA量约为其两倍。这表明RNAi在六倍体小麦中的作用是基因剂量依赖性的。EIN2 mRNA水平低的小麦幼苗对乙烯不敏感。因此,EIN2是小麦乙烯信号通路的正调控因子,与其在拟南芥和水稻中的同源物非常相似。我们的数据表明,RNAi导致稳定遗传的表型,因此是多倍体小麦功能基因组学研究的有效工具。